Evaluate Drug Response Research Articles

Abstract 1756: Genomic reclassification of endometrial carcinoma predicts drug response and facilitates refinement of current management based on PDX-guided efficacy outcomes

Abstract Endometrial cancer (EC) is the most common gynecological malignancy among American women. Steadily increasing numbers of newly diagnosed cases and deaths emphasize the necessity of improving current management strategies. Rather than the commonly known two types of EC based on histological classification, the Cancer Genome Atlas (TCGA) Research Network recently reported a reclassification that categorizes EC into four subtypes based on genomic characterization. Molecular similarities between these subtypes and other cancers, including colorectal, ovarian, and breast cancers, were identified. But to date, no prospective validation has delivered an improved understanding on how to translate these findings to the clinic in the form of treatments adapting genomic guidance. Objectives: To evaluate drug response according to TCGA classification using a panel of twenty-seven established endometrial cancer patient-derived xenografts (EC-PDXs). Methods: Twenty-seven EC-PDXs representing various stages of disease and histological subtypes were orthotopically transplanted and propagated over multiple generations. Upon thorough characterization including histology, metastatic status, and molecular features, EC-PDXs were further categorized into the four TCGA molecular subtypes on the basis of somatic copy number variations (CNV), microsatellite instability (MSI), and POLE mutations. A PDX clinical trial (PCT) was performed to re-evaluate current standard treatments in comparison to therapeutic agents adopted from treatment regimens for other cancer types based on their molecular similarities. Results: EC-PDXs established from patients with recurrent disease exhibited minimal activity to first-line chemotherapeutic drugs, as expected, but remained sensitive to other second-line drugs. None of the tested drugs exhibited sufficient activity in POLE EC-PDXs. MSI EC-PDXs were found to be less sensitive to platinum-based treatment and resistant to 5-FU as reported for MSI colorectal cancer. CNV-high EC-PDXs were more sensitive to drugs adopted from current ovarian and breast cancer treatment regimens. Conclusions: This is the first study that demonstrates the utility of TCGA classification for treatment guidance based on a PCT. Patients with recurrent and persistent disease are in desperate need of new therapeutic options. Our findings open up the possibility to utilize drugs which are currently used in the treatment of other cancers for the treatment of EC based on molecular similarities. In addition, this is the first study that successfully demonstrated the use of molecular classification to enable the prediction of drug response, and thus may facilitate the refinement of current EC management in a more precise and personalized way. Citation Format: Chieh-Hsiang Yang, David K. Gaffney, Theresa L. Werner, Elke A. Jarboe, Jason Gertz, Katherine E. Varley, Matthew Peterson, Margit M. Janat-Amsbury. Genomic reclassification of endometrial carcinoma predicts drug response and facilitates refinement of current management based on PDX-guided efficacy outcomes [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 1756. doi:10.1158/1538-7445.AM2017-1756

Abstract 2586: CellMinerCDB: Enabling cross-database exploration of molecular pharmacology data and response determinant discovery in cancer cell lines

Abstract Cancer cell line panels are widely used for evaluating drug response across diverse tissue types. A growing set of molecular profiling data complements measurements of chemosensitivity, providing novel avenues for response determinant discovery and clinical translation. Accessing and inter-relating data from different sources is essential for evaluating such determinants, but remains challenging. To enable wider access to cell line pharmacogenomic data, we have developed CellMinerCDB (CellMiner Cross-Database, discover.nci.nih.gov/cellminercdb), a web application integrating data from several widely studied cancer cell line panels, including the NCI-60 (NIH), GDSC (Sanger/MGH), and CCLE/CTRP (Broad). All together, our database spans over 1300 distinct cell lines, 400 clinically relevant cancer drugs, 20,000 experimental compounds, and molecular profiling data, such as gene/protein expression, DNA copy, methylation, and mutational status. Cell line and tested drug overlaps allow cross-database validation of genomic and drug data, and CellMinerCDB simplifies this by transparently matching differently named entities between sources. Data exploration can be additionally restricted to particular tissue types, with individual cell lines annotated to the OncoTree ontology for consistent treatment across sources. A range of analysis tools support interactive data exploration, from 2D plots of drug response and molecular profiling features to exhaustive correlation analyses and multivariate predictive models. We illustrate the power and utility of CellMinerCDB with examples of response determinant discovery and predictive modeling for Top1 and PARP inhibitors. Beginning with established individual determinants, such as SLFN11 mRNA expression, we show how both unbiased and biological knowledge network-based feature selection methods enable iterative refinement of a multivariate genomic signature of drug response. For Top 1 inhibitors, additional predictive features include expression of chromatin remodeling factors and genes modulating apoptosis capacity, while complementary PARP inhibitor response determinants include PARP1 and drug efflux pump expression. Pathway and process-based gene annotations allow biological interpretation of response predictive features. CellMinerCDB also includes ongoing algorithmic work to improve the construction of multivariate predictive models using constraints from biological networks. These approaches bridge a limiting gap in existing methods, which either ignore biological knowledge altogether or are limited to exploration within known pathways and processes. Citation Format: Vinodh N. Rajapakse, Augustin Luna, Chris Sander, William C. Reinhold, Yves Pommier. CellMinerCDB: Enabling cross-database exploration of molecular pharmacology data and response determinant discovery in cancer cell lines [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 2586. doi:10.1158/1538-7445.AM2017-2586

Targeted mass spectrometry: An emerging powerful approach to unblock the bottleneck in phosphoproteomics.

Following the rapid expansion of the proteomics field, the investigation of post translational modifications (PTM) has become extremely popular changing our perspective of how proteins constantly fine tune cellular functions. Reversible protein phosphorylation plays a pivotal role in virtually all biological processes in the cell and it is one the most characterized PTM up to date. During the last decade, the development of phosphoprotein/phosphopeptide enrichment strategies and mass spectrometry (MS) technology has revolutionized the field of phosphoproteomics discovering thousands of new site-specific phosphorylations and unveiling unprecedented evidence about their modulation under distinct cellular conditions. The field has expanded so rapidly that the use of traditional methods to validate and characterize the biological role of the phosphosites is not feasible any longer. Targeted MS holds great promise for becoming the method of choice to study with high precision and sensitivity already known site-specific phosphorylation events. This review summarizes the contribution of large-scale unbiased MS analyses and highlights the need of targeted MS-based approaches for follow-up investigation. Additionally, the article illustrates the biological relevance of protein phosphorylation by providing examples of disease-related phosphorylation events and emphasizes the benefits of applying targeted MS in clinics for disease diagnosis, prognosis and drug-response evaluation.

Proteogenomic integration reveals therapeutic targets in breast cancer xenografts

Recent advances in mass spectrometry (MS) have enabled extensive analysis of cancer proteomes. Here, we employed quantitative proteomics to profile protein expression across 24 breast cancer patient-derived xenograft (PDX) models. Integrated proteogenomic analysis shows positive correlation between expression measurements from transcriptomic and proteomic analyses; further, gene expression-based intrinsic subtypes are largely re-capitulated using non-stromal protein markers. Proteogenomic analysis also validates a number of predicted genomic targets in multiple receptor tyrosine kinases. However, several protein/phosphoprotein events such as overexpression of AKT proteins and ARAF, BRAF, HSP90AB1 phosphosites are not readily explainable by genomic analysis, suggesting that druggable translational and/or post-translational regulatory events may be uniquely diagnosed by MS. Drug treatment experiments targeting HER2 and components of the PI3K pathway supported proteogenomic response predictions in seven xenograft models. Our study demonstrates that MS-based proteomics can identify therapeutic targets and highlights the potential of PDX drug response evaluation to annotate MS-based pathway activities.

Involvement of microRNA-141-3p in 5-fluorouracil and oxaliplatin chemo-resistance in esophageal cancer cells via regulation of PTEN.

microRNAs (miRNAs) act as a major regulator of acquired chemo-resistance in various types of cancer therapeutics. This study investigated the contribution of miRNAs in influencing multiple drug resistance in esophageal squamous cell carcinoma (ESCC). The sensitivity of four ESCC cell lines (EC109, EC9706, TE-1 and KYSE-150) to 5-fluorouracil (5-FU) and oxaliplatin (OX) was determined by MTT assay. A 5-FU and OX-resistant subline, EC9706R, was established by continuous exposure to stepwise increasing concentration of 5-FU and OX. Microarray technology was used to compare the differential expression of miRNAs between resistant cells and parental cells. Chemo-sensitivity assay was performed to evaluate drug response in EC9706R cells transfected with miRNA mimic or inhibitor. The direct targets of miRNA were identified by employing pathway analysis and then confirmed with luciferase assay. Sixty ESCC tissue samples and their paired adjacent normal tissues were collected to validate the expression of identified miRNA. Mouse models were further utilized to investigate the function of miRNA on acquired chemo-resistance. MicroRNA panel results indicated that a total of 12 miRNAs were differentially expressed and miR-141-3p was highly over expressed in resistant cells. Inhibition of miR-141-3p reversed acquired chemo-resistance in EC9706R cells by stimulating apoptosis. The expression of miR-141-3p was significantly increased in ESCC tissue samples compared to their matched distant normal tissues. In addition, the elevated miR-141-3p expression was found to be associated with ESCC differentiation status and TNM stage. Moreover, Phosphatase and tensin homolog (PTEN) was identified as direct target of miR-141-3p. Western blot exhibited altered protein levels of PTEN, Akt, and PI3k with miR-141-3p inhibitor. An inverse correlation between PTEN expression and miR-141-3p expression was also observed in tissue samples. EC9706R xenograft mouse model became sensitized to 5-FU and OX treatment following miR-141-3p inhibitor transfection in vivo. Our study demonstrated that miR-141-3p contributed to an acquired chemo-resistance through PTEN modulation both in vitro and in vivo.

Abstract 4262: Combined 3D quantitative imaging and 3D cell culture for cancer drug discovery

Abstract Monitoring drug activity in multicellular tumor spheroid in vitro models is an important challenge as these 3D models have demonstrated their major relevance in pharmacological screening and anticancer drugs evaluation (1). Optical microscopy coupled to the selective plane illumination microscopy (SPIM)3D microscopy, and, and automatic automated images processing and quantitative analysis are promising approaches to process optimizes the evaluation of drug response in a 3D cell culture models with, in a high/mediumhigh throughput screening waycapabilities. BIOMIMESYS® used as a 3D cell model is based on hyaluronic to provide an in vivo like environment to the cells. The In this study the effects of the topoisomerase I inhibitor Topotecan were investigated on colon adenocarcinoma HCT-116 3D cell culture. Spheroids were prepared and grown in BIOMIMESYS® used as a 3D cell model isa matrix based on hyaluronic acid tothat provides an in vivo like environment to the cells. was monitored and coupled with mMorphometric parameters and with nuclei fluorescence markers were used to monitor analysis for drug activity. Data provideand to characterization characterize of both cytostatic and cytotoxic effects of this drug withon the basis of both quantitative 3D and imaging of the mechanisms. These data highlight the relevance of combining a biomimetic microenvironment for 3D cell culture recreating the the in vivo features of a tumor cell population biology, together with and high resolution content microscopy-based quantification for drug screening. This biomimetic model provides an innovative platform for the in-depth analysis of tumor development and for the discovery or the characterization of novel therapeutic targets. Ref: 1) Breslin S, O’Driscoll L. Drug Discov Today.18: 240-249 (2013) Citation Format: Guillaume Vidal, Valérie Lobjois, Zied Souguir, Pauline Pannetier, Elise Demange, Jean-Michel Lagarde. Combined 3D quantitative imaging and 3D cell culture for cancer drug discovery. [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 4262.

Abstract 616: High throughput robotic drug screening against microprinted tumor spheroids

Abstract Cancer cell spheroids present a biologically relevant model of avascular tumors and a unique tool for anti-cancer drug discovery. Despite being used in research laboratories for several decades, spheroids are not routinely used in the mainstream drug discovery pipeline primarily due to the difficulty of mass-producing uniformly sized spheroids and intense labor involved in handling, drug treatment, and analyzing of spheroids. We overcome this barrier using a novel technology to robotically microprint spheroids in standard 384-microwell plates. An aqueous drop containing cancer cells is dispensed into a bath of a second, immiscible aqueous phase to allow generation of a single spheroid. Using several cancer cells, we establish that this approach results in spheroids of well-defined size with less than 8% deviation from the mean diameter in 384-microwell plates. We demonstrate the feasibility of robotic, high throughput compound screening against tumor spheroids using of a collection of 25 standard chemotherapeutics and molecular inhibitors against three different cancer cells, HT-29 colon cancer cells, U-87 MG brain cancer cells, and MDA-MB-157 breast cancer cells. Dose-dependent experiments are done with each drug at a wide range of concentrations. After six days of incubation, viability of cancer cells in drug-treated spheroids is measured using a PrestoBlue assay that we have optimized for three-dimensional cell cultures. Post-treatment morphological changes are used as a secondary measure for analysis. This screening identifies specific MEK inhibitors including Trametinib and Selumetinib that potently inhibit growth of HT-29 spheroids at nanomolar concentrations, whereas several PI3K inhibitors such as pictilisib significantly block proliferation of U-87 MG spheroids at micromolar concentrations. Spheroids of MDA-MB-157 cells show resistance to most of the drugs. We identify few compounds such as a Survivin inhibitor, YM155, and doxorubicin that disintegrate spheroids and induce significant cell death in all three cells. To generate a scoring system, we use half-maximum inhibitory concentration (IC50), maximum inhibition (Emax), and area under the dose-response curve (AUC) to present a multi-parametric approach that takes into account both potency and efficacy parameters for evaluating drug responses in tumor spheroids. In conclusion, our robotic technology offers a low cost and convenient platform for high throughput screening of large collections of chemical compounds against tumor spheroids. Incorporation of this technology in drug discovery applications will make drug testing and screening with realistic tumor models a routine laboratory technique prior to expensive and tedious in vivo analyses, dramatically improving testing throughput and efficiency and reducing costs. Citation Format: Pradip Thakuri, Gary Luker, Hossein Tavana. High throughput robotic drug screening against microprinted tumor spheroids. [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 616.

Copy number deletion of RAD50 as predictive marker of BRCAness and PARP inhibitor response in BRCA wild type ovarian cancer

ObjectiveTo identify novel prognostic and therapeutic markers for PARP inhibitors in BRCA wild type ovarian cancer (OvCa). MethodsBRCAness status was defined by analyzing whole-exome deep sequencing data from 220 BRCAwt OvCa cases in TCGA. Thirty-three DNA-repair genes were screened in an integrated manner for BRCA-independent mechanism of BRCAness using multiple-dimensional genomic data. Publicly available databases and siRNA knock-down were used for external validation and evaluation of drug response in OvCa cell lines. ResultsIn 220 BRCAwt OvCa patients, tumors exhibiting the BRCAness signature have enhanced OS (HR [95% CI]=0.33 [0.15–0.69], P=0.004) and PFS (HR [95% CI]=0.51 [0.24–1.08], P=0.077), strongly suggesting a BRCA-independent mechanism of drug sensitivity in those patients. Systematic screening of driving molecular events of BRCAness revealed that RAD50 deletion is a marker of BRCAness. The RAD50 deletion occurred in 18% of BRCAwt OvCa patients. RAD50 deletion led to its decreased mRNA expression in tumors (fold change=0.63, P=3.56×10−13). In BRCAwt patients, RAD50 deletion was associated with significantly better OS (HR [95% CI]=0.44 [0.25–0.78], P=0.005) and PFS (HR [95% CI]=0.60 [0.37–0.99], P=0.044), adjusted by age and stage. Knockdown of RAD50 expression augmented OvCa cell's responses to cisplatin and olaparib. Among 19 OvCa cell lines, the RAD50 copy number deletion is significantly associated with better responses to two structurally distinct PARPis (i.e. olaparib and rucaparib). ConclusionOur study identified the copy number deletion of RAD50 as a candidate marker for survival and response to PARPis in BRCAwt OvCa tumors.

Mechanistic interrogation of combination bevacizumab/dual PI3K/mTOR inhibitor response in glioblastoma implementing novel MR and PET imaging biomarkers.

Resistance to bevacizumab (BEV) in glioblastoma is believed to occur via activation of molecular networks including the mTOR/PI3K pathway. Using an MR/PET molecular imaging biomarker approach, we investigated the response to combining BEV with the mTOR/PI3K inhibitor BEZ235. Tumours were established by orthotopically implanting U87MG-luc2 cells in mice. Animals were treated with BEZ235 and/or BEV, and imaged using diffusion-weighted-MRI, T2-weighted and T2*-weighted before and after administration of superparamagnetic iron oxide contrast agent. Maps for changes in relaxation rates (ΔR2, ΔR2* and apparent diffusion coefficient) were calculated. Vessel size index and microvessel density index were derived. 3'-Deoxy-3'-[(18)F]fluorothymidine ([(18)F]FLT) PET and O-(2-[(18)F]fluoroethyl)-L-tyrosine ([(18)F]FET) PET were further performed and tumour endothelium/proliferation markers assessed by immunohistochemistry. Treatment with BEV resulted in a pronounced decrease in tumour volume (T2-weighted MRI). No additive effect on tumour volume was observed with the BEV/BEZ235 combination compared with BEV monotherapy. The Ki67 proliferation index and [(18)F]FLT uptake studies were used to support the observations. Using ΔR2* and ΔR2 values, respectively, the BEV/BEZ235 combination significantly reduced tumour microvessel volume in comparison to BEV alone. Decreased microvessel density index was further observed in animals treated with the combination, supported by von Willebrand factor (vWF) immunohistochemistry. [(18)F]FET uptake was decreased following treatment with BEV alone, but was not further reduced following treatment with the combination. vWF immunohistochemistry analysis showed that the mean tumour vessel size was increased in all cohorts. Assessing MR imaging biomarker parameters together with [(18)F]FET and [(18)F]FLT PET provided information on mechanism of action of the drug combination and clues as to potential clinical responses. Following translation to clinical use, treatment with a BEV/BEZ235 combination could reduce peritumoral oedema obviating the requirement for steroids. The use of hypothesis-driven molecular imaging studies facilitates the preclinical evaluation of drug response. Studies of this kind may more accurately predict the clinical potential of the BEV/BEZ235 combinationregimen as a novel therapeutic approach in oncology.

Electrophysiological maturation and drug responses of human induced pluripotent stem cell-derived cortical neuronal networks over 1 years culture

Event Abstract Back to Event Electrophysiological maturation and drug responses of human induced pluripotent stem cell-derived cortical neuronal networks over 1 years culture Ikuro Suzuki1*, Aoi Odawara1, Hiroki Katoh1 and Naoki Matsuda1 1 Tohoku Institute of Technology, Department of Electronics, Japan Motivation Human induced pluripotent stem cell (hiPSC)-derived neurons may be effectively used for drug discovery and toxicity testing. However, the immaturity of cultured human iPSC-derived neurons and the lack of established functional evaluation methods are problematic. The purpose of this study was to evaluate the physiological maturation and drug responses depending on culture days using multi-electrode array (MEA) system, and to detect the epilepsy pehnomena and the effects of ani epilepsy drugs as an application example. Material and Methods Human induced PSC-derived cerebral cortical neurons (hyCCNs; ax0026F, Axol Bioscience Inc., UK) [1] were cultured at 1.0 ~ 106 cells/cm2 on 64-channel MEA chips (MED-P515A; Alpha Med Scientific) coated with Axol Sure Bond Coating Solution (Axol Sure Bond Coating solusion; ax0041, Axol Bioscience Inc.) at 37‹C in a 5% CO2/95% air atmosphere. Spontaneous and evoked extracellular field potentials were acquired at 37‹C under a 5% CO2 atmosphere using a 64-channel MEA system (MED64-Basic; Alpha Med Scientific) at a sampling rate of 20 kHz/channel. Signals were low-pass filtered at 100 Hz and stored on a personal computer. Spontaneous firing was recorded every week for over 600 days. To investigate pharmacological effects depending culture days, we administered 10 ƒÊM bicuculline, 5 ƒÊM kainic acid, 50 ƒÊM CNQX, and 50 ƒÊM AP-5 at 10-15 and 33-36 weeks culture respectively. To detect the epilepsy pehnomena and the effects of ani-epilepsy drugs (AEDs), we administerd pentylentetrazole (PTZ) and typical AEDs (phenytoin and sodium valproate (VPA). Results We have succeeded in detection of spontaneous firings over 600 days and the future of time course. Syncronized burst firings (SBFs), in which generated by chemical synapse transmission, were observed over 10-13 weeks. As the culture days has passed, the number of occurrences of SBFs were increased up to approximately 30 weeks. Both spontaneous firings and evoked responses, we detected the effects of typical synaptic-related drugs at 33-36 weeks compaird to 10-15 weeks culture, indicative of greater functional maturation. Moreover, we also detected the induction of epileptic seizure by PTZ, in which high frequency SBFs were generated by 0.1-1mM PTZ administration, and the effects of AEDs [2]. Discussion Synchronized spike bursts are critical for information transfer within the cortex and are indicators of functional maturation. Such SBFs were first observed at 10?13 weeks and increased in frequency up to approximately 30 weeks. In rat cortical neurons at 1/4 cell density, synchronized firing attributable to chemical synaptic transmission occurred after only 1 week in culture, suggesting that hiPSC-derived cortical neurons require much longer to achieve functional maturation in vitro. Nonetheless, this extra time is reasonable considering the advantages for translational research presented by human neurons. These spontaneous and evoked responses also showed progressive changes in pharmacological response. In particular, the detection of fully functional NMDA responses is important because the NMDA receptor mediates several forms of neuroplasticity related to learning. Moreover, NMDA overactivity is a key pathogenic event in numerous acute and chronic neurodegenerative diseases. We also suggest that the human iPSC-derived neuron culture is a useful model system to investigate the effects of common AEDs and an alternative to animal experiments for drug screening. In addition, our assay can be adapted to iPSC-derived neurons from epilepsy patients. Conclusion We found that functional maturation in hiPSC-derived coritical neurons need the long-term culture over 20-30 weeks. Evaluation of drug responses is suitable to the culture sample over 20- 30 weeks. We have proved the MEA coupled hiPSC-derived neuronal neurons is useful to neuropharmacological/neurotoxicological assays for drug screening by detecting the drug effects both spontaneous firings and evoked responses. Our results also provide an important indication for the international standardization of evaluation procedures using in vitro human central neurons [2]. Acknowledgements We thank Axol Bioscience and Alpha Med Scientific Company for supporting this research. This study was supported by JSPS KAKENHI Grant Number 26560247, 14J11194.

Facilitating Structure-Function Studies of CFTR Modulator Sites with Efficiencies in Mutagenesis and Functional Screening

There are nearly 2000 mutations in the CFTR gene associated with cystic fibrosis disease, and to date, the only approved drug, Kalydeco, has been effective in rescuing the functional expression of a small subset of these mutant proteins with defects in channel activation. However, there is currently an urgent need to assess other mutations for possible rescue by Kalydeco, and further, definition of the binding site of such modulators on CFTR would enhance our understanding of the mechanism of action of such therapeutics. Here, we describe a simple and rapid one-step PCR-based site-directed mutagenesis method to generate mutations in the CFTR gene. This method was used to generate CFTR mutants bearing deletions (p.Gln2_Trp846del, p.Ser700_Asp835del, p.Ile1234_Arg1239del) and truncation with polyhistidine tag insertion (p.Glu1172-3Gly-6-His*), which either recapitulate a disease phenotype or render tools for modulator binding site identification, with subsequent evaluation of drug responses using a high-throughput (384-well) membrane potential–sensitive fluorescence assay of CFTR channel activity within a 1 wk time frame. This proof-of-concept study shows that these methods enable rapid and quantitative comparison of multiple CFTR mutants to emerging drugs, facilitating future large-scale efforts to stratify mutants according to their “theratype” or most promising targeted therapy.

Personalized Drug Therapy in Cystic Fibrosis: From Fiction to Reality.

Personalized drug therapy for cystic fibrosis (CF) is a long-term dream for CF patients, caregivers, physicians and researchers. After years of study, the fiction of personalized treatment has turned to hope. Basic information about CFTR mutations classes and new treatments is needed if we are to deal properly with the new CF era. The problems involved in this issue, however, should be evaluated with greater care and attention. VX-770 is a new drug available to treat CF patients with some class III CFTR mutations and other drugs are being studied regarding other classes. The scientific literature has constantly given information about each therapy, both in vitro and in vivo. The hope is increasing. Nevertheless the "scientific world" still lacks information about patients' reality and daily health related practical needs. Clinical trials have showed good evaluation of some drugs so far, but clinical response is a wide spectrum yet to be analyzed: CFTR mutations spectrum, costs related to the treatment with new drugs (for VX-770 therapy), variability of CF clinical expression, limitations to test in vitro drugs, absence of good clinical markers to evaluate drug response, absence of long-term studies and with patients below six years old, multidrug treatment used to improve the expression response, and finally, the most important problem, who will benefit from the new drugs therapy, are issues that constitute a barrier that should be overcome. Personalized drug therapy may not be a fiction anymore, but it is not yet a reality for all CF patients.

Data Mining Approaches for Genomic Biomarker Development: Applications Using Drug Screening Data from the Cancer Genome Project and the Cancer Cell Line Encyclopedia.

Developing reliable biomarkers of tumor cell drug sensitivity and resistance can guide hypothesis-driven basic science research and influence pre-therapy clinical decisions. A popular strategy for developing biomarkers uses characterizations of human tumor samples against a range of cancer drug responses that correlate with genomic change; developed largely from the efforts of the Cancer Cell Line Encyclopedia (CCLE) and Sanger Cancer Genome Project (CGP). The purpose of this study is to provide an independent analysis of this data that aims to vet existing and add novel perspectives to biomarker discoveries and applications. Existing and alternative data mining and statistical methods will be used to a) evaluate drug responses of compounds with similar mechanism of action (MOA), b) examine measures of gene expression (GE), copy number (CN) and mutation status (MUT) biomarkers, combined with gene set enrichment analysis (GSEA), for hypothesizing biological processes important for drug response, c) conduct global comparisons of GE, CN and MUT as biomarkers across all drugs screened in the CGP dataset, and d) assess the positive predictive power of CGP-derived GE biomarkers as predictors of drug response in CCLE tumor cells. The perspectives derived from individual and global examinations of GEs, MUTs and CNs confirm existing and reveal unique and shared roles for these biomarkers in tumor cell drug sensitivity and resistance. Applications of CGP-derived genomic biomarkers to predict the drug response of CCLE tumor cells finds a highly significant ROC, with a positive predictive power of 0.78. The results of this study expand the available data mining and analysis methods for genomic biomarker development and provide additional support for using biomarkers to guide hypothesis-driven basic science research and pre-therapy clinical decisions.

Regulatory science for the proper evaluation of biomarkers (overview)

New drug development (NDD) for intractable diseases such as cancer and Alzheimer's disease has been challenging in recent years because it is difficult to evaluate the therapeutic efficacy of new drugs and the response of individual patients. Thus biomarkers might be a useful tool to facilitate NDD because they can be used to evaluate accurately drug responses. Biomarkers include proteins, metabolites, and genetic targets; imaging data and can also be used in pre-clinical studies, clinical trials, and post-marketing surveillance. In pre-clinical studies, biomarkers are used as an index of the pharmacological and toxicological effects of a new drug, which may help to predict the clinical response. In clinical studies, biomarkers are widely used as an index of clinical efficacy and safety for dose-adjustment and for patient selection. In post-clinical studies, biomarkers may facilitate the evaluation of drug responses, as well as aid improvements in drug efficacy. Several points should be considered for biomarker-guided NDD. First, the clinical study design is very important and must be suitable to permit the use of the relevant biomarkers. The analytical methods should be carefully evaluated, and evidence should be provided regarding the physiological significance and relevance of the biomarker with regard to its intended use. Regulatory sciences are required to resolve these issues and bridge the gap between basic science and clinical studies that involve biomarkers.

A tumor deconstruction platform identifies definitive end points in the evaluation of drug responses.

Tumor heterogeneity and the presence of drug-sensitive and refractory populations within the same tumor are almost never assessed in the drug discovery pipeline. Such incomplete assessment of drugs arising from spatial and temporal tumor cell heterogeneity reflects on their failure in the clinic and considerable wasted costs in the drug discovery pipeline. Here we report the derivation of a flow cytometry-based tumor deconstruction platform for resolution of at least 18 discrete tumor cell fractions. This is achieved through concurrent identification, quantification and analysis of components of cancer stem cell hierarchies, genetically instable clones and differentially cycling populations within a tumor. We also demonstrate such resolution of the tumor cytotype to be a potential value addition in drug screening through definitive cell target identification. Additionally, this real-time definition of intra-tumor heterogeneity provides a convenient, incisive and analytical tool for predicting drug efficacies through profiling perturbations within discrete tumor cell subsets in response to different drugs and candidates. Consequently, possible applications in informed therapeutic monitoring and drug repositioning in personalized cancer therapy would complement rational design of new candidates besides achieving a re-evaluation of existing drugs to derive non-obvious combinations that hold better chances of achieving remission.

Digital quantification of miRNA directly in plasma using integrated comprehensive droplet digital detection.

Quantification of miRNAs in blood can be potentially used for early disease detection, surveillance monitoring and drug response evaluation. However, quantitative and robust measurement of miRNAs in blood is still a major challenge in large part due to their low concentration and complicated sample preparation processes typically required in conventional assays. Here, we present the 'Integrated Comprehensive Droplet Digital Detection' (IC 3D) system where the plasma sample containing target miRNAs is encapsulated into microdroplets, enzymatically amplified and digitally counted using a novel, high-throughput 3D particle counter. Using Let-7a as a target, we demonstrate that IC 3D can specifically quantify target miRNA directly from blood plasma at extremely low concentrations ranging from 10s to 10 000 copies per mL in ≤3 hours without the need for sample processing such as RNA extraction. Using this new tool, we demonstrate that target miRNA content in colon cancer patient blood is significantly higher than that in healthy donor samples. Our IC 3D system has the potential to introduce a new paradigm for rapid, sensitive and specific detection of low-abundance biomarkers in biological samples with minimal sample processing.

Abstract A40: Development of a perfusion-based 3D human tri-culture breast microtumor

Abstract Background: The tumor microenvironment (TME) is a 3D, dynamic interaction between tumor and stromal cells, extracellular matrix, and soluble factors which can promote tumor progression and resistance to therapy. Significant efforts are being made to develop long-term in vitro TME models to study cancer biology, improve drug development, and guide clinical decisions. We have developed a cost-effective micro-bioreactor culture device which enables a wide variety of dynamic cellular and TME interactions that can be monitored with in situ, label free, and non-lytic microscopic analysis. Using the 3DKUBE™, we developed a 3D breast cancer model composed of MCF-7 cells and fibroblasts in segregated co-culture and identified a highly active PI3K inhibitor that had limited activity in 2D monoculture. Based on a more complex morphologic and functional 3D mammary gland model that incorporates multiple human primary cells including epithelial cells (HuMEC), mammary fibroblasts, and adipocytes, our long-term 3D perfused tri-culture breast cancer model is designed to evaluate drug response continuously using non-lytic analysis. We theorize that different breast cancer epithelial cells will be supported in a standard 3D stromal platform which supports mammary gland function. Herein, we describe the 3DKUBE™ modifications and early optimization of a defined stromal TME which supports HuMECs in 3D perfusion. Materials & Methods: Primary human cells were used to optimize seeding conditions for silk fibroin scaffolds containing Matrigel™/collagen hydrogels, including, media composition, cellular ratios, flow rates, and analytical methods for the 3DKUBE™. Destructive analytical methods included morphology (H&E), phenotype (histology), viability & survival (CyQuant, TUNEL, Ki-67), and ER, PR, and casein expression (RT-PCR). Non-lytic assays included metabolism (resazurin reduction), cytotoxicity (LDH release), soluble biomarker expression (xMAP™), and two-photon emission fluorescence via multiphoton microscopy (MPM). Results: The 3DKUBE™ imaging window (1.25 mm thick with a refractive index of 1.590, transmittance (~90%) at wavelengths >290 nm) was replaced with a No. 1.5 glass, high resolution window allowing non-destructive, in situ microscopic imaging. MPM images obtained provided cellular detail (granularity, nuclear-cytoplasmic ratio, size). Optimization of the media conditions and cell numbers revealed that a tri-culture ratio of 1:1:1 was stable in the silk-Matrigel-collagen scaffold during prolonged static and perfusion culture in mixed media. Differentiation of adipocytes had to be performed separately from the tri-culture due to the cytotoxicity of the differentiation media to the epithelial cells. This was achieved via a serial seeding and differentiation method. Our initial drug studies revealed that cytotoxicity could be monitored over the course of a week by multiple methods, including LDH secretion, Presto blue, and CyQuant. Though non-specific drug binding (CPT-11) was seen in the complex 3D matrix, it did not materially impact total drug concentrations. Conclusions: Our current optimization results indicate our ability to grow a bioengineered 3D human breast tri-culture model under perfusion using the 3DKUBE™ while monitoring tissue structure, function, and drug response through both lytic and non-lytic methods. Future Development: The optimized perfused stroma will be used to develop ex vivo breast microtumors of clinically relevant subtypes (ER+, Her+, triple negative) validated against targeted agents. If validated, these 3D microtumors may be applied as predictive phenotypic screens to improve preclinical and clinical breast cancer drug development. Citation Format: Teresa M. DesRochers, Stephen Shuford, Terri F. Bruce, Matthew R. Gevaert, Chaitra Cheluvaraju, Irene Georgakoudi, David L. Kaplan, Davie E. Orr, Howland E. Crosswell. Development of a perfusion-based 3D human tri-culture breast microtumor. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A40. doi:10.1158/1538-7445.CHTME14-A40

Abstract A37: Complex crosstalk between MAPK signaling and energy metabolism in melanoma

Abstract The notoriously high glucose uptake by metastatic melanoma (MM) lesions is commonly exploited clinically using PET/CT imaging to evaluate disease progression and treatment. The purpose of this study was to use patient-derived xenograft (PDX) MM models and a PDX derived (MM8.1) cell line, to determine how driver oncogene mutations and drugs targeting the RAS-RAF-MEK signaling pathways impact glucose uptake, ATP production and mitochondrial plasticity of MM cells both in vivo and in vitro and the implications on drug resistance. MM BRAF V600E+ patients are routinely treated targeting either mutant BRAF kinase (vemurafenib), or the MAPK downstream signaling intermediate; MEK. To begin exploring links between oncogenic BRAF and metabolism, we first conducted differential gene expression (DGE) profiling of 30 different MM lesions (n=15 patients: pre- and post-engraftment). Focusing on PGC1α; a master mitochondrial regulator promoting oxidative phosphorylation (OXPHOS) revealed lower expression levels in BRAF V600E+ tumors compared to BRAF V600V+ tumors. Further analysis of a BRAF V600E+ MM PDX model revealed strong glucose uptake by PET/CT imaging at baseline. However, 48 hrs. after treatment with vemurafenib triggered growth arrest, accompanied by increased PGC1α; mRNA levels and significant reduction in glucose uptake without change in tumor volume. Upon acquisition of acquired vemurafenib resistance in the PDX model (day 70), rapidly growing tumors displayed reversal to high glucose uptake; which was again diminished by targeting MEK (PD0325901). DGE analysis of vehicle vs. 48 hrs. day 50 and day 70 vemurafenib treated tumors revealed oscillation in the expression of genes regulating glycolysis and oxidative phosphorylation (OXPHOS) that is reflected in the PET/CT imaging. To gain deeper insight, a cell line derived from a vehicle treated tumor, was examined using the Seahorse metabolic analyzer. Vehicle treated MM8.1 cells display high glycolytic capacity and predominantly glycolytic-derived ATP production, which is reflected by only minor inhibition of ATP production with oligomycin (an inhibitor of ATP synthase, a component of the OXHPOS system). Upon treatment with PLX4032, there was rapid reduction in glycolytic capacity along with a concurrent increase in maximum oxygen consumption capacity. PLX4032 treatment additionally enhanced MM8.1 sensitivity to oligomycin, but only transiently (24 hrs. pre-treatment), as later exposure times did not induce oligomycin sensitivity despite DGE changes to indicate otherwise. In conclusion, caution is warranted in evaluating drug responses in patients using PET/CT depending on duration of treatment. Cross-talk involving MAPK signaling and energy metabolism is a dynamic process, with more of a rheostat than switch-type properties, requiring further evaluation to identify therapeutic approaches exploiting metabolic events within MM cells. Citation Format: Nathan J. Lanning, Pimiento Jose, Noel R. Monks, Paula J. Davidson, Andrew S. Borgman, Ting-Tung Chang, Anderson S. Peck, David J. Monsma, Chelsea A. Peterson, Mackeigan P. Jeff, Brian J. Nickoloff. Complex crosstalk between MAPK signaling and energy metabolism in melanoma. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A37. doi: 10.1158/1557-3125.RASONC14-A37

Anti-MET immunoPET for non-small cell lung cancer using novel fully human antibody fragments.

MET, the receptor of hepatocyte growth factor, plays important roles in tumorigenesis and drug resistance in numerous cancers, including non-small cell lung cancer (NSCLC). As increasing numbers of MET inhibitors are being developed for clinical applications, antibody fragment-based immunopositron emission tomography (immunoPET) has the potential to rapidly quantify in vivo MET expression levels for drug response evaluation and patient stratification for these targeted therapies. Here, fully human single-chain variable fragments (scFvs) isolated from a phage display library were reformatted into bivalent cys-diabodies (scFv-cys dimers) with affinities to MET ranging from 0.7 to 5.1 nmol/L. The candidate with the highest affinity, H2, was radiolabeled with (89)Zr for immunoPET studies targeting NSCLC xenografts: low MET-expressing Hcc827 and the gefitinib-resistant Hcc827-GR6 with 4-fold MET overexpression. ImmunoPET at as early as 4 hours after injection produced high-contrast images, and ex vivo biodistribution analysis at 20 hours after injection showed about 2-fold difference in tracer uptake levels between the parental and resistant tumors (P < 0.01). Further immunoPET studies using a larger fragment, the H2 minibody (scFv-CH3 dimer), produced similar results at later time points. Two of the antibody clones (H2 and H5) showed in vitro growth inhibitory effects on MET-dependent gefitinib-resistant cell lines, whereas no effects were observed on resistant lines lacking MET activation. In conclusion, these fully human antibody fragments inhibit MET-dependent cancer cells and enable rapid immunoPET imaging to assess MET expression levels, showing potential for both therapeutic and diagnostic applications.

Label-free, live optical imaging of reprogrammed bipolar disorder patient-derived cells reveals a functional correlate of lithium responsiveness.

Development of novel treatments and diagnostic tools for psychiatric illness has been hindered by the absence of cellular models of disease. With the advent of cellular reprogramming, it may be possible to recapitulate the disease biology of psychiatric disorders using patient skin cells transdifferentiated to neurons. However, efficiently identifying and characterizing relevant neuronal phenotypes in the absence of well-defined pathophysiology remains a challenge. In this study, we collected fibroblast samples from patients with bipolar 1 disorder, characterized by their lithium response (n=12), and healthy control subjects (n=6). We identified a cellular phenotype in reprogrammed neurons using a label-free imaging assay based on a nanostructured photonic crystal biosensor and found that an optical measure of cell adhesion was associated with clinical response to lithium treatment. This cellular phenotype may represent a useful biomarker to evaluate drug response and screen for novel therapeutics.