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Purpose: Originally isolated on the basis of its ability to induce p53, serdemetan showed potent activity in various preclinical models, inducing S-phase arrest and apoptosis in TP53 wild-type and mutant tumors. This study evaluated the safety and tolerability of serdemetan, determined the pharmacokinetic and pharmacodynamic profiles, and identified a recommended phase II dose.Patients and Methods: Patients (71) with refractory solid tumors were allocated to dose-escalating cohorts (3+3 patients each) and received oral serdemetan once daily in 21-day cycles to determine the maximum tolerated dose (MTD) and dose-limiting toxicities (DLT). Plasma was collected for pharmacokinetic analyses. Paired baseline and on-treatment skin and tumor biopsies were done; blood samples were collected for pharmacodynamic analyses, including p53 and macrophage inhibitory cytokine-1 induction.Results: The MTD of serdemetan was determined to be 350 mg once daily. During this study, grade 3 QTc prolongation was the most common DLT and nausea (66.2%) was the most frequent treatment-emergent adverse event. Serdemetan was rapidly absorbed after oral administration and exhibited dose-proportional pharmacokinetics. At steady state, mean maximum plasma concentration (Cmax) was 2,330 ng/mL and mean area under plasma concentration curve (AUC0–24h) was 43.0 μg.h/mL, with serdemetan 300 mg/d. There was a dose- and exposure-dependent p53 induction. One patient with breast cancer showed a partial response; 22 (38.6%) patients had stable disease.Conclusions: Serdemetan treatment was associated with p53 induction in both tumor and surrogate tissue pharmacodynamic studies and modest clinical activity. Although serdemetan was well tolerated with dose-proportional pharmacokinetics, exposure-related QTc liability was observed. Clin Cancer Res; 17(19); 6313–21. ©2011 AACR.

Supplementary Table 1 from Discovery of BMS-641988, a Novel and Potent Inhibitor of Androgen Receptor Signaling for the Treatment of Prostate Cancer

Supplementary Information and Figures 1-3 from Identification of Candidate Molecular Markers Predicting Sensitivity in Solid Tumors to Dasatinib: Rationale for Patient Selection

Supplementary Table 2 from Discovery of BMS-641988, a Novel and Potent Inhibitor of Androgen Receptor Signaling for the Treatment of Prostate Cancer

Supplementary Figure and Table Legends 1-2 from Discovery of BMS-641988, a Novel and Potent Inhibitor of Androgen Receptor Signaling for the Treatment of Prostate Cancer

The advent of immune oncology had a significant impact on the stratification of cancer patients in the past few years. Furthermore, immune cell phenotyping of the tumor microenvironment is becoming a tool not only for the identification of novel predictive biomarkers for cancer immunotherapy, but also for prognostic markers that may help to understand several mechanisms like invasion and epithelial-mesenchymal transition (EMT). We demonstrate how multiplexed immunofluorescence (mIF) assays and digital image analysis helped to investigate tumor budding in colorectal cancer by evaluating the tumor microenvironment and activated fibroblasts. Akoya Bioscience’s Opal detection system and fluorophores were optimized on LEICA Bond RX for use with human normal tonsil control tissues and FFPE colorectal cancer specimens. The 6-plex mIF panel included CD4 (clone SP35), CD8 (clone C8/144B), CD68 (clone PG-M1), FoxP3 (clone SP97), PD-L1 (clone SP263) and pan-Cytokeratin (clones AE1/AE3). In addition, FAP-alpha single-plex IHC was used to investigate reactivity of the stroma surrounding tumor cell buds. Each single marker of the mIF panel was independently validated for specificity regarding stripping efficacy and epitope stability, and for accuracy compared to single plex bright field immunohistochemistry. Automated image analysis and cellular phenotyping followed a workflow of custom Visiopharm apps. The tissue was segmented into regions of interest, such as areas with and without tumor cell budding, and bud microenvironment. Additional parameters like MSI (Microsatellite Instability) status and TNM classification were also taken into consideration for data analysis. The applied mIF immune cell panel and automated image analysis workflow identified a broad number of different immune cell phenotypes, including rare double- or triple-positive cell subtypes, as well as their spatial relation to tumor cell buds within the activated stroma region. This revealed new insights into the complexity of the tumor microenvironment in colorectal cancer around tumor cell buds, suggesting a potential effect of both the immune system and the stromal cells on tumor cell invasion into the surrounding tissue. Citation Format: Dirk Zielinski, Rebekka Vogtmann, Anne Siemon, Christina Koppel, Christina Schipper, Anastasiia Tereshchenko, Suso Platero, Hans-Ulrich Schildhaus. Multiplex immunofluorescence and image analysis to investigate the role of the immune contexture and fibroblast activation for tumor cell budding in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1018.

The landscape in immuno-oncology (I-O) has undergone profound changes since its early beginnings up through the rapid advances happening today. The current drug development pipeline consists of thousands of potential I-O therapies and therapy combinations, many of which are being evaluated in clinical trials. The efficient and successful development of these assets requires the investment in and utilization of appropriate tools and technologies that can facilitate the rapid transitions from preclinical evaluation through clinical development. These tools include (i) appropriate preclinical models, (ii) biomarkers of pharmacodynamic, predictive and monitoring utility, and (iii) evolving clinical trial designs that allow rapid and efficient evaluation during the development process. This article provides an overview of how novel discoveries and insights into each of these three areas have the potential to further address the clinical management needs for patients with cancer.

Bronchial premalignant lesions (PMLs) are precursors of lung squamous cell carcinoma, but have variable outcome, and we lack tools to identify and treat PMLs at risk for progression to cancer. Here we report the identification of four molecular subtypes of PMLs with distinct differences in epithelial and immune processes based on RNA-Seq profiling of endobronchial biopsies from high-risk smokers. The Proliferative subtype is enriched with bronchial dysplasia and exhibits up-regulation of metabolic and cell cycle pathways. A Proliferative subtype-associated gene signature identifies subjects with Proliferative PMLs from normal-appearing uninvolved large airway brushings with high specificity. In progressive/persistent Proliferative lesions expression of interferon signaling and antigen processing/presentation pathways decrease and immunofluorescence indicates a depletion of innate and adaptive immune cells compared with regressive lesions. Molecular biomarkers measured in PMLs or the uninvolved airway can enhance histopathological grading and suggest immunoprevention strategies for intercepting the progression of PMLs to lung cancer., Bronchial premalignant lesions can potentially progress to lung squamous cell carcinoma. Here, the authors profile bronchial biopsies from high-risk smokers by RNA sequencing and identify four molecular subtypes of premalignant lesions and an immune molecular signature that associates with lesion progression.

Fibroblast growth factor (FGF) signaling plays critical roles in key biological processes ranging from embryogenesis to wound healing and has strong links to several hallmarks of cancer. Genetic alterations in FGF receptor (FGFR) family members are associated with increased tumor growth, metastasis, angiogenesis, and decreased survival. JNJ-42756493, erdafitinib, is an orally active small molecule with potent tyrosine kinase inhibitory activity against all four FGFR family members and selectivity versus other highly related kinases. JNJ-42756493 shows rapid uptake into the lysosomal compartment of cells in culture, which is associated with prolonged inhibition of FGFR signaling, possibly due to sustained release of the inhibitor. In xenografts from human tumor cell lines or patient-derived tumor tissue with activating FGFR alterations, JNJ-42756493 administration results in potent and dose-dependent antitumor activity accompanied by pharmacodynamic modulation of phospho-FGFR and phospho-ERK in tumors. The results of the current study provide a strong rationale for the clinical investigation of JNJ-42756493 in patients with tumors harboring FGFR pathway alterations. Mol Cancer Ther; 16(6); 1010–20. ©2017 AACR.

Understanding the earliest molecular and cellular events associated with cancer initiation remains a key bottleneck to transforming our approach to cancer prevention and detection. While TCGA has provided unprecedented insights into the genomic events associated with advanced stage cancer, there have been few studies comprehensively profiling premalignant and early-stage disease or elucidating the role of the microenvironment in premalignancy and tumor initiation. In this article, we make a call for development of a “Pre-Cancer Genome Atlas (PCGA),” a concerted initiative to characterize the molecular alterations in premalignant lesions and the corresponding changes in the microenvironment associated with progression to invasive carcinoma. This initiative will require a multicenter coordinated effort to comprehensively profile (cellular and molecular) premalignant lesions and their corresponding “field of injury” collected longitudinally as the lesion progresses towards or regresses from frank malignancy across multiple tumor types. Genomic characterization of alterations in premalignant lesions and their microenvironment, for both bulk tissue and single cells, will enable development of biomarkers for early detection and risk stratification as well as allow for the development of novel targeted cancer interception strategies. The multi-institutional and multidisciplinary collaborative “big-data” effort underlying the PCGA will help usher in a new era of precision medicine for cancer detection and prevention. Cancer Prev Res; 9(2); 119–24. ©2016 AACR.

Purpose: Lung cancer is the leading cause of cancer-related death in the United States. The molecular events preceding the onset of disease are poorly understood, and no effective tools exist to identify smokers with premalignant lesions (PMLs) that will progress to invasive cancer. Prior work identified molecular alterations in the smoke-exposed airway field of injury associated with lung cancer. Here, we focus on an earlier stage in the disease process leveraging the airway field of injury to study PMLs and its utility in lung cancer chemoprevention. Experimental Design: Bronchial epithelial cells from normal appearing bronchial mucosa were profiled by mRNA-Seq from subjects with (n = 50) and without (n = 25) PMLs. Using surrogate variable and gene set enrichment analysis, we identified genes, pathways, and lung cancer–related gene sets differentially expressed between subjects with and without PMLs. A computational pipeline was developed to build and test a chemoprevention-relevant biomarker. Results: We identified 280 genes in the airway field associated with the presence of PMLs. Among the upregulated genes, oxidative phosphorylation was strongly enriched, and IHC and bioenergetics studies confirmed pathway findings in PMLs. The relationship between PMLs and squamous cell carcinomas (SCC) was also confirmed using published lung cancer datasets. The biomarker performed well predicting the presence of PMLs (AUC = 0.92, n = 17), and changes in the biomarker score associated with progression/stability versus regression of PMLs (AUC = 0.75, n = 51). Conclusions: Transcriptomic alterations in the airway field of smokers with PMLs reflect metabolic and early lung SCC alterations and may be leveraged to stratify smokers at high risk for PML progression and monitor outcome in chemoprevention trials. Clin Cancer Res; 23(17); 5091–100. ©2017 AACR.

Fibroblast growth factor receptor (FGFR) genetic alterations are frequently observed in cancer, suggesting that FGFR inhibition may be a promising therapy in patients harboring these lesions. Identification of predictive and pharmacodynamic biomarkers to select and monitor patients most likely to respond to FGFR inhibition will be the key to clinical development of this class of agents. Sensitivity to FGFR inhibition and correlation with FGFR pathway activation status were determined in molecularly annotated panels of cancer cell lines and xenograft models. Pathway inhibition in response to FGFR inhibitor treatment was assessed in cell lines (both in vitro and in vivo) and in samples from patients treated with the FGFR inhibitor JNJ-42756493 (erdafitinib). Frequency of FGFR aberrations was assessed in a panel of NSCLC, breast, prostate, ovarian, colorectal, and melanoma human tumor tissue samples. FGFR translocations and gene amplifications present in clinical specimens were shown to display potent transforming activity associated with constitutive pathway activation. Tumor cells expressing these FGFR activating mutants displayed sensitivity to the selective FGFR inhibitor erdafitinib and resulted in suppression of FGFR phosphorylation and downstream signal transduction. Clinically, patients receiving erdafitinib showed decreased Erk phosphorylation in tumor biopsies and elevation of serum phosphate. In a phase I study, a heavily pretreated bladder cancer patient with an FGFR3–TACC3 translocation experienced a partial response when treated with erdafitinib. This preclinical study confirmed pharmacodynamics and identified new predictive biomarkers to FGFR inhibition with erdafitinib and supports further clinical evaluation of this compound in patients with FGFR genetic alterations. Mol Cancer Ther; 16(8); 1717–26. ©2017 AACR.

Purpose: Originally isolated on the basis of its ability to induce p53, serdemetan showed potent activity in various preclinical models, inducing S-phase arrest and apoptosis in TP53 wild-type and mutant tumors. This study evaluated the safety and tolerability of serdemetan, determined the pharmacokinetic and pharmacodynamic profiles, and identified a recommended phase II dose. Patients and Methods: Patients (71) with refractory solid tumors were allocated to dose-escalating cohorts (3+3 patients each) and received oral serdemetan once daily in 21-day cycles to determine the maximum tolerated dose (MTD) and dose-limiting toxicities (DLT). Plasma was collected for pharmacokinetic analyses. Paired baseline and on-treatment skin and tumor biopsies were done; blood samples were collected for pharmacodynamic analyses, including p53 and macrophage inhibitory cytokine-1 induction. Results: The MTD of serdemetan was determined to be 350 mg once daily. During this study, grade 3 QTc prolongation was the most common DLT and nausea (66.2%) was the most frequent treatment-emergent adverse event. Serdemetan was rapidly absorbed after oral administration and exhibited dose-proportional pharmacokinetics. At steady state, mean maximum plasma concentration (Cmax) was 2,330 ng/mL and mean area under plasma concentration curve (AUC0–24h) was 43.0 μg.h/mL, with serdemetan 300 mg/d. There was a dose- and exposure-dependent p53 induction. One patient with breast cancer showed a partial response; 22 (38.6%) patients had stable disease. Conclusions: Serdemetan treatment was associated with p53 induction in both tumor and surrogate tissue pharmacodynamic studies and modest clinical activity. Although serdemetan was well tolerated with dose-proportional pharmacokinetics, exposure-related QTc liability was observed. Clin Cancer Res; 17(19); 6313–21. ©2011 AACR.

On 1–2 December 2010 worldwide leaders in the field of co-diagnostics gathered in Boston, MA, USA, to discuss the state of predictive medicine and the complexities of biomarker implementation and partnering. Flanking the primary conference were three workshops where participants could increase their understanding of companion diagnostic commercialization, best practices from well-known case studies and regulatory guidance and practice. The primary conference reviewed here consisted of 18 lectures and one round table discussion. Topics for the summit included biomarker discovery and validation, partnering between pharmaceutical development and diagnostics companies, commercialization models and evolving regulatory perspectives and guidance. While it is clear that there are many difficulties remaining in making personalized medicine a broad-based reality for all therapeutic areas, it is equally clear that much progress has already been made and that the conference participants are optimistic and dedicated to fulfilling the personalized medicine promise of bringing the right drug to the right patient at the right time.

Squamous cell carcinoma (SCC) of the lung is a leading cause of cancer mortality in the U.S. due to late-stage diagnosis and lack of effective treatments. Lung SCC arises in the epithelial layer of the bronchial airways and is often preceded by the development of premalignant lesions (PMLs). The molecular alterations involved in the progression of PMLs to lung SCC are not clearly understood as not all PMLs progress to carcinoma. We hypothesize that molecular characterization of PMLs and nonlesion areas will allow us to identify alterations associated with histology and lesion progression. We used mRNA sequencing to profile biopsies obtained from high-risk smokers undergoing lung cancer screening by auto-fluorescence bronchoscopy and CT at the Roswell Park Cancer Institute in Buffalo, NY. For each subject (n=49), a brushing of the airway field (normal fluorescing area) and endobronchial biopsies were collected over time in repeat locations with serial bronchoscopies. The discovery cohort, included 29 subjects, 197 biopsies, and 91 brushes, while the validation cohort included 20 subjects, 111 biopsies and 49 brushes. The mRNA-Seq data were aligned to hg19 using STAR, and gene/transcript levels were summarized using RSEM. Immune, stromal, and epithelial cell content were inferred using xCell. Biopsy molecular subtypes were discovered using consensus clustering in the discovery cohort and used to train a nearest centroid subtype predictor to assign subtypes in the validation cohort and the brushes. We identified four distinct molecular subtypes in the discovery cohort bronchial biopsies using genes (n=3936) co-expressed across the the discovery cohort brushes and biopsies and two additional RNA-seq lung SCC-related datasets. One of the four molecular subtypes is enriched (p We have identified four molecular subclasses of premalignant lung SCC lesions that may associate with prognosis. Molecular classification of PMLs may lead to biomarkers of future disease progression that could be used to stratify patients into prevention trials and to monitor efficacy of the treatment. Additionally, the results suggest that personalized lung cancer chemoprevention that targets specific cancer-related pathways or the immune system may have potential therapeutic benefits. Citation Format: Jennifer E. Beane, Sarah Mazzilli, Ania Tassinari, Joshua Campbell, Christopher Moy, Michael Schaffer, Catalina Perdomo, David Jenkins, Mary Beth Pine, Gang Liu, Sherry Zhang, Hangqio Lin, Jessica Vick, Evan Johnson, Suso Platero, Christopher Stevenson, Marc Lenburg, Mary Reid, Samjot Dhillon, Avrum Spira. Bronchial premalignant lesions have distinct molecular subtypes associated with future histologic progression [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr A05.

Background: Lung squamous cell carcinoma (SqCC) arises in the epithelial layer of the bronchial airway and is often preceded by the development of premalignant lesions. However, not all premalignant lesions progress to lung SqCC and many will regress spontaneously. Understanding the somatic alterations and molecular subtypes associated with progression will allow us to identify biomarkers for early detection and develop therapeutic strategies for disease prevention and interception. Methods: Biopsies were obtained from high-risk smokers undergoing lung cancer screening by auto-fluorescence bronchoscopy and CT at the Roswell Park Cancer Institute. For each subject, multiple sites were sampled repeatedly over time. One biopsy from each region was sent for pathological review while another biopsy was taken for molecular studies. Whole-exome sequencing (WES) was performed at Uniform Services University to 120x coverage and RNA-seq was performed at Boston University School of Medicine. Results: The median number of somatic mutations across all premalignant lesions that underwent DNA-seq (150 biopsies from 20 subjects) was 0.45 per megabase and displayed a modest association with histological grade (p=0.05). The most frequently mutated known lung cancer genes included NOTCH1 (14%), TP53 (6%), FAT1 (3%), PIK3CA (2%), KRAS ( Citation Format: Joshua D. Campbell, Xijun Zhang, Catalina Perdomo, Sarah Mazzilli, Yaron Geshalter, Samjot S. Dhillon, Gang Liu, Sherry Zhang, Hanqiao Liu, Jessica Vick, Christopher Moy, Stefano Monti, Evan Johnson, Matthew Meyerson, Matthew Wilkerson, Clifton Dalgard, Suso Platero, Chris Stevenson, Marc Lenburg, Mary Reid, Jennifer Beane, Avrum Spira. Genomic characterization of premalignant lung squamous cell carcinoma lesions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3248.

The process of neovascularization from preexisting blood vessels, referred to as angiogenesis, plays a critical role in both tumor growth and dissemination in multiple cancer types. Currently, there exists a need to identify biomarkers that can both indicate biological activity and predict efficacy at the molecular level for antiangiogenesis drugs which are anticipated to result in tumor stasis rather than regression. To identify such biomarkers, athymic mice bearing L2987 human tumor xenografts were treated with the antiangiogenic agent brivanib alaninate, which is currently under clinical evaluation. This is an orally available and selective tyrosine kinase inhibitor that targets the key angiogenesis receptors vascular endothelial growth factor receptor 2 (VEGFR-2) and fibroblast growth factor receptor 1. In the described studies, tumor samples were collected from these xenografts and RNA was extracted for gene expression profiling on Affymetrix 430A mouse GeneChips. Statistical analysis was done using a defined set of genes identified to be coexpressed with VEGFR-2 from a clinical tumor gene expression profiling database and between tumor samples isolated from brivanib alaninate–treated and untreated mice. Tyrosine kinase receptor 1 (Tie-1), collagen type IV α1 (Col4a1), complement component 1, q subcomponent receptor 1 (C1qr1), angiotensin receptor–like 1 (Agtrl1), and vascular endothelial-cadherin (Cdh5) were all identified to be significantly modulated by treatment with brivanib alaninate. These genes, which may be potentially useful as markers of brivanib alaninate activity, were further studied at the protein level in two separate in vivo human colon tumor xenograft models, HCT116 and GEO, using immunohistochemistry-based approaches. [Cancer Res 2007;67(14):6899–906]

Purpose JNJ-42756493 is an orally administered pan-fibroblast growth factor receptor (FGFR) tyrosine kinase inhibitor. This first-in-human study evaluates the safety, pharmacokinetics, and pharmacodynamics and defines the recommended phase II dose (RP2D) of JNJ-42756493. Patients and Methods Eligible patients with advanced solid tumors received escalating doses of JNJ-42756493 from 0.5 to 12 mg administered continuously daily or JNJ-42756493 10 or 12 mg administered intermittently (7 days on/7 days off). Results Sixty-five patients were enrolled. The most common treatment-emergent adverse events included hyperphosphatemia (65%), asthenia (55%), dry mouth (45%), nail toxicity (35%), constipation (34%), decreased appetite (32%), and dysgeusia (31%). Twenty-seven patients (42%) experienced grade ≥ 3 treatment-emergent adverse events, and one dose-limiting toxicity of grade 3 ALT elevation was observed at 12 mg daily. Maximum-tolerated dose was not defined. Nine milligrams daily was considered as the initial RP2D; however, tolerability was improved with intermittent schedules, and 10 mg administered on a 7-days-on/7-days-off schedule was considered the final RP2D. Pharmacokinetics were linear, dose proportional, and predictable, with a half-life of 50 to 60 hours. Dose-dependent elevations in serum phosphate, a manifestation of pharmacodynamic effect, occurred in all patients starting at 4 mg daily. Among 23 response-evaluable patients with tumor FGFR pathway alterations, four confirmed responses and one unconfirmed partial response were observed in patients with glioblastoma and urothelial and endometrial cancer (all with FGFR2 or FGFR3 translocations); 16 patients had stable disease. Conclusion JNJ-42756493 administered at 10 mg on a 7-days-on/7-days-off schedule achieved exposures at which clinical responses were observed, demonstrated pharmacodynamic biomarker activity, and had a manageable safety profile.

Background: Squamous cell carcinoma (SCC) of the lung is a leading cause of cancer mortality in the US due to late stage diagnosis and lack of effective treatments. Lung SCC arises in the epithelial layer of the bronchial airways and is often preceded by the development of premalignant lesions (PMLs). The molecular events involved in the progression of PMLs to lung SCC are not clearly understood, and not all PMLs go on to form carcinoma. By molecularly characterizing PMLs and non-lesion areas in the airway of individuals with PMLs, we hypothesize that we will be able to identify subgroups of PMLs that are more likely to progress. Methods: We used mRNA sequencing to profile biopsies obtained from high-risk smokers undergoing lung cancer screening by auto-fluorescence bronchoscopy and CT at the Roswell Park Cancer Institute in Buffalo, NY. For each subject (n=30), we sampled bronchial biopsies repeatedly over time (394 +/- 170 days) with serial bronchoscopies (6 +/- 5 biopsies/subject) as the biopsied area progressed towards or regressed away from frank malignancy. mRNA-Seq (n=197 biopsies) data was aligned to hg19 using STAR, and gene/transcript levels were summarized using RNA-Seq using RSEM and Ensembl 74 annotation. Immune, stromal, and epithelial cells content were inferred using the ESTIMATE algorithm and pathway activity of in vitro derived oncogenic signatures was estimated using GSVA for each sample. Molecular subtypes were derived using non-negative matrix factorization (NMF) and consensus clustering. Linear modeling was used to associate PML outcome metrics and pathway activity scores with subtype membership. Results: We identified six distinct molecular subtypes of bronchial biopsies using NMF across the 10% most variable genes (n=2,322 gene). One subtype contained samples with stable high-grade histology (p Conclusions: Molecular classification of premalignant lesions may lead to biomarkers of disease progression that could be used to stratify patients into prevention trials and to monitor efficacy of the treatment. Additionally, the results suggest that personalized interventions targeting specific cancer-related pathways or the immune system may be have potential therapeutic benefits. Citation Format: Jennifer E. Beane, Sarah Mazzilli, Joshua Campbell, Christopher Moy, Michael Schaffer, Catalina Perdomo, David Jenkins, Gang Liu, Sherry Zhang, Hanqiao Liu, Jessica Vick, Evan Johnson, Suso Platero, Marc Lenburg, Mary Reid, Samjot S. Dhillon, Avrum Spira. Premalignant squamous cell lung carcinoma lesions have distinct molecular subtypes associated with histologic progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5002. doi:10.1158/1538-7445.AM2017-5002

Background: Lung squamous cell carcinoma (SqCC) arises in the epithelial layer of the bronchial airway and is often preceded by the development of premalignant lesions. However, not all premalignant lesions progress to lung SqCC and many regress without therapeutic intervention. Understanding the somatic alterations that contribute to progression of premalignant lesions in the airway will allow us to identify biomarkers for early detection and develop therapeutic strategies for early intervention. Methods: Airway biopsies were obtained from high-risk smokers undergoing lung cancer screening by auto-fluorescence bronchoscopy and chest CT at the Roswell Park Cancer Institute. For each subject (n=30), multiple premalignant lesions were sampled repeatedly over time (n=144 samples). One biopsy from each region was sent for pathological review while another biopsy was taken for molecular studies. DNA was also isolated from the blood or cytologically normal bronchial brushings to serve as a matched normal control. Exome capture was performed using the Illumina TruSeq Rapid Exome kit and sequenced to a mean depth of coverage of 120x at Uniform Services University and Walter Reed National Military Medical Center. Results: The median number of somatic mutations across all premalignant lesions was 0.73 per megabase (range: 0.10 - 9.8 per Mb) and displayed a modest association with histological grade (p=0.07). The most frequently mutated lung cancer genes included KMT2C (12%), NOTCH1 (11%), FAT1 (6%), TP53 (5%), and CDKN2A (7/Mb) were taken from adjacent sites over two time points in the same individual with a prior history of lung squamous cell carcinoma. These lesions had a significantly overlapping set of mutations including FAT1 indicating a common evolutionary ancestor. Conclusions: The somatic alterations observed in known cancer genes such as TP53, KMT2C, NOTCH1, and FAT1 may be among the earliest driver events in lung SqCC development and may be useful as biomarkers for early detection as well as targets for lung cancer interception. Citation Format: Joshua Campbell, Xijun Zhang, Samjot S. Dhillon, Catalina Perdomo, Sarah Mazzilli, Yaron Geshalter, Gang Liu, Sherry Zhang, Hanqiao Lin, Jessica Vick, Christopher Moy, Stefano Monti, Evan Johnson, Matthew Meyerson, Steven Dubinett, Suso Platero, Matthew Wilkerson, Clifton Dalgard, Marc Lenburg, Mary Reid, Jennifer Beane, Avrum Spira. The genomic landscape of premalignant lung squamous cell carcinoma lesions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3259. doi:10.1158/1538-7445.AM2017-3259

Centromeres of most organisms are embedded within constitutive heterochromatin, the condensed regions of chromosomes that account for a large fraction of complex genomes. The functional significance of this centromere–heterochromatin relationship, if any, is unknown. One possibility is that heterochromatin provides a suitable environment for assembly of centromere components, such as special centromeric nucleosomes that contain distinctive histone H3-like proteins. We describe a Drosophila H3-like protein, Cid (for centromere identifier) that localizes exclusively to fly centromeres. When the cid upstream region drives expression of H3 and H2B histone–green fluorescent protein fusion genes in Drosophila cells, euchromatin-specific deposition results. Remarkably, when the cid upstream region drives expression of yeast, worm, and human centromeric histone–green fluorescent protein fusion proteins, localization is preferentially within Drosophila pericentric heterochromatin. Heterochromatin-specific localization also was seen for yeast and worm centromeric proteins constitutively expressed in human cells. Preferential localization to heterochromatin in heterologous systems is unexpected if centromere-specific or site-specific factors determine H3-like protein localization to centromeres. Rather, the heterochromatic state itself may help localize centromeric components.

We repeatedly released a distal block of heterochromatin lacking a natural centromere in mitotic cells and assayed its segregation. At anaphase, control acentric fragments typically remained unoriented between daughter nuclei and were subsequently lost. Fragments containing the brown Dominant ( bw D ) heterochromatic element displayed regular anaphase movement upon release. These fragments were found to segregate and function based on both cytological and phenotypic criteria. We also found that intact bw D -containing chromosomes normally display occasional dicentric behavior, suggesting that bw D has centromeric activity on the intact chromosome as well. Our findings suggest that centromere competence is innate to satellite-containing blocks of heterochromatin, challenging models for centromere identity in which competence is an acquired characteristic.

Over past two decades, an explosion in our molecular understanding of cancer has guided oncology therapeutic development and led to exciting breakthroughs in patient care. Throughout the pharmaceutical industry, the new paradigm for drug development now focuses on targeted therapies and related companion diagnostic assays. In contrast to nonspecific cytotoxic agents, these drugs harness our understanding of cancer pathways to precisely modulate oncogenic targets. In conjunction, assays to determine the most suitable patients for a particular therapy have ushered in the era of personalized medicine for oncology. Here, we explore the history of targeted drug development, how it has been shaped by pharmacogenomics, and the challenges and opportunities that lie ahead for pharmacogenomics in oncology.

Lung squamous cell carcinoma (SCC) arises in the epithelial layer of the bronchial airways and is preceded by the development of premalignant lesions (PMLs). The molecular events involved in the progression of PMLs to lung SCC are not clearly understood as not all PMLs that develop go on to form carcinoma. In addition, the majority of lung cancer chemoprevention agents tested to date are ineffective. Molecular characterization of the airway field of injury in individuals with PMLs could provide novel insights into the earliest molecular events associated with carcinogenesis and identify biomarkers to guide lung cancer detection and chemoprevention. RNA-sequencing was conducted on cytologically normal airway brushings from current and former smokers with (n = 50) and without (n = 25) PMLs as part of the British Columbia Lung Health Study. Linear modeling strategies were used to identify 280 differentialy expressed genes at FDR This is the first study to comprehensively profile gene expression changes in airway epithelial cells in the presence of PMLs. A subset of these changes reflects the earliest changes in the process of lung squamous cell carcinogenesis including the genes involved in cellular metabolism. However, the molecular alterations in the field of injury are dynamic as bronchial lesions either progress or regress these changes may be leveraged to monitor efficacy in chemoprevention trials. In addition monitoring molecular changes in high-risk smokers may identify smokers with PMLs that should receive lung cancer screening as well as lay the foundation for personalized lung cancer chemoprevention. Citation Format: Sarah A. Mazzilli, Ania Tassinari, Yaron Gethalter, Gang Lui, Mary Pine, Stephen Lam, Mary Reid, Suso Platero, Marc Lenburg, Avrum Spira, Jennifer Beane. The airway field of injury reflects gene expression changes associated with the presence of lung squamous premalignant lesions. [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 896.

Background: Lung squamous cell carcinoma (SqCC) arises in the epithelial layer of the bronchial airways and is often preceded by the development of premalignant lesions. However, not all premalignant lesions will progress to lung SqCC and many of these lesions will regress without therapeutic intervention. Understanding the molecular events that contribute to progression of premalignant lesions in the airway will allow us to identify biomarkers for early detection and develop therapeutic strategies for early intervention. Methods: Bronchial brushings and biopsies were obtained from high-risk smokers undergoing lung cancer screening by auto-fluorescence bronchoscopy and CT at the Roswell Park Cancer Institute. For each subject (n = 30), both premalignant lesions (PMLs) and the cytologically normal mainstem bronchus were sampled repeatedly over time (n = 288 samples). DNA and RNA were isolated from a total of 197 bronchial biopsies of PML (average of 5 per subject) and 91 bronchial brushings. DNA was also isolated from the blood to serve as a matched normal. Exome capture was performed using the Agilent SureSelect Human All Exon+UTR 70MB kit and sequenced to a mean depth of coverage of 75x (n = 85 samples from 22 subjects). RNA libraries were prepared with Illumina TruSeq (mRNA-Seq: n = 288 samples from 30 subjects and miRNA-Seq: n = 183 samples from 26 subjects). Results: We identified gene and miRNA expression changes associated with histological grade as well as progressive/stable disease. The Hippo pathway, Wnt signaling, p53 signaling, and immune-related pathways are modulated with histological grade and disease progression. Genes associated with histological grade in the cytologically normal airway and in the biopsies were significantly concordantly enriched (FDR3/Mb) were taken from adjacent sites over two time points in the same individual with a history of lung squamous cell carcinoma. These lesions had a significantly overlapping set of mutations (p = 2.2 × 10−17) indicating a common evolutionary ancestor, and contained mutations in CREBBP and FAT1, suggesting they are at increased risk for progressing to frank malignancy. Conclusions: We performed genomic profiling of PMLs in the airways of high-risk smokers. The gene expression and somatic alterations that were observed in known cancer genes may be among the earliest events in cancer development. Citation Format: Joshua D. Campbell, Catalina Perdomo, Sarah Mazzilli, Yaron Geshalter, Samjot S. Dhillon, Gang Liu, Sherry Zhang, Hangqio Lin, Jessica Vick, Christopher Moy, Evan Johnson, Matthew Meyerson, Suso Platero, Marc Lenburg, Mary Reid, Avrum Spira, Jennifer Beane. Genomic characterization of premalignant lung squamous cell carcinoma lesions. [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 895.

Identification of soluble biomarkers has become a critical non-invasive approach for disease diagnosis and monitoring in the treatment of solid tumors. Non-Small Cell Lung Cancer (NSCLC) is the most common form of lung cancer, accounting for approximately 85% of all lung cancers worldwide. The EGFR family of genes is known to be highly expressed in NSCLC tumors and play a critical role in the progression of the disease. Although patients initially respond well to EGFR TKI therapy, acquired resistance occurs through several mechanisms, including compensatory cMET pathway activation (Robinson et al. 2013). Previous reports have demonstrated that soluble forms of EGFR, Her2 and cMET are generated through either alternate splicing of mRNA or proteolytic cleavage of the full length receptors (Wilken et al. 2013). Serum concentrations of these receptors can be correlated with prognosis as well as treatment response (Gregorc, 2004, Kasahara, 2010 Heinmoller, 2003). Soluble receptor ligands, including Hepatocyte growth factor (HGF), have also been evaluated in NSCLC and a strong correlation has also been observed between the levels of serum HGF and disease outcome in patients treated with EGFR-TKIs (Kasahara et al. 2010). In this study, we evaluated concentrations of sEGFR, sMET, sHER2,, and HGF in NSCLC versus healthy normal serum samples via meso scale discovery (MSD) platform. Inventoried and custom MSD assays were optimized to measure these proteins in a training set consisting of 19 NSCLC and 16 normal healthy serum samples. Significantly lower concentrations of sEGFR and sHER2 were observed in the NSCLC serum samples compared to normal (p value = 0.0092 for both receptors), whereas, in contrast, the levels of HGF were significantly higher in the NSCLC patient samples (p value Citation Format: Dana S. Gaffney, Katherine Bell, Gabriela Martinez, Jayaprakash Karkera, Suso Platero. Detection of soluble protein biomarkers in NSCLC serum samples by meso scale discovery electrochemiluminescence platform. [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 5027.

Fibroblast growth factors (FGFs) are a family of homologous secreted glycoproteins involved in signaling pathways responsible for embryonic development, cell proliferation, survival, and migration. FGF activity is mediated by four transmembrane fibroblast growth factor receptors (FGFRs) which are receptor tyrosine kinases. Typically, FGF binding induces FGFR dimerization, leading to phosphorylation of the intracellular tyrosine kinase domain. This leads to downstream activation of multiple signaling pathways, including the mitogen-activated protein kinase (MAPK), PI3K/AKT, signal transducer and activator of transcription (STAT), and phospholipase-C-γ cascades. Deregulated FGFR activity, through mutations or translocations, is often associated with oncogenic events. Aberrations in FGFR genes have been observed in several tumor types including bladder, gastric, colorectal, ovarian, and hematologic cancers. To date, several FGFR3 mutations have been identified in bladder cancer. In this study, we developed a TaqMan qRT-PCR-based approach to detect four FGFR3 mutations in formalin-fixed paraffin embedded tissue (FFPET) samples. Cell lines overexpressing FGFR3 mutations were generated to determine impact on cell signaling, and sensitivity to the small-molecule pan-FGFR inhibitor JNJ-42756493. To determine the role and significance of these FGFR3 mutations in cancer, mutation expression constructs were designed and individually transfected into normal rat kidney epithelial cells. Cells harboring the FGFR3 mutations exhibited anchorage-independent growth, increased proliferation, and showed increased sensitivity to the FGFR inhibitor JNJ-42756493 in vitro compared to parental lines. These findings underline the oncogenic potential of the FGFR3 mutation genes and highlight their unique potential as predictive biomarkers in the selection of patients for FGFR-targeted therapy. Citation Format: Gabriela Martinez Cardona, Dana Gaffney, Katherine Bell, Joseph Portale, Matthew Dunworth, Matthew Lorenzi, Suso Platero, Jayaprakash Karkera. FGFR3 mutations as novel oncogenic targets. [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 3990.

Despite an excellent initial response to first-line hormonal treatment, most patients with metastatic prostate cancer will succumb to a hormone-refractory form of the disease. Because these tumors are still dependent on a functional androgen receptor (AR), there is a need to find novel and more potent antiandrogens. While searching for small molecules that bind to the AR and inhibit its transcriptional activity, BMS-641988 was discovered. This novel antiandrogen showed an increased (>1 log) potency compared with the standard antiandrogen, bicalutamide, in both binding affinity to the AR and inhibition of AR-mediated transactivation in cell-based reporter assays. In mature rats, BMS-641988 strongly inhibited androgen-dependent growth of the ventral prostate and seminal vesicles. In the CWR-22-BMSLD1 human prostate cancer xenograft model, BMS-641988 showed increased efficacy over bicalutamide (average percent tumor growth inhibition >90% versus

Preface. Contributors. 1 MOLECULAR PATHOLOGY AND DRUG DEVELOPMENT (Franz Fogt and J. Suso Platero). 1.1. General Pathology. 1.2. General Aspects. 1.3. Molecular Pathology, the Molecular Way. 1.4. Application of Molecular Pathology. 1.5. Molecular Pathology in Drug Development. 1.6. Pharmaceutical Drug Development. References. 2 MOLECULAR PATHOLOGY IN ONCOLOGY TARGET AND DRUG DISCOVERY (Rolf-P. Ryseck, Ricardo Attar, Matthew V. Lorenzi, and Brent A. Rupnow). 2.1. Introduction. 2.2. History of Chemotherapy and Cancer Drug Discovery. 2.3. Target-Based Drug Discovery. 2.4. Utilization of Molecular Pathology in the Discovery of Novel Cancer Targets. 2.5. Hit Identifi cation and In Vitro Lead Optimization. 2.6. Implications for Molecular Pathology in Cancer Drug Development and Use. 2.7. Summary and Future Considerations. References. 3 MOLECULAR PATHOLOGY AND TRANSCRIPTIONAL PROFILING IN EARLY DRUG DEVELOPMENT (Cornelia Liedtke, Lajos Pusztai, and W. Fraser Symmans). 3.1. Introduction. 3.2. Biomarkers in Clinical Setting and in Early Drug Development. 3.3. Advantages of Biomarker Implementation. 3.4. Changing Paradigm in Clinical Drug and Biomarker Development. 3.5. Promises of Transcriptional Profiling. 3.6. Biomarker Development and Validation Using Microarray Analysis. 3.7. Neoadjuvant Chemotherapy as an Intriguing Model for Biomarker Development. 3.8. Transcriptional Profi ling for Identifi cation of Individual Genes as Biomarkers. 3.9. Transcriptional Profi ling for the Definition of Multigene Predictors Using .Transcriptional Profiling. 3.10. Novel Tools for Pathway Analysis. 3.11. Implementation of Biomarkers into the Clinical Setting. 3.12. Conclusion. References. 4 MOLECULAR PATHOLOGY IN NONCLINICAL SAFETY ASSESSMENT (Richard A. Westhouse). 4.1. Introduction. 4.2. Drug Development. 4.3. Drug Discovery. 4.4. Biopharmaceuticals. 4.5. Summary. References. 5 TOXICOGENOMICS IN DRUG DEVELOPMENT (Wayne R. Buck and Eric A. G. Blomme). 5.1. Introduction. 5.2. Brief Overview of Large-Scale Gene Expression Technologies. 5.3. Analysis of Microarray Data. 5.4. Application of Toxicogenomics in Drug Development. 5.5. Considerations for Toxicogenomic Study Design. 5.6. Overview of Major Regulatory Developments Related to Use of Toxicogenomics in Drug Discovery and Development. 5.7. Summary. References. 6 MOLECULAR PATHOLOGY AS A WAY TO FIND THE RIGHT DOSE FOR A DRUG (F. Rojo, A. Rovira, S. Serrano, and J. Albanell). 6.1. Introduction. 6.2. Anti-EGFR-Targeted Therapies: The Pharmacodynamic Experience. 6.3. Molecular Pathology with Small Molecules Gefitinib and Erlotinib. 6.4. Molecular Pathology with Cetuximab and Other Monoclonal Antibodies to EGFR. 6.5. Proteasome Inhibitors: Pharmacodynamics on Blood Samples. 6.6. Pharmacodynamics with Rapamycin Analogs. 6.7. Second Generation of Targeted Therapies: Multitarget Agents. 6.8. Conclusions and Perspectives: Phase 0 Clinical Trials. References. 7 MOLECULAR PATHOLOGY IN LIFE-CYCLE MANAGEMENT IN DRUG DEVELOPMENT (Martha Quezado, Carlos A. Torres-Cabal, and David Berman). 7.1. Introduction. 7.2. Molecular Pathology Techniques. 7.3. Practical Applications of Molecular Pathology Biomarkers. 7.4. Conclusion. References. 8 MOLECULAR PATHOLOGY AND MOLECULAR THERAPY (Hewei Li). 8.1. Introduction. 8.2. Molecular Therapy Strategies. 8.3. Molecular Therapy Clinical Trials. References. 9 MOLECULAR PATHOLOGY: IMMUNOHISTOCHEMISTRY ASSAYS IN DRUG DEVELOPMENT PERFORMED BY A CONTRACT RESEARCH LABORATORY (Frank Lynch and Steve Bernstein). 9.1. Immunohistochemistry Is the Technique of Microscopic Visualization of Target Proteins in Tissue Sections Using Specific Antibodies. 9.2. Basics of the IHC Assay. 9.3. Immunohistochemistry Assay Development. 9.4. Sending a Study to a Contract Laboratory vs. Running In-house. 9.5. Choosing and Working with an Outside Laboratory- Keys for a Succe.ssful Relationship-What to Do before a Slide Is Stained. 9.6. Running and Managing Outsourced Clinical Studies 9.7. Applications of IHC in Drug Discovery and Development Process. 9.8. Conclusion. References. 10 QUANTIFICATION OF MOLECULAR PATHOLOGY: COLORIMETRIC IMMUNOHISTOCHEMISTRY (Raphael Marcelpoil). 10.1. Introduction. 10.2. Imaging Devices and Systems. 10.3. Quantifi cation: Introduction to Colorimetric Image Analysis. 10.4. Measuring Colorimetric Information. 10.5. Chromogen Separation. 10.6. Measuring Information. 10.7. Conclusion. References. 11 AQUA(R) TECHNOLOGY AND MOLECULAR PATHOLOGY (Mark Gustavson, Marisa Dolled-Filhart, Jason Christiansen, Robert Pinard, and David Rimm). 11.1. Introduction. 11.2. AQUA Technology-How It Works. 11.3. Standardization. 11.4. Quantification. 11.5. Localization. 11.6. Multiparametric Analysis. 11.7. Application of AQUA Technology to Drug Discovery and Companion Diagnostics. 11.8. Summary and Conclusions. References. Index.

The fibroblast growth factor receptors (FGFR) play a key role during development and in adult function. FGFRs belong to a family of receptor tyrosine kinases which are single-pass transmembrane receptors with extracellular ligand binding domains and an intracellular tyrosine kinase domain. Upon binding of the ligand the kinase domain activates intracellular signaling networks that coordinate cellular processes such as proliferation, growth, differentiation, migration and survival. Fibroblast growth factors (FGFs) are a family of 18 ligands which are able to bind and activate distinct FGFRs. Deregulated FGFR activity, through mutations or translocations, is often associated with oncogenic events. Overexpression of FGFR or FGF may lead to increased cell proliferation, growth, differentiation, migration or survival, thus making it an interesting target. In this study we optimized a TaqMan qRT-PCR-based approach to evaluate the copy number variation for several FGFRs (FGFR1, FGFR2, FGFR3 and FGFR4) and several FGF ligands (FGF1, FGF2, FGF3, FGF4, FGF10, FGF12, and FGF19) on a panel of 23 cell lines. The cell types covered a variety of diseases including bladder, breast, endometrial, gastric, kidney, liver, lymphoma, melanoma, sarcoma, small cell lung carcinoma and squamous cell carcinoma. These cell lines had been tested for sensitivity to the JNJ FGFR small molecule inhibitor JNJ42541707 which is a pan-FGFR inhibitor. We observed in the sensitive (IC50 Citation Format: Katherine Bell, Dana Gaffney, Gabriela Martinez-Cardona, Jayaprakash Karkera, Suso Platero. Co-amplification of FGF receptors and ligands in FGFR inhibitor-sensitive cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4326. doi:10.1158/1538-7445.AM2015-4326

Fusion genes are chromosomal aberrations that are found in many cancers and can be used as prognostic markers and drug targets in clinical practice. Gene fusions can lead to the production of oncogenic fusion proteins or to enhanced expression of oncogenes. Advances in next-generation sequencing technologies have made possible to identify more efficiently novel fusion proteins in cancer. Recently, several FGFR fusion genes with intact kinase domains have been identified in bladder, lung, breast, thyroid, oral, and prostate cancers, as well as other tumor types. To date, several FGFR3 partner genes have been identified. In this study, we focused on the FGFR3-BAIAP2L1 and FGFR3-TACC3 gene fusions and investigated their tumorigenic activity, mechanism of activation, and sensitivity to the FGFR inhibitor JNJ-42756493. To determine the role and significance of FGFR fusion genes in cancer, FGFR fusion expression constructs were designed and individually transfected into normal rat kidney epithelial cells. FGFR fusion overexpressing cells not only showed increased cell proliferation, but also exhibited anchorage-independent cell growth. Cells harboring the FGFR fusions showed increased sensitivity to the FGFR inhibitor JNJ-42756493 in vitro, whereas the wild-type FGFR3 did not in the absence of FGF ligands. In addition, Western blotting analyses indicated that the overexpression of the FGFR fusions resulted in highly activated proteins that induce signaling via the MAPK pathway. These findings underline the oncogenic potential of the FGFR fusion genes and highlight their unique potential as predictive biomarkers in the selection of patients for FGFR-targeted therapy. Citation Format: Gabriela Martinez Cardona, Katherine Bell, Dana Gaffney, Joseph Portale, Suso Platero, Matthew Lorenzi, Jayaprakash Karkera. The role of FGFR fusion genes as novel oncogenic targets. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4325. doi:10.1158/1538-7445.AM2015-4325

Squamous cell cancer (SCC) of the lung is a leading cause of cancer mortality in the US, due to late stage diagnosis and lack of effective treatments. Lung SCC arises in the epithelial layer of the bronchial airways and is often preceded by the development of premalignant lesions (PMLs). The molecular events involved in the progression of PMLs to lung SCC are not clearly understood and not all PMLs go on to form carcinoma. By molecularly characterizing PMLs and non-lesion areas in the airway of individuals with PMLs we hypothesize that we will be able to identify early events in the process of lung carcinogenesis that lead to SCC. We used next-generation sequencing to profile bronchial brushings and biopsies obtained from high-risk smokers undergoing lung cancer screening by auto-fluorescence bronchoscopy and CT at the Roswell Park Cancer Institute in Buffalo, NY. For each subject (n = 26), we sampled the PML(s) and the mainstem bronchus repeatedly over time (394 +/- 170 days) with serial bronchoscopies (5 +/- 3 biopsies/subject) as the PML progressed towards or regressed away from frank malignancy. mRNA-Seq (n = 192) and miRNA-Seq (n = 183) were performed on the endobronchial biopsies and brushings and exome-Seq was performed on blood DNA from these subjects. RNA-seq data was aligned to the hg19 and gene/transcript levels were summarized using RSEM/Ensembl 74 or Bedtools/ mirBase 18. Single nucleotide variants were quantified using a modified PRADA pipeline and GATK. We identified gene and miRNA expression changes as well as pathways that are associated with biopsy histological grade as well as progressive/stable disease. H&E stains of PMLs clustering with lesions of dissimilar grades were examined to show that molecular classification provides complementary information to pathological grading. Genes altered in the normal airway in the presence of a PML were significantly concordantly enriched with genes identified in the biopsies demonstrating a strong relationship between the PMLs and the field of injury. In addition, cancer-associated missense and loss-of-function mutations were detected in PMLs. One individual had cancer-associated mutations at the initial time point in a single moderate dysplastic PML. Upon resampling ∼40 months later, the PML had progressed to an in situ carcinoma while gaining additional mutations. Furthermore, an adjacent PML at this second time point, also in situ carcinoma, had a significantly overlapping set of mutations with the original lesion suggesting that clonal expansion and invasion occurred between temporal samplings. The multiomic characterization of PMLs reveals gene and miRNA expression changes and somatic mutations that are associated with the lesion severity and progressive disease. These studies provide insights into the early molecular events in the process of lung carcinogenesis and may be predictors of lung cancer risk and lesion progression as well as novel candidates for targeted or preventive therapy. Citation Format: Jennifer E. Beane, Joshua Campbell, Christopher Moy, Catalina Perdomo, Michael Schaffer, Sarah Mazzilli, Yaron Geshalter, Jacob Kantrowitz, Liye Zhang, David Jenkins, Mary Beth Pine, Samjot Dhillon, Gang Liu, Hanqiao Liu, Sherry Zhang, Jessica Vick, Stefano Monti, Evan Johnson, Suso Platero, Marc Lenburg, Mary Reid, Avrum Spira. Development of the pre-cancer genome atlas (PCGA) for squamous cell lung carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2878. doi:10.1158/1538-7445.AM2015-2878

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: JNJ-42756493 is an FGFR 1, 2, 3, and 4 inhibitor with nanomolar affinity, orally bioavailable, and has demonstrated a broad spectrum antitumor activity in cell line, xenograft and patient-derived explant models with abnormality in FGFR signaling pathway such as FGFR gene amplification, mutation and translocation. Methods: A multipart phase 1 first in human study of JNJ-42756493 was initiated in advanced solid tumor patients ([NCT01962532][1]) including, Part 1 dose-escalation to determine the recommended phase 2 dose (RP2D) based on safety, pharmacokinetic, and pharmacodynamic data; Part 2 biopsy cohort to confirm the RP2D and Part 3 expansion phase to evaluate anti-tumor activity in NSCLC, SCLC, Breast cancer and other solid tumor patients with FGFR gene amplification, mutation or translocation at RP2D. Multiple biomarkers from tissue (including phospho-FGFR, phospho-Erk and phospho-S6) and serum (phosphate, calcium, Vitamin D, PTH and FGF23) were also assessed in the study. Results: At data cut-off (22 October 2013), total of 28 patients had been treated at 5 dose levels (0.5, 2, 4, 6 and 9 mg daily continuously) in Part 1 of the study. JNJ-42756493 appeared safe, did not generate any dose-limiting toxicities or any drug related severe adverse events. One patient from 4 mg cohort died after receiving 6 doses in Cycle 1, autopsy indicated a serious adverse event (SAE) of pulmonary edema which not related to the study drug. The most common adverse events (AEs) were hyperphosphatemia (57%), asthenia (46%), dry mouth (32%), abdominal pain (29%), diarrhea (25%), vomiting (25%), decreased appetite (21%) and constipation (21%). Grade 1 or 2 hyperphosphatemia was managed by co-administration of phosphate lowering therapy and by treatment interruption, other AEs were generally mild to moderate. No clinically significant cardiac safety findings were observed. Pharmacokinetics was linear and predictable with a half-life of ∼50 hours. Dose levels ≥ 6mg achieved plasma concentrations predicted to be efficacious from preclinical experiments. Dose dependent changes in biomarkers including increases in phosphate, FGF23 and calcium and decreases in PTH were observed in blood, evaluation of biomarker response in tissue is ongoing. A bladder cancer patient with lung metastasis harboring a FGFR3-TACC3 translocation treated at 9 mg had a confirmed PR. 9 mg was declared as the first RP2D, but safety evaluation of JNJ-42756493 at higher doses is ongoing. Conclusions: JNJ-42756493 has excellent pharmaceutical properties and appeared safe with manageable side effects at dose levels that elicit anti-tumor activity. Citation Format: Rodrigo Dienstmann, Rastilav Bahleda, Barbara Adamo, Jordi Rodon, Andrea Varga, Anas Gazzah, Suso Platero, Hans Smit, Timothy Perera, Bob Zhong, Kim Stuyckens, Yusri Elsayed, Chris Takimoto, Vijay Peddareddigari, Josep Tabernero, Feng Roger Luo, Jean-Charles Soria. First in human study of JNJ-42756493, a potent pan fibroblast growth factor receptor (FGFR) inhibitor in patients with advanced solid tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT325. doi:10.1158/1538-7445.AM2014-CT325 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01962532&atom=%2Fcanres%2F74%2F19_Supplement%2FCT325.atom

Autocrine or paracrine constitutive Wnt pathway activation occurs at a high frequency in several tumor types. The R-spondin (RSPO) protein family is comprised of four secreted growth factors. The four paralogs share 40-60% pairwise amino acid sequence identity and are predicted to share substantial structural homology. RSPO proteins are involved in vertebrate development and their ligand-type activities overlap substantially with those of canonical Wnt ligands. A characteristic feature of all four RSPO members is their ability to activate β-catenin signaling and enhance WNT-mediated β-catenin activation. It has recently been described that recurrent gene fusions involving RSPO family members RSPO2 and RSPO3 occur in ∼10% of colon tumors. In this study we developed a TaqMan qRT-PCR-based approach to evaluate the expression of these three (3) RSPO fusion transcripts in formalin-fixed paraffin embedded tissue (FFPET) samples. We examined 324 lung cancer, 81 colorectal cancer, 71 head & neck, 11 esophageal, 92 ovarian cancer, and 103 breast cancer FFPET samples for the presence of EIF3E(e1)-RSPO2(e1), PTPRK(e1)-RSPO3(e2), and PTPRK(e7)-RSPO3(e2). EIF3E(e1)-RSPO2(e1), a fusion which is expected to produce a functional RSPO2 protein driven by the EIF3E promoter, was identified in ∼1-2% of most of cancer types with the exception of breast cancer. The PTPRK(e1)-RSPO3(e2) fusion was expressed by ∼1-11% of the samples in the different cancers, making it the most prevalent of the fusions. PTPRK(e1)-RSPO3(e2) fusion is an in-frame fusion that preserves the entire coding sequence of RSPO3 and replaces its secretion signal sequence with that of PTPRK. The PTPRK(e7)-RSPO3(e2) fusion is also an in-frame fusion in which the RSPO3 native signal peptide is replaced by the secretion signal of PTPRK. The PTPRK(e7)-RSPO3(e2) was the least prevalent of all the fusions, positive samples were found exclusively in the head and neck (∼2%) and breast cancer samples (∼2%). All of the fusions detected were mutually exclusive. The RSPO gene fusions identified may provide new potential opportunities for therapeutic intervention. Citation Format: Gabriela Martinez Cardona, Katherine Bell, Joseph Portale, Dana Gaffney, Christopher Moy, Suso Platero, Matthew V. Lorenzi, Jayaprakash Karkera. Identification of R-Spondin fusions in various types of human cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2408. doi:10.1158/1538-7445.AM2014-2408

The fibroblast growth factor (FGF) signaling axis is increasingly implicated in tumorigenesis and chemoresistance. Alterations in FGFR family members including focal amplification of FGF receptor 1 (FGFR1), mutations in FGFR 2, 3 and 4, translocations involving FGFR 2 and FGFR3, as well as amplification or transcriptional upregulation of various ligand family members have been associated with tumor growth and survival, suggesting that FGFR inhibitors may be a viable therapeutic option in subsets of various disease settings. A number of agents targeting the FGF signaling axis including small-molecule FGFR targeted agents, with diverse kinase inhibitory and pharmacological profiles, are currently in clinical development. JNJ-42756493 (first disclosure of the structure) has a pharmacological profile that is differentiated from other agents in this class currently under investigation. JNJ-42756493 displays single digit nanomolar FGFR (1, 2, 3 4) tyrosine kinase inhibitory activity. JNJ-42756493 inhibited recombinant FGFR kinase activity in vitro and suppressed FGFR signaling and growth in engineered cell lines and tumor cell lines dependent upon deregulated FGFR expression. JNJ-42756493 demonstrated highly specific tumor inhibitory effects in FGFR1-4 dependent cell lines, in vitro cell lines based xenografts and direct patient derived xenografts, with no discernible activity in models that were not dependent on FGFR signaling. JNJ-42756493 showed favorable drug like properties and displayed a high distribution to lung, liver and kidney tissue. JNJ-42756493 was well tolerated at efficacious doses and resulted in potent dose-dependent antitumor activity accompanied by pharmacodynamic modulation of tumor FGFR and downstream pathway components. Data presented here highlights JNJ-42756493 as a novel, highly potent and selective small-molecule inhibitor of all four known active FGFR kinase family members with potent antitumor activity against FGFR-dependent tumor models. These data, together with emerging observations from our ongoing Phase 1 clinical trial, position JNJ-42756493 as a differentiated FGFR 1, 2, 3 and 4 kinase inhibitor and support its continued clinical development in lung cancer and other malignancies associated with aberrant FGFR signaling. Citation Format: Timothy Perera, Eleanora Jovcheva, Jorge Vialard, Tinne Verhulst, Norbert Esser, Berthold Wroblowski, Ron Gilissen, Eddy Freyne, Peter King, Suso Platero, Olivier Querolle, Laurence Mevellec, Christopher Murray, Lynsey Fazal, Gordon Saxty, George Ward, Matthew Squires, Neil Thompson, David Newell, Patrick Angibaud. JNJ-42756493 is an inhibitor of FGFR-1, 2, 3 and 4 with nanomolar affinity for targeted therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1738. doi:10.1158/1538-7445.AM2014-1738

Non-Small Cell Lung Cancer (NSCLC) is the most common form of lung cancer, accounting for approximately 85% of all lung cancers worldwide. Standard therapy options include surgical resection followed by radiation and/or chemotherapy. More recently, analysis of the genomic profile of NSCLC has led to the development of molecularly targeted treatment strategies designed to enhance survival of select subsets of patients containing pre-determined genetic alterations, including gene mutations and aberrant gene expression profiles. In this study, we have developed a TaqMan-based approach to evaluate the genomic profile of 339 NSCLC FFPE tumors, consisting of 152 adenocarcinoma and 187 squamous cell carcinoma (SCC), stages I-IV. Using a custom qRT-PCR somatic mutation array, we have identified the frequencies of key mutations prevalent in NSCLC- associated genes, including EGFR: 5% SCC , 29% adenocarcinoma, cMET: 6% SCC, 4% adenocarcinoma, and KRAS: 3% SCC, 19% adenocarcinoma. Overall, these frequencies are concordant with previous reports. Additionally, we have evaluated the gene expression and IHC profiles of NSCLC-driver signaling pathways including EGFR, cMET and HGF. Various levels of expression for EGFR, cMET and HGF were observed across all samples, with a significant association detected between the presence of EGFR mutation and high EGFR expression in adenocarcinoma (p value = 0.0046). Overall, our findings represent a comprehensive catalog of common genetic aberrations with concurrent gene and protein expression profiles in NSCLC. Furthermore, this data provides insight into correlations observed across these molecular characteristics contributing to NSCLC tumor development. These insights can provide guidance for patient stratification and novel therapeutic strategies for select targeted therapies in NSCLC. Citation Format: Dana S. Gaffney, Katherine Bell, Gabriela Martinez, Yashoda Rajpurohit, Jayaprakash Karkera, Christopher Moy, Suso Platero. Genomic and molecular profiling of NSCLC formalin-fixed paraffin-embedded tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1548. doi:10.1158/1538-7445.AM2014-1548

Mesothelin is a 40 kDa secreted glycoprotein expressed in normal mesothelial cells and over-expressed in several histological types of tumors. Detection of mesothelin by immunohistochemistry (IHC) may assist in the diagnosis of mesothelioma. Mesotheliomas are positive for mesothelin staining, but carcinomas of the lung may also be positive for this marker. Mesothelin positivity in adenocarcinoma of the lung reportedly ranges from 22% to 71% of cases depending upon the specific subtype. Mesothelin positivity in squamous cell carcinomas (SCC) of lung is reported to be 16% in non-keratinizing and 31% in keratinizing subtypes. We performed mesothelin IHC on a cohort of 50 lung carcinoma samples (16 adenocarcinomas and 34 SCC). Mesothelin expression was observed in 10 out of 16 (62.5%) lung adenocarcinoma samples, of which 8 showed greater than 10% of tumor cells with positive membrane staining of any intensity. Mesothelin expression was observed in 19 out of 34 (55.9%) lung SCC samples, of which only 3 samples showed greater than 10% of tumor cells with positive membrane staining of any intensity. These findings are in concordance with previous reports which show a higher prevalence of mesothelin protein expression in lung adenocarcinoma than in lung SCC. To correlate RNA expression with protein expression, we performed gene expression profiling on a subcohort of these lung cancer specimens. Out of 50 lung carcinoma samples, 28 samples (10 adenocarcinomas and 18 SCCs) provided adequate RNA yield for gene expression profiling for mesothelin. A total mean relative gene expression (mRGE) value of 13.64 with a standard deviation (SD) of ±3.71 was obtained for the adenocarcinoma samples and a mRGE value of 14.78 with a SD of 2.64 was obtained for the SCC samples. We next arbitrarily assigned samples with greater than 10% mesothelin stained cells as “positive” and the remainder as “negative”. Six negative adenocarcinoma samples yielded a mRGE value of 12.98 with a SD of ±3.84, and four positive adenocarcinoma samples yielded a mRGE value of 14.63 with SD of ±3.82. Sixteen negative SCC samples resulted in a mRGE 14.63 with SD of ± 2.75 and two positive SCC samples yielded a mRGE of 15.98 with a SD of ±1.37. There was no apparent correlation between mRGE values and IHC positivity. We also correlated gene expression with p53 mutation status. Sixteen wild type samples, composed of equal number of adenocarcinoma and SSC had a total mRGE of 14.29 with SD of ±3.32. Seven samples with p53 mutations had a total mRGE of 16.09 with SD ±1.93. These data indicate that mesothelin gene expression is not associated with p53 mutation status. Future studies with an increased number of samples may yield significant associations between protein expression, gene expression and mutation status. Citation Format: Jackson Wong, Dana Gaffney, Michael Sharp, Brenda Hertzog, Jayaprakash Karkera, Suso Platero, John Alvarez. Profiling mesothelin protein expression by immunohistochemistry and gene expression in adenocarcinoma and squamous cell carcinoma of lung. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4666. doi:10.1158/1538-7445.AM2014-4666

2501 Background: JNJ-42756493 is an orally bioavailable FGFR 1, 2, 3 and 4 inhibitor with nanomolar antitumor activity in cell lines and in vivo models with FGFR pathway aberration. Methods: This f...

We examined the heterochromatic binding of GAGA factor and proliferation disrupter (Prod) proteins during the cell cycle in Drosophila melanogaster and sibling species. GAGA factor binding to the brownDominant AG-rich satellite sequence insertion was seen at metaphase, however, no binding of GAGA factor to AG-rich sequences was observed at interphase in polytene or diploid nuclei. Comparable mitosis-specific binding was found for Prod protein to its target satellite in pericentric heterochromatin. At interphase, these proteins bind numerous dispersed sites in euchromatin, indicating that they move from euchromatin to heterochromatin and back every cell cycle. The presence of Prod in heterochromatin for a longer portion of the cell cycle than GAGA factor suggests that they cycle between euchromatin and heterochromatin independently. We propose that movement of GAGA factor and Prod from high affinity sites in euchromatin occurs upon condensation of metaphase chromosomes. Upon decondensation, GAGA factor and Prod shift from low affinity sites within satellite DNA back to euchromatic sites as a self-assembly process.

Prostate Cancer (PCa) is the third most common cause of death from cancer in men of all ages. Surgical or medical castration is one of the most common treatments for patients with advanced PCa; however a majority of patients develop castration resistant prostate cancer (CRPC), tumor relapse, which remains to be the second leading cause of cancer-related deaths of men in the US. Androgen receptor (AR) signaling is shown to play a critical role in the development and progression of PCa. Genetic aberrations within AR, including constitutively active AR splice variants and AR point mutations have been identified in CRPC. The most common AR splice variants lack the ligand-binding domain (LBD), which is often the target of CRPC therapies. Therefore, presence of these variants may act as a mechanism of resistance to AR-targeted therapies leading to the progression of prostate tumor growth. Additional PCa specific genetic aberrations include fusions between the androgen-related gene, TMPRSS2 and the ETS transcription factors, ERG (predominant) and ETV1. These fusion events are frequently associated with more aggressive prostate cancers leading to poorer prognosis. In this study, we developed TaqMan qRT-PCR assays to evaluate the presence of several previously identified AR splice variants, including ARV1, ARV3/V7, ARV567 and ARV8, AR somatic mutations, including L701H, V715M, H874Y and T877A, along with TMPRSS2 fusion genes, TMPRSS2:ERG and TMPRSS2:ETV1, in two independent PCa FFPET sample sets. The first sample set consisted of 42 Prostate adenocarcinomas ranging from stage II to stage IV. Results showed that ARV1 and ARV3/V7 were the most prevalent variants with 92% of all samples showing expression of either or both variant. TMPRSS2: ERG was present in 72% of all samples tested, with a high concordance to AR variant expression, prevalent in later stage (III/IV) PCa samples. The second sample set consisted of 8 prostate adenocarcinomas, including matched adjacent normal FFPET. Similar expression of the AR variants was observed in both the tumor and matched normal samples, however tumor prostate samples showed a higher and more prevalent expression (66.67%) of the TMPRSS2: ERG fusion gene than in the matched normal samples (33%). None of the four AR mutations evaluated were detected in either sample set. Overall, these findings demonstrate a strong presence of both AR splice variants and the TMPRSS2: ERG fusion gene in the prostate cancer patient population, supporting evidence for a functional role of these markers in PCa diagnosis and disease progression. Furthermore, presence of LBD negative AR splice variants indicates an attractive biomarker for stratification of the patient population resistant to AR targeted therapies. Citation Format: Dana S. Gaffney, Gabriela Martinez, Katherine Bell, Suso Platero, Deborah Ricci, Jayaprakash Karkera. Identification of androgen receptor (AR) splice variants, AR somatic mutations and TMPRSS2:ETS fusion genes in prostate cancer FFPET by qRT-PCR. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5. doi:10.1158/1538-7445.AM2013-5

Many advances have been made in the diagnosis and treatment of breast cancer in recent years, but it still remains one of the leading causes of death in women. It is well known that the growth of breast cells is regulated by the interactions of different steroid hormones, in particular androgens and estrogens that are related due to their connected metabolic pathways. A number of novel inhibitors of steroidogenic enzymes have been developed that target pre-receptor events_those pertaining to the production, transport, and conversion of steroid ligands. Critical pre-receptor steps include the conversion of pregnenolone-like steroids into androgens, mediated largely by the 17α-hydroxylase/lyase (CYP17) enzyme complex, and the conversion of testosterone and androstenedione to estradiol and estrone, mediated by aromatase (CYP19). Both conversions are implicated in the emergence of tumor resistance and thus are targets for intervention. Androgen Receptor (AR) is the sex hormone receptor most frequently found in both primary and secondary breast tumors, which is indicitative of the importance of AR in regulating the growth of breast cancer cells. It is estimated that 90% of human genes undergo alternative splicing and AR is no exception. Alternative splicing of the AR could culminate in a receptor that is capable of translocation, or can bind DNA without ligand, leading current AR therapies to be less efficacious. Using TaqMan qRT-PCR we examined 213 female breast-cancer FFPET samples, 80 ER- PR- Her2- samples, 68 ER- PR- Her2+ samples, and 64 ER+ PR+ Her2- samples, as well as 8 breast-cancer cell lines for the presence of ESR1, CYP17, CYP19, full length AR and AR splice variants ARV1, ARV3/V7, ARV567, and Delta3AR. ARV3/V7 and Delta3AR were the most prevalent variants in the ER+ PR+ Her2- and ER- PR- Her2+ sample sets, with >85% of these samples showing expression of either or both of these variants. On the other hand, ARV1, ARV3/V7, and ARV567 were the most prevalent variants in the ER- PR- Her2- sample set, with >90% of these samples showing expression of one or a combination of these variants. Lower expression values of most of the AR variants were observed in higher grade ER+ PR+ Her2- and ER- PR- Her2+ samples as compared to the lower grade samples. CYP19 was highly prevalent in all sample sets with >75% of all samples showing expression, while CYP17 expression was observed in Citation Format: Gabriela Martinez, Dana Gaffney, Katherine Bell, Suso Platero, Deborah Ricci, Weimin Li, Jayaprakash D. Karkera. Identification of androgen receptor splice variants, ESR1, CYP17, and CYP19 in human breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3467. doi:10.1158/1538-7445.AM2013-3467

Lung cancer is the leading cause of death in cancer worldwide. There are two major forms of lung cancer, including small cell lung cancer (SCLC) which accounts for approximately 20% of all lung cancers and non-small cell lung cancer (NSCLC) which accounts for approximately 80% of lung cancers. Around 25% of these lung cancer patients are never smokers and these cancers tend to be the result of single somatic mutation events. Several somatic events have been reported in NSCLC, including mutations in EGFR and KRAS along with an EML4-ALK fusion gene, however more than 40% of these cancers are the result of unknown genetic events. Recently several papers have reported a novel fusion gene resulting from a 10.6 Mb inversion on chromosome 10 which leads to a fusion between the KIF5B and RET genes. The RET gene is a well-known tyrosine-kinase proto-oncogene which has been linked to papillary thyroid carcinomas and its expression is generally very low in lung. RET tyrosine kinase stimulates autophosphorylation of the tyrosine kinase unit which activates several pathways including STAT3, RAS/ERK, MAPK, PI3K/AKT and SRC. In the KIF5B-RET fusion KIF5B retains its coiled-coil domain necessary for homodimerization and the RET retains its kinase function leading to aberrant activation of several kinase pathways. Several fusion genes between the exons of KIF5B and RET have been previously reported including KIF5B15:RET12, KIF5B16:RET12, KIF5B22:RET12, KIF5B23:RET12, KIF5B15:RET11, KIF5B24:RET8 and KIF5B24:RET11. In this study we developed a TaqMan qRT-PCR-based approach to evaluate the expression of these seven (7) KIF5B-RET fusion transcripts in 64 NSCLC fresh frozen biopsies, ranging from stage I to stage III, including 25 adenocarcinoma and 37 squamous cell carcinoma samples, respectively. Our findings confirm the presence of the fusion between KIF5B15 (exon 15) and RET12 (exon 12) at a frequency of 1.56% in all subtypes. The clinicopathological background of the KIF5B/RET fusion-positive patient agrees with previously reported trends for this fusion event consisting of a caucasian female, non smoker, with adenocarcinoma subtype. Although this percentage is relatively small, it still represents around 12,000 individuals worldwide that express this fusion transcript, presenting a promising biomarker for targeted therapeutics in the treatment of NSCLC disease. Citation Format: Katherine Bell, Dana Gaffney, Gabriela Martinez, Suso Platero, Jayaprakash Karkera. A real-time qPCR approach to detect fusions between the KIF5B and RET genes in non-small cell lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3174. doi:10.1158/1538-7445.AM2013-3174

The ability of a chimeric HP1-Polycomb (Pc) protein to bind both to heterochromatin and to euchromatic sites of Pc protein binding was exploited to detect stable protein-protein interactions in vivo. Previously, we showed that endogenous Pc protein was recruited to ectopic heterochromatic binding sites by the chimeric protein. Here, we examine the association of other Pc group (Pc-G) proteins. We show that Posterior sex combs (Psc) protein also is recruited to heterochromatin by the chimeric protein, demonstrating that Psc protein participates in direct protein-protein interaction with Pc protein or Pc-associated protein. In flies carrying temperature-sensitive alleles of Enhancer of zeste[E(z)] the general decondensation of polytene chromosomes that occurs at the restrictive temperature is associated with loss of binding of endogenous Pc and chimeric HP1-Polycomb protein to euchromatin, but binding of HP1 and chimeric HP1-Polycomb protein to the heterochromatin is maintained. The E(z) mutation also results in the loss of chimera-dependent binding to heterochromatin by endogenous Pc and Psc proteins at the restrictive temperature, suggesting that interaction of these proteins is mediated by E(z) protein. A myc-tagged full-length Suppressor 2 of zeste [Su(z)2] protein interacts poorly or not at all with ectopic Pc-G complexes, but a truncated Su(z)2 protein is strongly recruited to all sites of chimeric protein binding. Trithorax protein is not recruited to the heterochromatin by the chimeric HP1-Polycomb protein, suggesting either that this protein does not interact directly with Pc-G complexes or that such interactions are regulated. Ectopic binding of chimeric chromosomal proteins provides a useful tool for distinguishing specific protein-protein interactions from specific protein-DNA interactions important for complex assembly in vivo.