Co-clinical Trials Research Articles

Abstract 38: NSCLC patient-derived organoids to guide personalized therapy

Abstract Purpose: Translational research in non-small cell lung cancer (NSCLC) has been largely hampered by relatively low success rates for establishing patient-derived cell model or patient-derived xenograft model. Recently, NSCLC patient-derived organoid model (PDO) has been introduced, showing a high take rate and stable growth in long-term in vitro cultures. Translational relevance of NSCLC PDOs remains unclear. In this study, we evaluated whether NSCLC PDOs can predict anti-cancer treatment responses and provide therapeutic strategies to overcome drug resistance. Experimental design: Nineteen malignant effusions and 13 biopsied/surgical specimens were obtained from 31 patients with lung adenocarcinoma. Eleven patients (35%) had no prior therapy, 7 (23%) chemotherapy or radiotherapy, 6 (19%) EGFR-tyrosine kinase inhibitors (TKI), 4 (13%) ALK/ROS1-TKIs, and 3 (10%) immunotherapy. Samples were processed and cultured as reported previously. In brief, specimens were reviewed by pathologists to confirm malignancy. Then, samples were cultured in Advanced DMEM/F12 containing 10% R-spondin 1 conditioned medium, 25 ng/mL FGF7, 100 ng/mL FGF10, 100 ng/mL noggin, 500 nM A83-01, 500 nM SB202190, 1X B27, 1.25 mM N-acetylcysteine, 5 mM nicotinamide, 1X glutamax, 10 mM HEPES, and 1X primocin. For efficient establishment, we stained organoids with H&E at early passages to confirm histological features of NSCLC. Cell viability assay was performed using CellTiter-Glo 3D. PDOs (<P10) were analyzed by whole-exome sequencing and RNA-seq and cryopreserved to establish a biobank. Results: A total of 22 PDOs derived from NSCLC patients was established. Take rates from malignant effusions and biopsied/surgical specimens were 89% and 38%. Of these established PDOs, 9 were wild-type, 9 EGFR-mutant, 3 ROS1-rearranged, and 1 ALK-rearranged. Genomic and transcriptomic profile of established PDOs were concordant to those of matching parental tumors. Of note, we established clinically important models progressing to targeted therapy or immunotherapy as follows: osimertinib-resistant EGFR mutant PDOs (n=4), repotrectinib-resistant ROS1-rearranged PDO (n=1), and pembrolizumab-resistant PDO (n=1). For example, YUO-2 was established from progressing pleural effusion after 5 months of osimertinib and had lost EGFR T790M but maintained EGFR exon 19 deletion. YUO-2 was resistant to gefitinib, afatinib, and osimertinib with IC50 values of 27300 nM, 9296 nM, and 5691 nM, respectively, implicating clinical relevance of NSCLC PDO models. TKI-resistant PDOs were screened with combinations of TKI targeting the driver mutation of the established models and each drug from the FDA-approved drug library. Conclusions: NSCLC PDO models could recapitulate the characteristics of corresponding NSCLC tumors and clinical response. PDO-based co-clinical trial will accelerate translational research in NSCLC. Further results will be presented at 2019 AACR meeting. Citation Format: Seok-Young Kim, Dong Hwi Kim, Hyeong-Seok Joo, Mi Ran Yun, Ji Yeon Lee, Sang Min Kim, Hyunki Kim, Min Hee Hong, Hye Ryun Kim, Byoung Chul Cho. NSCLC patient-derived organoids to guide personalized therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 38.

Abstract 2934: AKT mutant allele-specific activation dictates pharmacologic sensitivities

Abstract The objective of this study is to explore the biochemical characteristics and therapeutic sensitivity profiles of novel, non-E17K, less frequent AKT1 mutations, and thereby expand the biomarker of sensitivity to AKT inhibition in molecularly defined cancer patients. AKT is a critical signaling node that translates phosphoinositide 3-kinase (PI3K) pathway stimulation into diverse cellular effects. Gain of function AKT1 mutations arise in diverse human cancers, of which E17K is the most common. Presence of AKT1 E17K mutation renders these tumors susceptible to AKT inhibition. Nevertheless, the long tail of potentially activating mutations in AKT is largely uncharacterized, thereby limiting our ability to act clinically in prospectively sequenced advanced cancer patients. We performed a population-scale candidate driver mutation discovery analysis in AKT1, AKT2 and AKT3 in a cohort of 41,075 retrospectively and prospectively sequenced primary and metastatic cancers, and explored both their functional and biological impact as well as their therapeutic sensitivity. Our results demonstrated that some, but not all of the identified AKT missense mutations activated PI3K signaling in a growth factor-independent manner, and sensitized tumor cells to diverse AKT inhibitors. By contrast, we discovered a different class of small in-frame paralogous AKT duplication mutants that induced distinctive structural changes, leading to a far greater degree of membrane affinity, AKT activation, pathway dependence, and hyper-sensitivity to ATP-competitive AKT inhibitors, while conferring resistance to allosteric AKT inhibitors. Leveraging a co-clinical trial framework, we are now enrolling patients on the basis of these mutations in a basket study involving AKT alterations. One such case was that of a castration-resistant metastatic prostate cancer patient who harbored AKT2 duplication mutant, and subsequently responded to AKT inhibition. Collectively, our data indicate that the degree and mechanism of activation of oncogenic AKT mutants vary, thereby dictating allele-specific pharmacological sensitivities to AKT inhibition. Citation Format: Tripti Shrestha Bhattarai, Tambudzai Shamu, Swati Patel, Alexander Gorelick, Matthew T. Chang, Elena I. Gavrila, JianJong Gao, Mark T. Donoghue, Paul S. Gao, Tara Soumerai, Wassim Abida, Lilian M. Smyth, David M. Hyman, David B. Solit, Barry S. Taylor. AKT mutant allele-specific activation dictates pharmacologic sensitivities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2934.

Systematic Review of Patient-Derived Xenograft Models for Preclinical Studies of Anti-Cancer Drugs in Solid Tumors

Patient-derived xenograft (PDX) models are used as powerful tools for understanding cancer biology in PDX clinical trials and co-clinical trials. In this systematic review, we focus on PDX clinical trials or co-clinical trials for drug development in solid tumors and summarize the utility of PDX models in the development of anti-cancer drugs, as well as the challenges involved in this approach, following the preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines. Recently, the assessment of drug efficacy by PDX clinical and co-clinical trials has become an important method. PDX clinical trials can be used for the development of anti-cancer drugs before clinical trials, with their efficacy assessed by the modified response evaluation criteria in solid tumors (mRECIST). A few dozen cases of PDX models have completed enrollment, and the efficacy of the drugs is assessed by 1 × 1 × 1 or 3 × 1 × 1 approaches in the PDX clinical trials. Furthermore, co-clinical trials can be used for personalized care or precision medicine with the evaluation of a new drug or a novel combination. Several PDX models from patients in clinical trials have been used to assess the efficacy of individual drugs or drug combinations in co-clinical trials.

Bridging the translational gap: Implementation of multimodal small animal imaging strategies for tumor burden assessment in a co-clinical trial.

In designing co-clinical cancer studies, preclinical imaging brings unique challenges that emphasize the gap between man and mouse. Our group is developing quantitative imaging methods for the preclinical arm of a co-clinical trial studying immunotherapy and radiotherapy in a soft tissue sarcoma model. In line with treatment for patients enrolled in the clinical trial SU2C-SARC032, primary mouse sarcomas are imaged with multi-contrast micro-MRI (T1 weighted, T2 weighted, and T1 with contrast) before and after immune checkpoint inhibition and pre-operative radiation therapy. Similar to the patients, after surgery the mice will be screened for lung metastases with micro-CT using respiratory gating. A systems evaluation was undertaken to establish a quantitative baseline for both the MR and micro-CT systems against which others systems might be compared. We have constructed imaging protocols which provide clinically-relevant resolution and contrast in a genetically engineered mouse model of sarcoma. We have employed tools in 3D Slicer for semi-automated segmentation of both MR and micro-CT images to measure tumor volumes efficiently and reliably in a large number of animals. Assessment of tumor burden in the resulting images was precise, repeatable, and reproducible. Furthermore, we have implemented a publicly accessible platform for sharing imaging data collected during the study, as well as protocols, supporting information, and data analyses. In doing so, we aim to improve the clinical relevance of small animal imaging and begin establishing standards for preclinical imaging of tumors from the perspective of a co-clinical trial.

Patient-derived xenografts: a valuable platform for clinical and preclinical research in pancreatic cancer.

Pancreatic cancer (PC) remains a highly lethal cancer, which still requires profound clinical and preclinical research. The predictive power of patient-derived xenograft (PDX) model determines that it will become an important tool for preclinical and clinical research. PDX is not only widely used in preclinical studies of PC drugs, but also can be used to predict the result of human clinical trials. The PDX model and the corresponding patients' co-clinical trial can quickly obtain the drug response, optimize the combination, explore drug resistance mechanisms, and screen for biomarkers. Meanwhile, PDX can be used for personalized medicine as patient's avatar model. With the development of novel PDX models: patient-derived orthotopic xenografts (PDOX) model, humanized PDX model and CTC-derived PDX model, cooperation of various research teams is needed to build PDX encyclopedia of PC.

Translating Human Cancer Sequences Into Personalized Porcine Cancer Models.

The global incidence of cancer is rapidly rising, and despite an improved understanding of cancer molecular biology, immune landscapes, and advancements in cytotoxic, biologic, and immunologic anti-cancer therapeutics, cancer remains a leading cause of death worldwide. Cancer is caused by the accumulation of a series of gene mutations called driver mutations that confer selective growth advantages to tumor cells. As cancer therapies move toward personalized medicine, predictive modeling of the role driver mutations play in tumorigenesis and therapeutic susceptibility will become essential. The development of next-generation sequencing technology has made the evaluation of mutated genes possible in clinical practice, allowing for identification of driver mutations underlying cancer development in individual patients. This, combined with recent advances in gene editing technologies such as CRISPR-Cas9 enables development of personalized tumor models for prediction of treatment responses for mutational profiles observed clinically. Pigs represent an ideal animal model for development of personalized tumor models due to their similar size, anatomy, physiology, metabolism, immunity, and genetics compared to humans. Such models would support new initiatives in precision medicine, provide approaches to create disease site tumor models with designated spatial and temporal clinical outcomes, and create standardized tumor models analogous to human tumors to enable therapeutic studies. In this review, we discuss the process of utilizing genomic sequencing approaches, gene editing technologies, and transgenic porcine cancer models to develop clinically relevant, personalized large animal cancer models for use in co-clinical trials, ultimately improving treatment stratification and translation of novel therapeutic approaches to clinical practice.

A Coclinical Radiogenomic Validation Study: Conserved Magnetic Resonance Radiomic Appearance of Periostin-Expressing Glioblastoma in Patients and Xenograft Models.

Radiomics is the extraction of multidimensional imaging features, which when correlated with genomics, is termed radiogenomics. However, radiogenomic biological validation is not sufficiently described in the literature. We seek to establish causality between differential gene expression status and MRI-extracted radiomic-features in glioblastoma. Radiogenomic predictions and validation were done using the Cancer Genome Atlas and Repository of Molecular Brain Neoplasia Data glioblastoma patients (n = 93) and orthotopic xenografts (OX; n = 40). Tumor phenotypes were segmented, and radiomic-features extracted using the developed radiome-sequencing pipeline. Patients and animals were dichotomized on the basis of Periostin (POSTN) expression levels. RNA and protein levels confirmed RNAi-mediated POSTN knockdown in OX. Total RNA of tumor cells isolated from mouse brains (knockdown and control) was used for microarray-based expression profiling. Radiomic-features were utilized to predict POSTN expression status in patient, mouse, and interspecies. Our robust pipeline consists of segmentation, radiomic-feature extraction, feature normalization/selection, and predictive modeling. The combination of skull stripping, brain-tissue focused normalization, and patient-specific normalization are unique to this study, providing comparable cross-platform, cross-institution radiomic features. POSTN expression status was not associated with qualitative or volumetric MRI parameters. Radiomic features significantly predicted POSTN expression status in patients (AUC: 76.56%; sensitivity/specificity: 73.91/78.26%) and OX (AUC: 92.26%; sensitivity/specificity: 92.86%/91.67%). Furthermore, radiomic features in OX were significantly associated with patients with similar POSTN expression levels (AUC: 93.36%; sensitivity/specificity: 82.61%/95.74%; P = 02.021E-15). We determined causality between radiomic texture features and POSTN expression levels in a preclinical model with clinical validation. Our biologically validated radiomic pipeline also showed the potential application for human-mouse matched coclinical trials.

Patient-derived xenograft mouse models: A high fidelity tool for individualized medicine.

Patient-derived xenograft (PDX) mouse models involve the direct transfer of fresh human tumor samples into immunodeficient mice following surgical resection or other medical operations. Gene expression in tumors may be maintained by serial passages of tumors from mouse to mouse. These models aid research into tumor biology and pharmacology without manual manipulation of cell cultures in vitro. and are widely used in individualized cancer therapy/translational medicine, drug development and coclinical trials. PDX models exhibit higher predictive values for clinical outcomes than cell line-derived xenograft models and genetically engineered mouse models. However, PDX models are associated with certain challenges in clinical application. The present study reviewed current collections of PDX models and assessed the challenges and future directions of this field.

Oral Cavity Squamous Cell Carcinoma Xenografts Retain Complex Genotypes and Intertumor Molecular Heterogeneity

SUMMARYHerein, we report an oral cavity squamous cell carcinoma (OCSCC) patient-derived xenograft (PDX) platform, with genomic annotation useful for co-clinical trial and mechanistic studies. Genomic analysis included whole-exome sequencing (WES) and transcriptome sequencing (RNA-seq) on 16 tumors and matched PDXs and additional whole-genome sequencing (WGS) on 9 of these pairs as a representative subset of a larger OCSCC PDX repository (n = 63). In 12 models with high purity, more than 90% of variants detected in the tumor were retained in the matched PDX. The genomic landscape across these PDXs reflected OCSCC molecular heterogeneity, including previously described basal, mesenchymal, and classical molecular subtypes. To demonstrate the integration of PDXs into a clinical trial framework, we show that pharmacological intervention in PDXs parallels clinical response and extends patient data. Together, these data describe a repository of OCSCC-specific PDXs and illustrate conservation of primary tumor genotypes, intratumoral heterogeneity, and co-clinical trial application.

Case study: patient-derived clear cell adenocarcinoma xenograft model longitudinally predicts treatment response

There has been little progress in the use of patient-derived xenografts (PDX) to guide individual therapeutic strategies. In part, this can be attributed to the operational challenges of effecting successful engraftment and testing multiple candidate drugs in a clinically workable timeframe. It also remains unclear whether the ancestral tumor will evolve along similar evolutionary trajectories in its human and rodent hosts in response to similar selective pressures (i.e., drugs). Herein, we combine a metastatic clear cell adenocarcinoma PDX with a timely 3 mouse x 1 drug experimental design, followed by a co-clinical trial to longitudinally guide a patient’s care. Using this approach, we accurately predict response to first- and second-line therapies in so far as tumor response in mice correlated with the patient’s clinical response to first-line therapy (gemcitabine/nivolumab), development of resistance and response to second-line therapy (paclitaxel/neratinib) before these events were observed in the patient. Treatment resistance to first-line therapy in the PDX is coincident with biologically relevant changes in gene and gene set expression, including upregulation of phase I/II drug metabolism (CYP2C18, UGT2A, and ATP2A1) and DNA interstrand cross-link repair (i.e., XPA, FANCE, FANCG, and FANCL) genes. A total of 5.3% of our engrafted PDX collection is established within 2 weeks of implantation, suggesting our experimental designs can be broadened to other cancers. These findings could have significant implications for PDX-based avatars of aggressive human cancers.

Abstract 2972: Co-clinical trial of olaparib and temozolomide in SCLC PDX models uncovers new biomarkers of sensitivity

Abstract Introduction: Small cell lung cancer (SCLC) is a common and rapidly fatal malignancy for which no biomarker-targeted therapies have been developed. Despite a critical need, progress suffers from (1) scarcity of cutting-edge laboratory models and (2) absence of promising targets. Patient-derived xenografts (PDX) may faithfully model the clinical disease, but because SCLC is rarely biopsied or resected, specimens for PDX generation are scarce. PARP inhibition has recently emerged as a compelling strategy for SCLC, and an ongoing phase 1/2 trial of combination olaparib tablets and temozolomide (O/T) has shown promising activity in patients. However, biomarkers for patient selection remain elusive. Methods: We generated SCLC PDX models from circulating tumor cells (CTCs), biopsies and malignant effusions. CTCs were enriched with an automated microfluidic device, the CTC-iChip. To assess the genomic fidelity of the models, we performed comparative whole exome sequencing (WES) and RNA-seq on 6 sets of corresponding patient biopsies, founder (P0) PDX tumors, and early-passage PDXs. We then assessed the activity of combination O/T in a panel of PDX models, and compared PDX responses with molecular profiles to identify candidate biomarkers. Results: 44 PDXs were generated from 32 patients, including 6 sets of serial models and 4 synchronous CTC- and biopsy-derived models. PDXs were derived with high efficiency from both CTCs (35% per blood draw) and biopsies/effusions (82% per implant). WES demonstrated that somatic alterations in tumor biopsies were stably maintained in both CTC and biopsy-derived models, without significant accumulation of new mutations, and transcriptional profiles remained consistent through early passages. Six models were derived from O/T trial patients, including two sets of serial models before and after durable responses. The serial models faithfully recapitulated patient responses to O/T: pre-trial models were highly sensitive and post-relapse were highly resistant. The co-clinical trial was expanded to 30 models, using the models derived from trial patients to delineate the margins of clinical sensitivity (6 models), intermediate sensitivity (6 models) and resistance (18 models). Among the molecular features evaluated, basal protein PARylation best distinguished the O/T-sensitive category from both intermediate (p < 0.001) and resistant models (p < 0.0001). In addition, PARylation decreased after relapse in serial models from O/T trial patients. Conclusions: Both biopsy- and CTC-derived SCLC PDX models faithfully recapitulate the genomic and functional features of the donor patient tumor. O/T sensitivity in this panel correlated with basal PARylation. The value of the co-clinical trial is the potential to refine the clinical application of O/T in real time, to optimize follow-on clinical trials and to develop biomarker-directed therapy for SCLC. Citation Format: Benjamin J. Drapkin, Julie George, Marcello Stanzione, Beow Y. Yeap, Mari Mino-Kenudson, Camilla L. Christensen, Ruben Dries, Sarah Phat, Jun Zhong, David T. Myers, Joseph A. Licausi, Tilak Sundaresan, Marina Kem, Nima Abedpour, Leica V. Sequist, Alice T. Shaw, Aaron N. Hata, Mehmet Toner, Shyamala Maheswaran, Daniel A. Haber, Martin Peifer, Roman K. Thomas, Anna F. Farago, Nicholas J. Dyson. Co-clinical trial of olaparib and temozolomide in SCLC PDX models uncovers new biomarkers of sensitivity [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 2972.

Abstract 1036: Establishment and molecular profiling of patient-derived xenograft models of Korean patients with primary lung cancer

Abstract Non-small cell lung cancer is the most prevalent type of lung cancer, and one of its subtype, squamous cell carcinoma (SCC) has the genomic complexity and high overall mutation due to the chemical carcinogens so that limited therapeutic options are available in comparison with adenocarcinoma. We constructed the patient-derived xenograft models of Korean primary lung cancer and then focused on the SCC PDX models for this particular ethnic subgroup. PDX models were established using the tissues of patients who underwent surgery as primary lung cancer at Samsung Medical Center during the period between October, 2014 and September, 2017. Briefly, tumor tissues from patients were subcutaneously engrafted and passaged two more times in NOD-scid-IL2Rγnull mouse. Models were selected whose tumor tissues showed the histopathology and genomics similar to the patient tumor tissues. Statistics of established PDX models are summarized in the Table. Successful engraftmentSuccess rate (%)Primary lung cancer (n=532)19937.41Histology--Non-small cell lung cancer (n=524)19437.02- Adenocarcinoma (n=355)8925.07- Squamous cell carcinoma (n=135)8361.48- Other NSCLC (n=34)2264.71Small cell carcinoma (n=8)562.5Type of resection--Curative lung resection (n=458)17437.99Biopsy (n=74)2533.78Pathologic staging of ADC (n=355)--I (n=162)4024.69II (n=58)1525.86III (n=68)1927.94IV (n=67)1522.39 In order to help facilitate the target drug development of SCC, we concentrated on the construction of comprehensive database of fourteen SCC PDX models integrating the information from whole exome sequencing, whole transcriptome sequencing and clinical profiles. We detected mutations commonly reported in SCC genes including EGFR, BRAF, CUL3, DDR2, TP53, MYD88, PIK3CA, PTEN, MED12, SMAD4, AKT1, CTNNB1, IDH1, HRAS, KRAS, NRAS, SMARCA4, GNAS, CDKN2A, APC, CDKN2A and RRAS2. Specifically potential therapeutic targets such as EFGR, BRAF, CUL3, DDR2, TP53 and KRAS which are currently under several clinical trials were found in our SCC PDX models, and intriguingly one model had five mutations in CUL3, KRAS, TP53, and EGFR simultaneously. We expect these SCC PDX models could be valuable resource for target drug development and interpretation of their therapeutic responses in the pre- or co-clinical trials. Detailed information on pathology and genomic information on all PDX models will be available at DNA Link PDX webpage. Citation Format: Tae Ho Kim, Hyunjin Heo, Soo Jung Lee, Seungje Lee, Eunjoo Hwang, Jinseon Lee, Heekyoung Lee, Kevin Koo, Hanna Lee, Seungjae Lee, Hwanseok Rhee, Jong Eun Lee, Jhingook Kim. Establishment and molecular profiling of patient-derived xenograft models of Korean patients with primary lung cancer [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 1036.

Abstract 5013: Rapid generation of phenomic and functional profiles of patient-derived 3D cell culture models for identification of treatment vulnerabilities of breast cancer: Early results of the EFRE-PoP project

Abstract Targeted treatment for breast cancer subsets currently relies on the occurrence of estrogen, progesterone and Her2/neu receptors. For triple negative breast cancer (TNBC) there is no identified targeted therapy. The mutational landscape for breast cancer subsets has been characterized, but drug development has been limited due to the lack of appropriate preclinical models. Development of patient-derived xenograft models (PDX) has been difficult with take rates of below 30%.To establish a series of patient-derived 3D (PD3D) cell culture models as a versatile resource for ex vivo drug sensitivity screens as well as secondary establishment of PDX. We obtained breast cancer specimens either by biopsy or surgical resection. Upon arrival tumor tissue was minced and enzymatically digested. After subsequent filtering, respective tissue size fractions were seeded as Matrix-droplets into 24-well plates and incubated under standard conditions. Growth of PD3Ds was monitored daily by microscopy. PD3Ds were splitted when their diameter reached appropriate size. Organoids were than fixed and embedded. FFPE sections of donor-tissues and derived PD3D models were than used for IHC and inspected by a pathologist. In parallel, mutational profiling of snap-frozen tumor tissue and cell cultures was performed. All models were subjected to a semi-automated multi-drug response assay in a 384-well format to assess individual compound sensitivities.We established a scalable workflow for culturing and screening of PD3D cell cultures from limited quantities of breast cancer tissue from true-cut and vacuum biopsies as well as surgical specimen. Tissue fragments of ca. 3mm in diameter were sufficient to successfully establish PD3D cell cultures at a high yield. The time needed to expand PD3D cell cultures to obtain a sufficient amount of cells for subsequent molecular analyses varies from 2-6 weeks depending on amount and quality of samples as well as grade and stage of the donor tumor tissue. At the time, we can report a rate of culture establishment rate of 75% (11 models from 15 patient samples) for various clinical relevant subgroups, including TNBC, HR+, Her 2 pos. BC and one DCIS sample. The immunohistology and mutational profiles of these samples are currently being confirmed. The established workflow for culturing PD3D cell cultures offers high yield rates within a potentially clinically relevant time-frame for HTP cytotoxicity screenings. We are encouraged that this method is suitable for various molecular subtypes of breast cancer. Once adequately validated in co-clinical trials, PD3D models would make for an intriguing tool in supporting clinical decision-making for individual patients. Particularly patients with breast cancer refractory to standard of care compounds could benefit from this approach. Citation Format: Verena I. Kiver, Guido Gambara, Olga Gorea, Jens-Uwe Blohmer, Philipp Jurmeister, Carsten Denkert, Alessandra Silvestri, Caroline M. Schweiger, Maxine Silvestrov, Ulrich Keilholz, Christian R. Regenbrecht. Rapid generation of phenomic and functional profiles of patient-derived 3D cell culture models for identification of treatment vulnerabilities of breast cancer: Early results of the EFRE-PoP project [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 5013.

Abstract 3105: Oncopig carcinoma cell lines: A foundation for co-clinical trials

Abstract The Oncopig cancer model (OCM) was developed to support a broad range of solid and liquid tumor malignancies while providing the appropriate anatomical and physiological models for preclinical and co-clinical trials to expedite human clinical trials. The OCM develops tumors following Cre recombinase induced expression of KRASG12D and TP53R167H transgenes in a temporally and spatially controlled manner. To date, in vitro transformation and in vivo tumor formation of soft tissue sarcomas (STS), hepatocellular carcinoma (HCC), and pancreatic ductal adenocarcinoma (PDAC) have been demonstrated. However, the OCM was designed to generate cancers of all tissue origins. To demonstrate the potential importance of the OCM, we have successfully isolated and transformed multiple OCM cell types, each a progenitor of clinically relevant human disease. Cell type specific cell isolations were performed, and cells cultured under appropriate conditions. Within the first 48 hours, isolated cells were treated with adenoviral vector encoding Cre recombinase (AdCre) at 200-400 MOI for 5 hours in low serum (5%) medium. Both control and transformed (AdCre treated) cells underwent basic histopathologic screening (H&E staining, immunohistochemical staining for vimentin and cytokeratin), and verification of transgene expression (RT-PCR) and tumorigenicity (xenograft into SCID mice). Cell types isolated include fibroblasts, hepatocytes, pancreatic ductal cells, dermal epithelial cells, splenocytes, ovarian surface epithelial cells, fallopian tube secretory epithelial cells, renal proximal tubule epithelial cells, urinary bladder epithelial cells, bone marrow (without specific cell isolation), and testis (no specific cell isolation). Each cell type was successfully transformed, as shown by in vitro phenotypic changes and tumor formation in vivo. Additional gene expression analysis (RNA-Seq) of the STS and HCC cell lines verified the OCM transformed cells recapitulate transcriptional hallmarks of human cancers. Consistent with human STS, OCM STS cell lines display altered TP53 signaling, Wnt signaling activation, and evidence of epigenetic reprogramming. OCM HCC cell lines show transcriptional evidence of TERT reactivation, apoptosis evasion, angiogenesis activation, and Wnt signaling activation, all similar to human HCC. While not an exhaustive list of Oncopig cell types, we have not encountered cells resistant to malignant transformation through Cre recombinase exposure. These results support the unprecedented potential for modeling of additional cancer types, including colorectal, ovarian, fallopian tube, uterine, renal, bladder, skin, and hematological cancers to support and expand preclinical, translational, and co-clinical investigations. Citation Format: Laurie A. Rund, Kyle M. Schachtschneider, Ron C. Gaba, Daniel R. Principe, Paul Grippo, Regina M. Schwind, Howard Ozer, Lawrence B. Schook. Oncopig carcinoma cell lines: A foundation for co-clinical trials [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 3105.

Abstract 1150: Acute myeloid leukemia human/mouse co-clinical trial feasibility study optimized in human transgenic IL-3/GMCSF NOD/Shi-scid-IL2rγnull mice

Abstract Acute myeloid leukemia (AML) co-clinical modeling has been optimized with peripheral blood mononuclear cells (PBMCs) collected from low volume (14 mL) patient samples to establish an algorithm for efficiently co-clinically modeling AML patients. Methods: PBMCs were ficoll gradient purified and viably cryopreserved. Intrahepatic (i.h.) inoculation of AML PBMCs in neonate NOD/Shi-scid-IL2rγnull (NOG) mice and intravenous (i.v.) inoculation in both juvenile NOG mice and juvenile human transgenic IL-3/GMCSF NOD/Shi-scid-IL2rγnull mice (NOG-EXL) were evaluated. Bone marrow (BM) aspirates, splenocytes and PBMCs from mice were evaluated by fluorescence-activated cell sorting (FACS) at 12 weeks post AML inoculation for engraftment as determined by % ratio of human CD33+ cells to total CD45+ cells (human + murine cells). Humerus bones from inoculated animals were also evaluated by human CD33 immunohistochemistry (IHC). Results: Cells from 2/6 AML patient samples (CTG-2224 and CTG-2357) successfully engrafted into neonate mice. Animals were dosed with vehicle, cytarabine, ABBV-075 (clinical trial-staged BET family bromodomain (BD) inhibitor), or ABBV-744 (a preclinical BDII selective inhibitor) and evaluated for tumor burden six weeks post drug treatment initiation. ABBV-075 and ABBV-744 treated animals had lower tumor burden in the CTG-2224 model, 17% (p<0.05) and 4% (p<0.01); respectively. Similar trends, albeit at lower engraftment (10%), were observed for both models in spleen and blood compartments. In an effort to improve engraftment efficiency NOG-EXL mice were evaluated as hosts for engraftment with a patient PBMC sample (CTG-2357) and compared with juvenile NOG mice. Efficiency in the number of mice engrafted (8/8 vs. 3/7) and the extent of BM engraftment (51% vs. 14%) following a 0.5 X 106 PBMC inoculation was improved with the NOG-EXL mice. Furthermore, a patient inoculum (CTG-2241) that had previously shown no engraftment in juvenile NOG mice exhibited a 92% take rate with 93% BM tumor burden in NOG-EXL mice. Discussion: Prior advances in AML modeling show engraftment in IL2rγnul mouse models including i.h. and i.v. inoculation of BM clinical isolates in neonate NSG mice, i.v. inoculation of leukapheresis clinical isolates in juvenile NSG and NSG-SGM3 mice, and i.v. inoculation of BM aspirates in MISTRG mice. Each approach has limitations in terms of tedious neonate mice husbandry, ease of clinical sample collection with respect to clinical patient compliance, and mouse strain availability. Here we examined the feasibility of efficiently engrafting AML regardless of mutation or clinical stage from 14 ml PBMC samples. Results showed that i.v. inoculation of AML clinical samples in juvenile NOG-EXL mice was advantageous. This research is prerequisite for conducting translational co-clinical in vivo pharmacology studies for ABBV-075. Citation Format: Neal C. Goodwin, Daniel H. Albert, Angela M. Davies, Jenny Rowe, Gerold Feuer, Michael Boyiadzis, Kathleen A. Dorritie, Maria Mancini, Regina Gandour-Edwards, Warren M. Kati, Mark D. McKee, Keith F. McDaniel, David J. Frost. Acute myeloid leukemia human/mouse co-clinical trial feasibility study optimized in human transgenic IL-3/GMCSF NOD/Shi-scid-IL2rγnull mice [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 1150.

Liquid biopsy and its role in an advanced clinical trial for lung cancer.

Liquid biopsy methodologies, for the purpose of plasma genotyping of cell-free DNA (cfDNA) of solid tumors, are a new class of novel molecular assays. Such assays are rapidly entering the clinical sphere of research-based monitoring in translational oncology, especially for thoracic malignancies. Potential applications for these blood-based cfDNA assays include: (i) initial diagnosis, (ii) response to therapy and follow-up, (iii) tumor evolution, and (iv) minimal residual disease evaluation. Precision medicine will benefit from cutting-edge molecular diagnostics, especially regarding treatment decisions in the adjuvant setting, where avoiding over-treatment and unnecessary toxicity are paramount. The use of innovative genetic analysis techniques on individual patient tumor samples is being pursued in several advanced clinical trials. Rather than using a categorical treatment plan, the next critical step of therapeutic decision making is providing the "right" cancer therapy for an individual patient, including correct dose and timeframe based on the molecular analysis of the tumor in question. Per the 21st Century Cures Act, innovative clinical trials are integral for biomarker and drug development. This will include advanced clinical trials utilizing: (i) innovative assays, (ii) molecular profiling with cutting-edge bioinformatics, and (iii) clinically relevant animal or tissue models. In this paper, a mini-review addresses state-of-the-art liquid biopsy approaches. Additionally, an on-going advanced clinical trial for lung cancer with novelty through synergizing liquid biopsies, co-clinical trials, and advanced bioinformatics is also presented. Impact statement Liquid biopsy technology is providing a new source for cancer biomarkers, and adds new dimensions in advanced clinical trials. Utilizing a non-invasive routine blood draw, the liquid biopsy provides abilities to address perplexing issues of tumor tissue heterogeneity by identifying mutations in both primary and metastatic lesions. Regarding the assessment of response to cancer therapy, the liquid biopsy is not ready to replace medical imaging, but adds critical new information; for instance, through a temporal assessment of quantitative circulating tumor DNA (ctDNA) assay results, and importantly, the ability to monitor for signs of resistance, via emerging clones. Adjuvant therapy may soon be considered based on a quantitative cfDNA assay. As sensitivity and specificity of the technology continue to progress, cancer screening and prevention will improve and save countless lives by finding the cancer early, so that a routine surgery may be all that is required for a definitive cure.

Primary Patient-Derived Cancer Cells and Their Potential for Personalized Cancer Patient Care

SummaryPersonalized cancer therapy is based on a patient’s tumor lineage, histopathology, expression analyses, and/or tumor DNA or RNA analysis. Here, we aim to develop an in vitro functional assay of a patient’s living cancer cells that could complement these approaches. We present methods for developing cell cultures from tumor biopsies and identify the types of samples and culture conditions associated with higher efficiency of model establishment. Toward the application of patient-derived cell cultures for personalized care, we established an immunofluorescence-based functional assay that quantifies cancer cell responses to targeted therapy in mixed cell cultures. Assaying patient-derived lung cancer cultures with this method showed promise in modeling patient response for diagnostic use. This platform should allow for the development of co-clinical trial studies to prospectively test the value of drug profiling on tumor-biopsy-derived cultures to direct patient care.

Preclinical and Coclinical Studies in Prostate Cancer.

Men who develop metastatic castration-resistant prostate cancer (mCRPC) will invariably succumb to their disease. Thus there remains a pressing need for preclinical testing of new drugs and drug combinations for late-stage prostate cancer (PCa). Insights from the mCRPC genomic landscape have revealed that, in addition to sustained androgen receptor (AR) signaling, there are other actionable molecular alterations and distinct molecular subclasses of PCa; however, the rate at which this knowledge translates into patient care via current preclinical testing is painfully slow and inefficient. Here, we will highlight the issues involved and discuss a new translational platform, "the co-clinical trial project," to expedite current preclinical studies and optimize clinical trial and experimental drug testing. With this platform, in vivo preclinical and early clinical studies are closely aligned, enabling in vivo testing of drugs using genetically engineered mouse models (GEMMs) in defined genetic contexts to personalize individual therapies. We will discuss the principles and essential components of this novel paradigm, representative success stories and future therapeutic options for mCRPC that should be explored.

Long noncoding RNA EGFR-AS1 mediates epidermal growth factor receptor addiction and modulates treatment response in squamous cell carcinoma

Targeting EGFR is a validated approach in the treatment of squamous-cell cancers (SCCs), although there are no established biomarkers for predicting response. We have identified a synonymous mutation in EGFR, c.2361G>A (encoding p.Gln787Gln), in two patients with head and neck SCC (HNSCC) who were exceptional responders to gefitinib, and we showed in patient-derived cultures that the A/A genotype was associated with greater sensitivity to tyrosine kinase inhibitors (TKIs) as compared to the G/A and G/G genotypes. Remarkably, single-copy G>A nucleotide editing in isogenic models conferred a 70-fold increase in sensitivity due to decreased stability of the EGFR-AS1 long noncoding RNA (lncRNA). In the appropriate context, sensitivity could be recapitulated through EGFR-AS1 knockdown in vitro and in vivo, whereas overexpression was sufficient to induce resistance to TKIs. Reduced EGFR-AS1 levels shifted splicing toward EGFR isoform D, leading to ligand-mediated pathway activation. In co-clinical trials involving patients and patient-derived xenograft (PDX) models, tumor shrinkage was most pronounced in the context of the A/A genotype for EGFR-Q787Q, low expression of EGFR-AS1 and high expression of EGFR isoform D. Our study reveals how a 'silent' mutation influences the levels of a lncRNA, resulting in noncanonical EGFR addiction, and delineates a new predictive biomarker suite for response to EGFR TKIs.

The Generation and Application of Patient-Derived Xenograft Model for Cancer Research.

Establishing an appropriate preclinical model is crucial for translational cancer research. The most common way that has been adopted by far is grafting cancer cell lines, derived from patients. Although this xenograft model is easy to generate, but has several limitations because this cancer model could not represent the unique features of each cancer patient sufficiently. Moreover, accumulating evidences demonstrate cancer is a highly heterogeneous disease so that a tumor is comprised of cancer cells with diverse characteristics. In attempt to avoid these discrepancies between xenograft model and patients’ tumor, a patient-derived xenograft (PDX) model has been actively generated and applied. The PDX model can be developed by the implantation of cancerous tissue from a patient’s tumor into an immune-deficient mouse directly, thereby it preserves both cell-cell interactions and tumor microenvironment. In addition, the PDX model has shown advantages as a preclinical model in drug screening, biomarker development and co-clinical trial. In this review, we will summarize the methodology and applications of PDX in detail, and cover critical issues for the development of this model for preclinical research.