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The success of targeted or immune therapies is often hampered by the emergence of resistance and/or clinical benefit in only a subset of patients. We hypothesized that combining targeted therapy with immune modulation would show enhanced antitumor responses. Here, we explored the combination potential of erdafitinib, a fibroblast growth factor receptor (FGFR) inhibitor under clinical development, with PD-1 blockade in an autochthonous FGFR2K660N/p53mut lung cancer mouse model. Erdafitinib monotherapy treatment resulted in substantial tumor control but no significant survival benefit. Although anti–PD-1 alone was ineffective, the erdafitinib and anti–PD-1 combination induced significant tumor regression and improved survival. For both erdafitinib monotherapy and combination treatments, tumor control was accompanied by tumor-intrinsic, FGFR pathway inhibition, increased T-cell infiltration, decreased regulatory T cells, and downregulation of PD-L1 expression on tumor cells. These effects were not observed in a KRASG12C-mutant genetically engineered mouse model, which is insensitive to FGFR inhibition, indicating that the immune changes mediated by erdafitinib may be initiated as a consequence of tumor cell killing. A decreased fraction of tumor-associated macrophages also occurred but only in combination-treated tumors. Treatment with erdafitinib decreased T-cell receptor (TCR) clonality, reflecting a broadening of the TCR repertoire induced by tumor cell death, whereas combination with anti–PD-1 led to increased TCR clonality, suggesting a more focused antitumor T-cell response. Our results showed that the combination of erdafitinib and anti–PD-1 drives expansion of T-cell clones and immunologic changes in the tumor microenvironment to support enhanced antitumor immunity and survival.

Supplemental figure legends 1-8, Supplementary tables 1-5, and Supplementary methods

Supplemental Figures 1-8

A) Structure of JNJ-42541707, a structurally related compound to JNJ-42756493. B) 72h growth inhibition (IC50) of JNJ-42541707 against 236 cancer cell lines from multiple origins color coded based on FGFR1,2,4 mRNA overexpression and FGFR WT.

The MEK1 and MEK2 inhibitor GSK1120212 is currently in phase II/III clinical development. To identify predictive biomarkers, sensitivity to GSK1120212 was profiled for 218 solid tumor cell lines and 81 hematologic malignancy cell lines. For solid tumors, RAF/RAS mutation was a strong predictor of sensitivity. Among RAF/RAS mutant lines, co-occurring PIK3CA/PTEN mutations conferred a cytostatic response instead of a cytotoxic response for colon cancer cells that have the biggest representation of the comutations. Among KRAS mutant cell lines, transcriptomics analysis showed that cell lines with an expression pattern suggestive of epithelial-to-mesenchymal transition were less sensitive to GSK1120212. In addition, a proportion of cell lines from certain tissue types not known to carry frequent RAF/RAS mutations also seemed to be sensitive to GSK1120212. Among these were breast cancer cell lines, with triple negative breast cancer cell lines being more sensitive than cell lines from other breast cancer subtypes. We identified a single gene DUSP6, whose expression was associated with sensitivity to GSK1120212 and lack of expression associated with resistance irrelevant of RAF/RAS status. Among hematologic cell lines, acute myeloid leukemia and chronic myeloid leukemia cell lines were particularly sensitive. Overall, this comprehensive predictive biomarker analysis identified additional efficacy biomarkers for GSK1120212 in RAF/RAS mutant solid tumors and expanded the indication for GSK1120212 to patients who could benefit from this therapy despite the RAF/RAS wild-type status of their tumors. Mol Cancer Ther; 11(3); 720–9. ©2011 AACR.

In vitro kinase inhibition by DiscoverX KinomeScan assay. For details see (19)

Inhibitory activity of Brivanib and JNJ-42756943 in kinase (top) and BaF3 kinase dependent proliferation (bottom) assays and ratio of FGFRs/VEGFR2 activities.

JNJ-42756493 anti-proliferative activity against cancer cells lines from multiple origins.. Detailed data supporting Figure 2.

PDF file - 388K

Polo-like kinases are a family of serine threonine kinases that are critical regulators of cell cycle progression and DNA damage response. Predictive biomarkers for the Plk1-selective inhibitor GSK461364A were identified by comparing the genomics and genetics of a panel of human cancer cell lines with their response to a drug washout followed by an outgrowth assay. In this assay, cell lines that have lost p53 expression or carry mutations in the TP53 gene tended to be more sensitive to GSK461364A. These more sensitive cell lines also had increased levels of chromosome instability, a characteristic associated with loss of p53 function. Further mechanistic studies showed that p53 wild-type (WT) and not mutant cells can activate a postmitotic tetraploidy checkpoint and arrest at pseudo-G1 state after GSK461364A treatment. RNA silencing of WT p53 increased the antiproliferative activity of GSK461364A. Furthermore, silencing of p53 or p21/CDKN1A weakened the tetraploidy checkpoint in cells that survived mitotic arrest and mitotic slippage. As many cancer therapies tend to be more effective in p53 WT patients, the higher sensitivity of p53-deficient tumors toward GSK461364A could potentially offer an opportunity to treat tumors that are refractory to other chemotherapies as well as early line therapy for these genotypes. Mol Cancer Ther; 9(7); 2079–89. ©2010 AACR.

Supplementary Tables 1-5 from Sensitivity of Cancer Cells to Plk1 Inhibitor GSK461364A Is Associated with Loss of p53 Function and Chromosome Instability

Supplementary Figure 1 from Sensitivity of Cancer Cells to Plk1 Inhibitor GSK461364A Is Associated with Loss of p53 Function and Chromosome Instability

Supplementary Figure 1 from Molecular Target Class Is Predictive of In vitro Response Profile

Supplementary Methods, Figure Legend from Molecular Target Class Is Predictive of In vitro Response Profile

Supplementary Table 1 from Molecular Target Class Is Predictive of In vitro Response Profile

Supplementary Table 3 from Molecular Target Class Is Predictive of In vitro Response Profile

Supplementary Table 2 from Molecular Target Class Is Predictive of In vitro Response Profile

The success of targeted or immune therapies is often hampered by the emergence of resistance and/or clinical benefit in only a subset of patients. We hypothesized that combining targeted therapy with immune modulation would show enhanced antitumor responses. Here, we explored the combination potential of erdafitinib, a fibroblast growth factor receptor (FGFR) inhibitor under clinical development, with PD-1 blockade in an autochthonous FGFR2K660N/p53mut lung cancer mouse model. Erdafitinib monotherapy treatment resulted in substantial tumor control but no significant survival benefit. Although anti–PD-1 alone was ineffective, the erdafitinib and anti–PD-1 combination induced significant tumor regression and improved survival. For both erdafitinib monotherapy and combination treatments, tumor control was accompanied by tumor-intrinsic, FGFR pathway inhibition, increased T-cell infiltration, decreased regulatory T cells, and downregulation of PD-L1 expression on tumor cells. These effects were not observed in a KRASG12C-mutant genetically engineered mouse model, which is insensitive to FGFR inhibition, indicating that the immune changes mediated by erdafitinib may be initiated as a consequence of tumor cell killing. A decreased fraction of tumor-associated macrophages also occurred but only in combination-treated tumors. Treatment with erdafitinib decreased T-cell receptor (TCR) clonality, reflecting a broadening of the TCR repertoire induced by tumor cell death, whereas combination with anti–PD-1 led to increased TCR clonality, suggesting a more focused antitumor T-cell response. Our results showed that the combination of erdafitinib and anti–PD-1 drives expansion of T-cell clones and immunologic changes in the tumor microenvironment to support enhanced antitumor immunity and survival.

The protein arginine methyltransferase 5 (PRMT5) methylates a variety of proteins involved in splicing, multiple signal transduction pathways, epigenetic control of gene expression, and mechanisms leading to protein expression required for cellular proliferation. Dysregulation of PRMT5 is associated with clinical features of several cancers, including lymphomas, lung cancer, and breast cancer. Here, we describe the characterization of JNJ-64619178, a novel, selective, and potent PRMT5 inhibitor, currently in clinical trials for patients with advanced solid tumors, non-Hodgkin's lymphoma, and lower-risk myelodysplastic syndrome. JNJ-64619178 demonstrated a prolonged inhibition of PRMT5 and potent antiproliferative activity in subsets of cancer cell lines derived from various histologies, including lung, breast, pancreatic, and hematological malignancies. In primary acute myelogenous leukemia samples, the presence of splicing factor mutations correlated with a higher ex vivo sensitivity to JNJ-64619178. Furthermore, the potent and unique mechanism of inhibition of JNJ-64619178, combined with highly optimized pharmacological properties, led to efficient tumor growth inhibition and regression in several xenograft models in vivo, with once-daily or intermittent oral-dosing schedules. An increase in splicing burden was observed upon JNJ-64619178 treatment. Overall, these observations support the continued clinical evaluation of JNJ-64619178 in patients with aberrant PRMT5 activity-driven tumors. ispartof: MOLECULAR CANCER THERAPEUTICS vol:20 issue:12 pages:2317-2328 ispartof: location:United States status: published

Introduction The programmed death-ligand 1 inhibitor atezolizumab improves progression-free survival (PFS) and overall survival (OS) for patients with previously treated advanced NSCLC. Preclinical studies indicate that targeting CD38-positive cells with daratumumab may synergistically enhance atezolizumab's antitumor activity by increasing the effector T-cell activity. Methods This phase 1b-2 study included a safety run-in (one cycle of daratumumab plus atezolizumab) and randomized phases (daratumumab plus atezolizumab versus atezolizumab alone). The primary objective of the randomized phase was to compare overall response rates. The secondary objectives included evaluations of safety, clinical benefit rate (stable disease or better), PFS, OS, and pharmacokinetics. Results In total, 99 patients were enrolled (safety run-in, n = 7; randomized, n = 46 per arm). In the randomized phase, the overall response rate was 4.3% for daratumumab plus atezolizumab and 13.0% for atezolizumab alone (OR: 0.30; 95% confidence interval: 0.03–1.92). The respective clinical benefit rates were 52.2% and 43.5%. No improvements were observed in the median PFS or median OS for combination therapy. The study was terminated because of the limited efficacy of daratumumab plus atezolizumab. Conclusions Daratumumab plus atezolizumab therapy did not improve efficacy versus atezolizumab monotherapy for patients with previously treated advanced NSCLC.

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.

We completed targeted exome sequencing of the tumors of 50 patients with pTis–pT4b bladder cancer. Mutations were categorized by type, stratified against previously identified cancer loci in the Catalogue of Somatic Mutations in Cancer and The Cancer Genome Atlas databases, and evaluated in pathway analysis and comutation plots. We analyzed mutation associations with receipt of neoadjuvant chemotherapy, nodal involvement, metastatic disease development, and survival. Compared with The Cancer Genome Atlas, we found higher mutation rates in genes encoding products involved in epigenetic regulation and cell cycle regulation. Of the pathways examined, PI3K/mTOR and Cell Cycle/DNA Repair exhibited the greatest frequencies of mutation. RB1 and TP53 , as well as NF1 and PIK3CA were frequently comutated. We identified no association between mutations in specific genes and key clinical outcomes of interest when corrected for multiple testing. Discovery phase analysis of the somatic mutations in 50 high-risk bladder cancer patients revealed novel mutations and mutational patterns, which may be useful for developing targeted therapy regimens or new biomarkers for patients at very high risk of disease metastasis and death. Patient summary In this report we found known, as well as previously unreported, genetic mutations in the tumors of patients with high-risk bladder cancer. These mutations, if validated, may serve as actionable targets for new trials.

TPS480 Background: The pan- FGFR tyrosine kinase inhibitor erdafitinib is approved by the US Food and Drug Administration for adults with locally advanced or metastatic urothelial carcinoma and susceptible FGFR3/2 genetic alterations who have progressed during or after ≥ 1 line of prior platinum-containing chemotherapy. FGFR gene alterations are potential oncogenic drivers that have been reported in many solid tumors in adult and pediatric pts. Because of limited response to standard of care options in pts failing systemic therapy, there is strong rationale to assess the safety and efficacy of erdafitinib in adolescent and adult pts with advanced solid tumors and FGFR alterations. Methods: This phase 2, open-label study (RAGNAR/42756493CAN2002; NCT04083976) will include pts aged ≥ 12 years with histologically confirmed unresectable, locally advanced, or metastatic solid tumors (except urothelial tumors) harboring predefined FGFR mutations or fusions. Eligibility screening includes molecular screening for FGFR alterations by central or local next-generation sequencing assays, and other clinical criteria. Pts will enroll into either a broad panel cohort (BPC) of target FGFR alterations or an exploratory cohort (EC) for FGFR alterations that do not meet criteria for BPC. Approximately 280 pts (BPC, n = 240; EC, n = 40) will be enrolled. The primary efficacy end point is overall response rate (ORR) as assessed by the independent review committee. Secondary end points include investigator-assessed ORR, duration of response, disease control rate, progression-free survival, overall survival, safety, pharmacokinetics, and health-related quality of life. Safety assessments include adverse events, vital signs, electrocardiograms, physical examinations, laboratory tests, performance status assessment, growth assessments in adolescents, and ophthalmologic examination. As of December 2019, pts are being enrolled at ~158 sites in 15 countries. Results of this study will provide efficacy and safety data for erdafitinib across multiple solid tumors with FGFR alterations and evaluate the potential benefit of targeting the underlying altered biology of FGFR irrespective of tumor histology in adult and adolescent pts. Clinical trial information: NCT04083976.

PRMT5 is a type II methyltransferase that specifically adds methyl groups to arginine as a long-lasting post-translational modification. The PRMT5/MEP50 complex regulates a plethora of cellular processes, such as epigenetics and splicing, which are notable events involved in tumorigenesis. Although not frequently mutated or amplified in tumors, elevated PRMT5 protein levels in lung and hematologic cancers are correlated with poorer survival. The PRMT5 inhibitor JNJ-64619178 has been selected as a clinical candidate based on its high selectivity and potency (subnanomolar range) under different in vitro and cellular conditions, paired with favorable pharmacokinetics and safety properties. JNJ-64619178 binds into the SAM binding pocket and reaches the substrate binding pocket to inhibit PRMT5/MEP50 function in a time-dependent manner. Broad cell line panel profiling of JNJ-64619178 revealed a wide range of sensitivity, which is indicative of a genomic dependency instead of a general cytotoxic on-target consequence of PRMT5 inhibition. Further investigations indicate a synthetic lethal correlation between PRMT5 inhibition and key cancer driver pathways. JNJ-64619178, dosed orally (10 mg/kg, every day), showed selective and efficient blockage of the methylation of SMD1/3 proteins, which are crucial components of the spliceosome and substrates of PRMT5/MEP50. JNJ-64619178 also demonstrated tumor regression in a biomarker-driven human small cell lung cancer xenograft model (NCI-H1048) and prolonged tumor growth inhibition after dosing cessation. In rodent and nonrodent toxicology studies, a tolerated dose of JNJ-64619178 has been identified, with the observed toxicity consistent with on-target activity. In summary, JNJ-64619178 has a favorable preclinical package that supports clinical testing in patients diagnosed with lung cancer and hematologic malignancies. Citation Format: Dirk Brehmer, Tongfei Wu, Geert Mannens, Lijs Beke, Petra Vinken, Dana Gaffney, Weimei Sun, Vineet Pande, Jan-Willem Thuring, Hillary Millar, Italo Poggesi, Ivan Somers, An Boeckx, Marc Parade, Erika van Heerde, Thomas Nys, Carol Yanovich, Barbara Herkert, Tinne Verhulst, Marc Du Jardin, Lieven Meerpoel, Christopher Moy, Gaston Diels, Marcel Viellevoye, Wim Schepens, Alain Poncelet, Joannes T. Linders, Edward C. Lawson, James P. Edwards, Dushen Chetty, Sylvie Laquerre, Matthew V. Lorenzi. A novel PRMT5 inhibitor with potent in vitro and in vivo activity in preclinical lung cancer models [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 DDT02-04. doi:10.1158/1538-7445.AM2017-DDT02-04

420 Background: Plasma samples from a Phase 2 study of the pan-FGFR inhibitor erdafitinib in advanced UC patients (pts) with FGFR mutations (mut) or fusions, were tested using next generation sequencing (NGS) for circulating tumor DNA (ctDNA), and results compared to central FGFR status determination from tissue. Methods: FGFR alterations were measured in archival tissue using an RNA-based RT-PCR test and compared with FGFR alterations identified in pre-treatment plasma specimens using the Guardant360 ctDNA test. Sensitivity of the ctDNA test to detect the FGFR alterations identified by RT-PCR from tissue, the proportion of pts with a study-eligible FGFR alteration in ctDNA, and the ability to detect a tissue FGFR alteration in ctDNA in relation to clinical response were assessed. Results: Samples from 155 pts with detectable baseline ctDNA were included. Overall, concordance between ctDNA and tissue-based FGFR results was 56% (87/155): 63% (77/122) for tissue mut-positive pts vs 24% (7/29) in fusion-positive pts. 61% of pts (94/155) were positive for a study-eligible FGFR alteration from blood-based testing in the tissue-positive population. The response rate was 47% (36/77) for patients for which FGFR mutations could be detected in blood (ctDNA- FGFR-positive) compared with 32% (14/44) in patients for which mutations were not detected in blood (ctDNA- FGFR-negative), with an estimated odds ratio of 1.882 (95% CI: 0.866; 4.090) Conclusions: The 63% concordance rate for detecting FGFR mut in temporally unmatched blood and tissue supports potential for patient selection with blood-based testing, while ctDNA FGFR fusion detection may require further optimization. Although differences in clinical response rate were observed between ctDNA- FGFR-positive and negative patients, the results were not statistically significant. Importantly, a proportion of ctDNA-negative pts responded to erdafitinib, indicating that tissue-based testing may be warranted for pts negative for FGFR alterations via blood-based testing. (BLC2001, NCT02365597). Clinical trial information: NCT02365597.

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.

Inhibition of centromere-associated protein-E (CENP-E) has demonstrated preclinical anti-tumor activity in a number of tumor types including neuroblastoma. A potent small molecule inhibitor of the kinesin motor activity of CENP-E has recently been developed (GSK923295). To identify an effective drug combination strategy for GSK923295 in neuroblastoma, we performed a screen of siRNAs targeting a prioritized set of genes that function in therapeutically tractable signaling pathways. We found that siRNAs targeted to extracellular signal-related kinase 1 (ERK1) significantly sensitized neuroblastoma cells to GSK923295-induced growth inhibition (p = 0.01). Inhibition of ERK1 activity using pharmacologic inhibitors of mitogen-activated ERK kinase (MEK1/2) showed significant synergistic growth inhibitory activity when combined with GSK923295 in neuroblastoma, lung, pancreatic and colon carcinoma cell lines. Synergistic growth inhibitory activity of combined MEK/ERK and CENP-E inhibition was a result of increased mitotic arrest and apoptosis. There was a significant correlation between ERK1/2 phosphorylation status in neuroblastoma cell lines and GSK923295 growth inhibitory activity (r = 0.823, p = 0.0006). Consistent with this result we found that lung cancer cell lines harboring RAS mutations, which leads to oncogenic activation of MEK/ERK signaling, were significantly more resistant than cell lines with wild-type RAS to GSK923295-induced growth inhibition (p = 0.047). Here we have identified (MEK/ERK) activity as a potential biomarker of relative GSK923295 sensitivity and have shown the synergistic effect of combinatorial MEK/ERK pathway and CENP-E inhibition across different cancer cell types including neuroblastoma.

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.

PRMT5 is a type II methyltransferase that symmetrically di-methylates arginine residues on proteins involved in signal transduction and cellular transcription. For example, PRMT5 acts as the enzymatic machinery of the methylosome complex, crucial for spliceosome assembly and activity. Although not frequently mutated or amplified in tumors, an elevated PRMT5 protein level that leads to higher methylosome activity and promotes epithelial–mesenchymal transition, has recently been correlated with a poor survival of cancer patients. The PRMT5 inhibitor JNJ-64619178 has been selected as a clinical candidate based on its high selectivity and potency, paired with favorable oral pharmacokinetics and safety properties. JNJ-64619178 binds simultaneously to the SAM- and protein substrate- binding pockets of the PRMT5/MEP50 complex with a pseudo-irreversible mode-of-action. Chemical proteomics, methylomics and RNA-sequencing analyses of PRMT5 inhibitor treated cell line samples support the current biological understanding of PRMT5 as a regulator of alternative splicing events. JNJ-64619178 showed potent and broad inhibition of cellular growth, observed in several cell line panels that represent diverse cancer histologies. Ongoing investigations will explore the potential synthetic lethal correlation between PRMT5 inhibition and cancer driver pathways, including those addicted to altered splicing. Oral administration of JNJ-64619178 resulted in efficient inhibition of di-methylation of SMD1/3 proteins, components of the splicing machinery and direct substrates of the methylosome, in several human NSCLC and SCLC cancer mouse xenograft models. JNJ-64619178 demonstrated dose-dependent tumor growth inhibition and regression in several human NSCLC and SCLC cancer mouse xenograft models with sustained blockage of tumor re-growth after dosing cessation. In summary, JNJ-64619178 has a favorable pre-clinical profile supporting clinical testing in patients with lung cancer and other malignancies. Citation Format: Tongfei Wu, Hillary Millar, Dana Gaffney, Lijs Beke, Geert Mannens, Petra Vinken, Ivan Sommers, Jan-Willem Thuring, Weimei Sun, Christopher Moy, Vineet Pande, Junguo Zhou, Nahor Haddish-Berhane, Mark Salvati, Sylvie Laquerre, Matthew V. Lorenzi, Dirk Brehmer. JNJ-64619178, a selective and pseudo-irreversible PRMT5 inhibitor with potent in vitro and in vivo activity, demonstrated in several lung cancer models [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 4859.

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.

Polo-like kinases are a family of serine threonine kinases that are critical regulators of cell cycle progression and DNA damage response. Predictive biomarkers for the Plk1-selective inhibitor GSK461364A were identified by comparing the genomics and genetics of a panel of human cancer cell lines with their response to a drug washout followed by an outgrowth assay. In this assay, cell lines that have lost p53 expression or carry mutations in the TP53 gene tended to be more sensitive to GSK461364A. These more sensitive cell lines also had increased levels of chromosome instability, a characteristic associated with loss of p53 function. Further mechanistic studies showed that p53 wild-type (WT) and not mutant cells can activate a postmitotic tetraploidy checkpoint and arrest at pseudo-G1 state after GSK461364A treatment. RNA silencing of WT p53 increased the antiproliferative activity of GSK461364A. Furthermore, silencing of p53 or p21/CDKN1A weakened the tetraploidy checkpoint in cells that survived mitotic arrest and mitotic slippage. As many cancer therapies tend to be more effective in p53 WT patients, the higher sensitivity of p53-deficient tumors toward GSK461364A could potentially offer an opportunity to treat tumors that are refractory to other chemotherapies as well as early line therapy for these genotypes. Mol Cancer Ther; 9(7); 2079–89. ©2010 AACR.

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

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.

Summary Background MEK is a member of the MAPK signalling cascade that is commonly activated in melanoma. Direct inhibition of MEK blocks cell proliferation and induces apoptosis. We aimed to analyse safety, efficacy, and genotyping data for the oral, small-molecule MEK inhibitor trametinib in patients with melanoma. Methods We undertook a multicentre, phase 1 three-part study (dose escalation, cohort expansion, and pharmacodynamic assessment). The main results of this study are reported elsewhere; here we present data relating to patients with melanoma. We obtained tumour samples to assess BRAF mutational status, and available tissues underwent exploratory genotyping analysis. Disease response was measured by Response Evaluation Criteria in Solid Tumors, and adverse events were defined by common toxicity criteria. This study is registered with ClinicalTrials.gov, number NCT00687622. Findings 97 patients with melanoma were enrolled, including 81 with cutaneous or unknown primary melanoma (36 BRAF mutant, 39 BRAF wild-type, six BRAF status unknown), and 16 with uveal melanoma. The most common treatment-related adverse events were rash or dermatitis acneiform (n=80; 82%) and diarrhoea (44; 45%), most of which were grade 2 or lower. No cutaneous squamous-cell carcinomas were recorded. Of 36 patients with BRAF mutations, 30 had not received a BRAF inhibitor before; two complete responses (both confirmed) and ten partial responses (eight confirmed) were noted in this subgroup (confirmed response rate, 33%). Median progression-free survival of this subgroup was 5·7 months (95% CI 4·0–7·4). Of the six patients who had received previous BRAF inhibition, one unconfirmed partial response was recorded. Of 39 patients with BRAF wild-type melanoma, four partial responses were confirmed (confirmed response rate, 10%). Interpretation Our data show substantial clinical activity of trametinib in melanoma and suggest that MEK is a valid therapeutic target. Differences in response rates according to mutations indicate the importance of mutational analyses in the future. Funding GlaxoSmithKline.

The MEK1 and MEK2 inhibitor GSK1120212 is currently in phase II/III clinical development. To identify predictive biomarkers, sensitivity to GSK1120212 was profiled for 218 solid tumor cell lines and 81 hematologic malignancy cell lines. For solid tumors, RAF/RAS mutation was a strong predictor of sensitivity. Among RAF/RAS mutant lines, co-occurring PIK3CA/PTEN mutations conferred a cytostatic response instead of a cytotoxic response for colon cancer cells that have the biggest representation of the comutations. Among KRAS mutant cell lines, transcriptomics analysis showed that cell lines with an expression pattern suggestive of epithelial-to-mesenchymal transition were less sensitive to GSK1120212. In addition, a proportion of cell lines from certain tissue types not known to carry frequent RAF/RAS mutations also seemed to be sensitive to GSK1120212. Among these were breast cancer cell lines, with triple negative breast cancer cell lines being more sensitive than cell lines from other breast cancer subtypes. We identified a single gene DUSP6, whose expression was associated with sensitivity to GSK1120212 and lack of expression associated with resistance irrelevant of RAF/RAS status. Among hematologic cell lines, acute myeloid leukemia and chronic myeloid leukemia cell lines were particularly sensitive. Overall, this comprehensive predictive biomarker analysis identified additional efficacy biomarkers for GSK1120212 in RAF/RAS mutant solid tumors and expanded the indication for GSK1120212 to patients who could benefit from this therapy despite the RAF/RAS wild-type status of their tumors. Mol Cancer Ther; 11(3); 720–9. ©2011 AACR.

Background Aurora kinases play critical roles in mitosis and are being evaluated as therapeutic targets in cancer. GSK1070916 is a potent, selective, ATP competitive inhibitor of Aurora kinase B and C. Translation of predictive biomarkers to the clinic can benefit patients by identifying the tumors that are more likely to respond to therapies, especially novel inhibitors such as GSK1070916. Methods 59 Hematological cancer-derived cell lines were used as models for response where in vitro sensitivity to GSK1070916 was based on both time and degree of cell death. The response data was analyzed along with karyotype, transcriptomics and somatic mutation profiles to determine predictors of response. Results 20 cell lines were sensitive and 39 were resistant to treatment with GSK1070916. High chromosome number was more prevalent in resistant cell lines (p-value = 0.0098, Fisher Exact Test). Greater resistance was also found in cell lines harboring polyploid subpopulations (p-value = 0.00014, Unpaired t-test). A review of NOTCH1 mutations in T-ALL cell lines showed an association between NOTCH1 mutation status and chromosome number (p-value = 0.0066, Fisher Exact Test). Conclusions High chromosome number associated with resistance to the inhibition of Aurora B and C suggests cells with a mechanism to bypass the high ploidy checkpoint are resistant to GSK1070916. High chromosome number, a hallmark trait of many late stage hematological malignancies, varies in prevalence among hematological malignancy subtypes. The high frequency and relative ease of measurement make high chromosome number a viable negative predictive marker for GSK1070916.

Preclinical cellular response profiling of tumor models has become a cornerstone in the development of novel cancer therapeutics. As efforts to predict clinical efficacy using cohorts of in vitro tumor models have been successful, expansive panels of tumor-derived cell lines can recapitulate an “all comers” efficacy trial, thereby identifying which tumors are most likely to benefit from treatment. The response profile of a therapy is most often studied in isolation; however, drug treatment effect patterns in tumor models across a diverse panel of compounds can help determine the value of unique molecular target classes in specific tumor cohorts. To this end, a panel of 19 compounds was evaluated against a diverse group of cancer cell lines (n = 311). The primary oncogenic targets were a key determinant of concentration-dependent proliferation response, as a total of five of six, four of four, and five of five phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway, insulin-like growth factor-I receptor (IGF-IR), and mitotic inhibitors, respectively, clustered with others of that common target class. In addition, molecular target class was correlated with increased responsiveness in certain histologies. A cohort of PI3K/AKT/mTOR inhibitors was more efficacious in breast cancers compared with other tumor types, whereas IGF-IR inhibitors more selectively inhibited growth in colon cancer lines. Finally, specific phenotypes play an important role in cellular response profiles. For example, luminal breast cancer cells (nine of nine; 100%) segregated from basal cells (six of seven; 86%). The convergence of a common cellular response profile for different molecules targeting the same oncogenic pathway substantiates a rational clinical path for patient populations most likely to benefit from treatment. Cancer Res; 70(9); 3677–86. ©2010 AACR.

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

Gerald Falchook and colleagues show clinical activity of the MEK inhibitor trametinib in patients with either BRAF-mutant or wild-type melanoma. Recently, two essential topics for melanoma have been reported: hepatocyte growth factor (HGF)-dependent resistance to therapy and new driver mutations in melanoma. Stroma-mediated resistance to treatment of BRAF-mutant melanoma is common and HGF secretion from stromal cells seems to be responsible for this drug resistance. Analysis of biopsy samples from patients with BRAF-mutant melanoma suggests that patients with abundant HGF from stromal cells have poor responses to treatment. Furthermore, an inverse relation has been reported between plasma HGF concentration and response to treatment in patients with BRAF-mutant melanoma. HGF secretion leads to activation of the HGF receptor MET, and dual inhibition of RAF and either HGF or MET results in reversal of drug resistance in BRAFmutant melanoma. In Falchook and colleagues’ study, examination by immuno histochemistry of whether HGF expression in melanoma sections correlated with poor responses to the MEK inhibitor trametinib would have been informative, not only in patients with the BRAF mutation, but also in patients with wild-type BRAF. These results would clarify the possibility that a combination of the MEK inhibitor and MET inhibitors have a synergistic eff ect. Moreover, although EGF is less able than HGF to reactivate ERK in most melanoma cell lines, EGF and EGFR mediated resistance might be of interest in melanoma and in colon cancers. For driver mutations, six genes related to melanoma have been newly identifi ed (PPP6C, RAC1, SNX31, TACC1, STK19, and ARID2), with RAC1 mutations also reported. The Illumina platform was used in Falchook and colleagues’ study to analyse the mutational and copy number status of 78 diff erent genes commonly implicated in tumorigenesis. Within these data, is there any information about the six new melanoma genes? If these driver mutations are present, examination of whether they correlate with reduced responses to therapy with trametinib would be interesting, because such examination would not only contribute to drug response predictions but also might help select other treatment options, including combination therapies. Furthermore, data on amplifi cation of BRAF, either with or without mutations, could provide information on whether copy number of this gene plays a role in response to therapy? In the context of treatment with the MEK inhibitor trametinib, an integrated examination including HGF secretion and other gene mutations would open up the possibility of prediction of drug response and combination therapy.

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

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