Carcinoma Patient-derived Xenograft Model Research Articles

Abstract 945: In vivo anti-tumor activity of onvansertib, a PLK1 inhibitor, combined with gemcitabine or carboplatin in platinum- resistant ovarian carcinoma patient-derived xenograft models

Abstract Background: The standard treatment for high grade ovarian carcinoma (HGOC) is cytoreductive surgery followed by a platinum based therapy. Despite an initial high rate of complete remission, most of the patients relapse with a platinum resistant disease. Patients with resistant tumors have few options, including monotherapy with gemcitabine, and new effective therapeutic alternatives are needed. The Polo-like kinase 1 (PLK1) is a master regulator of mitosis and recent evidences suggest its role in interfering with several DNA repair mechanisms. We investigated the effect of onvansertib (ONVA), a highly selective ATP-competitor PLK1 inhibitor, in combination with carboplatin (CARBO) and gemcitabine (GEM) in platinum-resistant HGOC patient-derived xenografts (PDXs). Methods: The combination of ONVA with either CARBO or GEM was investigated in two platinum-resistant HGOC PDXs, one intrinsically resistant to platinum (MNHOC#315), the other made resistant after repeated in vivo treatments with cisplatin (MNHOC#266R). Tumors were subcutaneously (s.c.; MNHOC#315) or orthotopically (i.p.; MNHOC#266R) transplanted in nude mice, mice were treated for 4 weeks with vehicle, single agents or drug combinations and followed for tumor growth (s.c. model) and survival. Results: CARBO- and GEM-ONVA combinations were well tolerated, with a maximum body weight loss of 6.6%. Both combinations showed strong anti-tumor activities in the two tested models. In MNHOC#315 PDX, CARBO- and GEM-ONVA combinations caused a tumor stabilization that extended beyond the dosing period and resulted in a higher tumor growth inhibition as compared to vehicle and single agents treated mice. In i.p. MNHOC#266R PDX, single agents were inactive, while a statistically significant increase in survival was observed in CARBO/ONVA and in GEM/ONVA treated mice as compared to vehicle treated mice, with a 6.6- and 7-fold increase in mean survival time, respectively. Pharmacodynamic studies are ongoing to decipher the mechanisms underlying the synergic effects of both combinations. Conclusions: The combinations of ONVA with either CARBO or GEM showed potent anti-tumor activities in two platinum-resistant HGOC PDXs. Additional studies in other HGOC platinum-resistant models, both in vivo and in vitro, as well pharmacodynamic studies are ongoing to validate these results and to get insights in the mechanisms underlying their efficacy. Citation Format: Michela Chiappa, Federica Guffanti, Ilaria Mengoli, Maya Ridinger, Giovanna Damia. In vivo anti-tumor activity of onvansertib, a PLK1 inhibitor, combined with gemcitabine or carboplatin in platinum- resistant ovarian carcinoma patient-derived xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 945.

Abstract 2836: Assessment of therapeutic antibody efficacy without the interference of murine Fc receptors allows for investigation of human antibody-dependent cellular cytotoxicity mediated by NK cells in the FcResolv™ hIL-15 NOG mouse model

Abstract Targeted antibody therapy is applied to treat various cancer types. In addition to the primary mode of action (MOA), which involves direct binding to the tumor antigen, indirect MOA acting through the constant region (Fc) of the antibody can enhance anti-tumor efficacy. Indirect mechanisms engage the innate immune system, mediated by both the complement system (complement-dependent cytotoxicity (CDC)) and immune cells (antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent cellular cytotoxicity (ADCC)). These indirect mechanisms can complicate the evaluation and accurate assessment of antibody-induced ADCC by human NK cells in current mouse models. In immune-deficient mouse strains (e.g. NOG), false positives and/or negatives may occur due to interactions with murine Fc receptors. These can either result in anti-tumor responses via activation of the murine innate immune system or can interfere with the human-targeted therapy's primary MOA. To study the response to anti-cancer antibodies without the interference of these murine Fc receptor interactions and to investigate ADCC mediated by human NK cells, a novel mouse model deficient in Fc receptors and expressing human IL-15 (FcResolv™ hIL-15 NOG) was employed for testing antibody therapies. Methods: Patient-derived xenograft (PDX) tumor models were transplanted into hIL-15 NOG and FcResolv™ hIL-15 NOG mice. A human head and neck squamous cell carcinoma and a lung adenocarcinoma PDX model were both treated with cetuximab. Treatment with pertuzumab and trastuzumab was applied in a breast ductal carcinoma PDX model. Rituximab treatment was tested in two diffuse large B cell lymphoma PDX models. Based on growth kinetics, the lung cancer PDX model was chosen for further testing of ADCC in the NK cell-humanized FcResolv™ hIL-15 NOG mouse. Results and Conclusion: There was no difference in percent tumor growth inhibition between the FcResolv™ hIL-15 NOG and hIL-15 NOG mice with regards to cetuximab treatment in the lung and head and neck cancer or for trastuzumab treatment of breast ductal carcinoma. However, pertuzumab treatment revealed a false positive efficacy, with the false positive effect more pronounced in hIL-15 NOG mice than in FcResolv™ hIL-15 NOG mice. In one of the lymphoma models, a false negative result was observed. Here, rituximab did not show notable tumor inhibition in the hIL-15 NOG mice but did in the FcResolv™ hIL-15 NOG mice. These results demonstrate that FcResolv™ hIL-15 NOG mice serve as a suitable mouse model for a more accurate assessment of the therapeutic efficacy of anti-tumor antibodies. Additionally, evaluation of human-mediated ADCC of therapeutic antibodies in NK cell-humanized FcResolv™ hIL-15 NOG allows detection of effects specifically mediated by human NK cells. Citation Format: Simone Rhein, Maria Stecklum, Monika Buczek, Janell Richardson, Jens Hoffmann. Assessment of therapeutic antibody efficacy without the interference of murine Fc receptors allows for investigation of human antibody-dependent cellular cytotoxicity mediated by NK cells in the FcResolv™ hIL-15 NOG mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2836.

Abstract B127: Novel anti-CD147 antibody DS-1471a exerts antitumor effect in hepatocellular carcinoma Patient Derived Xenograft models and its efficacy correlates with the expression of CD147

Abstract Background: CD147 is an immunoglobulin-like receptor that is highly expressed in various cancers and involved in the growth, metastasis, and activation of inflammatory pathways via interactions with various functional molecules, such as integrins, CD44, and monocarboxylate transporters. Through screening of CD147-targeting antibodies with antitumor efficacy, we developed a novel humanized IgG4-formatted monoclonal anti-CD147 antibody, DS-1471a. Our previous studies revealed this antibody showed potent antitumor efficacy superior to standard of care drugs in various cell line xenograft mouse models such as hepatocellular carcinoma (HCC), pancreatic invasive ductal adenocarcinoma, and chronic myelogenous leukemia. In this study, we further examined antitumor efficacy and biomarker expression in 18 patient derived xenograft (PDX) models established from HCC patients. Methods: DS-1471a or vehicle was administered intravenously once per week at 10 mg/kg doses in HCC-PDX models and tumor volumes were measured every 2 or 3 days. After tumors were harvested, samples were analyzed by simple western blotting (sWB, capillary based western blotting system) and immunohistochemistry (IHC). H-scores were calculated from IHC results and correlation between H-score, protein expression by sWB, and tumor growth inhibition (TGI) ratio were examined. Results: Seven out of 18 PDX models showed over 50% TGI with DS-1471a treatment in comparison to vehicle, regardless of gender, tumor grade, and hepatitis virus type. Protein expression analysis by sWB showed good correlation of several biomarker candidates with efficacy of DS-1471a. Among the biomarker candidates tested, CD147, the DS-1471a target molecule, showed the strongest correlation with efficacy (r = 0.78). SMAD4, which regulates the downstream signaling of the CD147 complex, and FBXO22, reported to contribute to CD147 degradation, also showed good correlation with DS-1471a efficacy (r = 0.66 and r = 0.77, respectively). By both sWB and IHC analysis, all PDX models showed negative or weak KLF5 expression, another biomarker candidate for DS-1471a that is known to be a suppressor for SMAD signaling pathway. A clear correlation between CD147 H-score and TGI was observed (r= 0.74) in these patient derived PDX models. Correlation between CD147 H-score and protein expression levels by sWB was also observed (r = 0.71). Of note, all PDX models showed moderate to high expression of CD147. Conclusion: These pre-clinical study results suggest DS-1471a may be potentially beneficial for HCC treatment. In addition, our results show correlation of DS-1471a efficacy with expression of CD147. Further studies are needed to further understand the full potential benefit of DS-1471a in clinical trials. Citation Format: Hiroshi Yuita, Ryuichi Nakamura, Jun Tsukada, Hideo Yukinaga, Huynh The Hung, Keisuke Fukuchi. Novel anti-CD147 antibody DS-1471a exerts antitumor effect in hepatocellular carcinoma Patient Derived Xenograft models and its efficacy correlates with the expression of CD147 [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B127.

Gastroenteropancreatic neuroendocrine carcinoma tumor spheroid drug screen reveals vulnerability to tyrosyl-DNA phosphodiesterase 1 inhibitors

BackgroundGastroenteropancreatic neuroendocrine carcinomas are rare neoplasms with no effective treatments and poor prognosis. Few reliable preclinical models exist for the study of gastroenteropancreatic neuroendocrine carcinomas, limiting investigation of novel treatments. We used tumor spheroids from our recently established gastroenteropancreatic neuroendocrine carcinoma patient-derived xenograft models to systematically screen for compounds with diverse structures to identify potential new categories of therapeutic agents that can target gastroenteropancreatic neuroendocrine carcinomas. MethodsTumor spheroids were derived from our NEC913 and NEC1452 gastroenteropancreatic neuroendocrine carcinoma patient-derived xenograft models. Gastroenteropancreatic neuroendocrine carcinoma spheroids were screened against a library of 885 compounds from the National Cancer Institute Diversity Set VII collection. Cell viability was measured via AlamarBlue assay. After identification of potential therapeutic compounds, synergy screening of a selected group with temozolomide and doxorubicin was performed, and these combinations were further analyzed for γH2AX and phosphorylated-ERK proteins. ResultsWe identified 16 compounds that inhibit over 75% of gastroenteropancreatic neuroendocrine carcinoma spheroid survival. Seven are inhibitors of tyrosyl-DNA phosphodiesterase 1, a DNA repair enzyme working closely with the topoisomerase I complex. When combined with temozolomide or doxorubicin, the tyrosyl-DNA phosphodiesterase 1 inhibitor cytarabine increased the cytotoxic effects of these drugs on NEC1452 cells which was further evidenced by increasing γH2AX and decreasing phosphorylated-ERK in combination treatment compared to temozolomide alone. ConclusionBoth NEC913 and NEC1452 gastroenteropancreatic neuroendocrine carcinoma spheroid lines are useful preclinical models for drug testing. Our library screen revealed these gastroenteropancreatic neuroendocrine carcinoma spheroids are highly sensitive to a novel class of anti-cancer drugs that target nuclear genome stability.

Abstract 1293: AZD0171 (anti-LIF) combines productively with chemotherapy and anti-PD-L1 in mouse models of cancer

Abstract Leukemic inhibitory factor (LIF) is an IL-6 family member involved in embryonic stem cell maintenance and protection from maternal immune-attack. Emerging evidence indicates that LIF biology is hijacked by some tumors to promote immunosuppressive tumor associated macrophages, cancer initiating cells and epithelial-mesenchymal transition (EMT). In patients treated with anti-PD-1/anti-PD-L1 antibodies (IO), baseline high LIF expression correlated with poor outcome (Loriot et al. 2021). In preclinical models, LIF blockade has separately been reported to sensitize tumors to IO and to chemotherapy (chemo), but the impact of LIF blockade on chemo/IO combination activity is unknown. We therefore evaluated the hypothesis that LIF blockade sensitizes tumors to chemo/IO combinations. AZD0171 is a monoclonal antibody that binds to LIF and prevents signaling through LIF receptor. A murine surrogate of AZD0171 (mAZD0171) was generated and tested in vivo using three syngeneic murine cancer models, and AZD0171 was tested in patient derived xenograft models. Monotherapy mAZD0171 induced tumor gene expression changes consistent with its proposed mechanism of action in the syngeneic CT26 colon tumor model; including elevated CD8 attracting chemokines (CXCL9/10), cDC1 transcription factor (Batf3) and reduced stem cell (CD44), EMT (CY61) and hypoxic/angiogenic signatures, including CXCL12. Flow cytometry analysis highlighted changes consistent with suppressive macrophage re-education (reduced CD206/elevated MHCII). mAZD0171 sensitized mouse tumors to both IO and chemo. mAZD0171 + anti-PD-L1 treatment reversed CD8 T cell exclusion in the D2A1-m2 model; a LIF expressing cancer associated fibroblast rich, anti-PD-L1 resistant mouse mammary tumor syngeneic model (Jungwirth et al. 2018). mAZD0171 + anti-PD-L1 treatment further inhibited D2A1-m2 growth versus (vs) anti-PD-L1 treatment alone. In a non-small cell lung carcinoma patient derived xenograft model, AZD0171 improved the tumor growth inhibitory effect of Carboplatin + Paclitaxel. The triplet combination of mAZD0171 plus chemo and IO had the most marked impact on both tumor microenvironment and tumor growth. mAZD0171 addition to oxaliplatin (OHP) or Docetaxel (DTX) chemotherapy plus anti-PD-L1 significantly elevated CD8 abundance and granzyme B expression in MC38 colon tumors. mAZD0171 + anti-PD-L1 + OHP or DTX improved inhibition of MC38 tumor growth (63% for OHP triplet vs 34% for anti-PD-L1 + OHP; 49% for DTX triplet vs 36% for anti-PD-L1 + DTX) and extended median survival times (P<0.05 vs anti-PD-L1+OHP/DTX). Together these data support the hypothesis that AZD0171 has the potential to sensitize solid tumors to chemotherapy/IO combinations. The safety and activity of AZD0171 is currently being assessed in a Ph2 clinical trial in combination with durvalumab (anti-PD-L1) and chemotherapy in metastatic pancreatic cancer (NCT04999969). Citation Format: Juliana Candido, Gozde Kar, Bairu Zhang, Grace Opoku-Ansah, Amit Grover, Elizabeth A. Kuczynski, Tim Slidel, Doug Palmer, Robert W. Wilkinsom, Nadia Luheshi, Jim Eyles. AZD0171 (anti-LIF) combines productively with chemotherapy and anti-PD-L1 in mouse models of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1293.

Abstract 3112: Metabolic hallmarks of rare renal cell carcinoma patient-derived xenograft models

Abstract Renal medullary carcinoma (RMC) and fumarate hydratase (FH)-deficient renal cell carcinoma (RCC) are rare but highly aggressive malignancies that are often refractory to the therapies used for the more common RCCs. FH-deficient RCC most often occurs in individuals with hereditary leiomyomatosis and renal cell carcinoma syndrome (HLRCC) and is characterized by loss of FH activity, a key metabolic enzyme. Bevacizumab plus erlotinib (B+E) is a preferred first-line therapy for FH-deficient RCC. RMC is characterized by loss of SMARCB1, which is involved in ATP-dependent chromatin remodeling. Platinum-based chemotherapy consisting of either cisplatin or carboplatin plus paclitaxel is the preferred first-line therapy for RMC. We developed patient-derived xenograft (PDX) models of tumors exposed to the commonly used first-line therapies to allow functional characterization of the metabolic hallmarks of treatment-experienced RMC and FH-deficient RCC. Methods: Resected tumor tissue was immediately implanted into NSG male and female mice. After tumor engraftment, animals were euthanized, and the tumor tissue implanted into another set of NSG mice. PDX tissues were confirmed to be genetically identical to the original patient tissue by short tandem repeat analysis. Histology was confirmed to be identical between original patient tumor and PDX by a trained clinical pathologist. Four tumors from each PDX model and four normal human kidney tissue samples were analyzed using reverse phase protein array (RPPA). Expression of proteins shown to be highly expressed by RPPA were further validated by Western blots. Results: We successfully generated one PDX model for RMC and one for FH-deficient RCC, respectively. The RMC model was generated from a 28-year old male with sickle cell trait, who had previously received cisplatin plus paclitaxel followed carboplatin plus paclitaxel. His lesion was characterized as ypT3apN1pMx and histology was consistent with RMC. The FH-deficient RCC model was generated from a 24-year-old female with HLRCC, who had been previously treated with B+E. Her lesion was characterized as pT3apN1pM1 and histology consistent with FH-deficient RCC. Germline testing revealed a R233H pathogenic mutation in the FH gene. RPPA revealed higher expression of hexokinase II (10x HLRCC, 16x RMC), lactate dehydrogenase A (27x HLRCC, 17x RMC), and glutaminase (4x HLRCC) in PDX tissue compared to normal kidney tissue. Western blots confirmed higher expression of glutaminase 1 in both HLRCC and RMC compared to normal kidney. Furthermore, we observed substantially higher hypoxia inducible factor 2α (HIF2α) protein expression in PDX tissue from FH-deficient RCC compared to RMC PDX tumors and normal kidney. Conclusions: Our study revealed distinct metabolic features in PDX models of FH-deficient RCC following treatment with B+E, and of platinum-experienced RMC, these differences may confer targetable vulnerabilities. Citation Format: Manuel Ozambela, Alberto Pieretti, Graciela M. Nogueras Gonzalez, Tapati Maity, Lei Wang, Carolyn De La Cerda, Breanna Alonzo, Luis Segarra, Angelita Alaniz, Priya Rao, Nizar Tannir, Jose A. Karam, Christopher G. Wood, Pavlos Msaouel, Niki M. Zacharias. Metabolic hallmarks of rare renal cell carcinoma patient-derived xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3112.

Establishment of Novel Neuroendocrine Carcinoma Patient-Derived Xenograft Models for Receptor Peptide-Targeted Therapy.

Gastroenteropancreatic neuroendocrine neoplasms (GEP NENs) are rare cancers consisting of neuroendocrine carcinomas (NECs) and neuroendocrine tumors (NETs), which have been increasing in incidence in recent years. Few cell lines and pre-clinical models exist for studying GEP NECs and NETs, limiting the ability to discover novel imaging and treatment modalities. To address this gap, we isolated tumor cells from cryopreserved patient GEP NECs and NETs and injected them into the flanks of immunocompromised mice to establish patient-derived xenograft (PDX) models. Two of six mice developed tumors (NEC913 and NEC1452). Over 80% of NEC913 and NEC1452 tumor cells stained positive for Ki67. NEC913 PDX tumors expressed neuroendocrine markers such as chromogranin A (CgA), synaptophysin (SYP), and somatostatin receptor-2 (SSTR2), whereas NEC1452 PDX tumors did not express SSTR2. Exome sequencing revealed loss of TP53 and RB1 in both NEC tumors. To demonstrate an application of these novel NEC PDX models for SSTR2-targeted peptide imaging, the NEC913 and NEC1452 cells were bilaterally injected into mice. Near infrared-labelled octreotide was administered and the fluorescent signal was specifically observed for the NEC913 SSTR2 positive tumors. These 2 GEP NEC PDX models serve as a valuable resource for GEP NEN therapy testing.

Hexavalent TRAIL Fusion Protein Eftozanermin Alfa Optimally Clusters Apoptosis-Inducing TRAIL Receptors to Induce On-Target Antitumor Activity in Solid Tumors.

TRAIL can activate cell surface death receptors, resulting in potent tumor cell death via induction of the extrinsic apoptosis pathway. Eftozanermin alfa (ABBV-621) is a second generation TRAIL receptor agonist engineered as an IgG1-Fc mutant backbone linked to two sets of trimeric native single-chain TRAIL receptor binding domain monomers. This hexavalent agonistic fusion protein binds to the death-inducing DR4 and DR5 receptors with nanomolar affinity to drive on-target biological activity with enhanced caspase-8 aggregation and death-inducing signaling complex formation independent of FcγR-mediated cross-linking, and without clinical signs or pathologic evidence of toxicity in nonrodent species. ABBV-621 induced cell death in approximately 36% (45/126) of solid cancer cell lines in vitro at subnanomolar concentrations. An in vivo patient-derived xenograft (PDX) screen of ABBV-621 activity across 15 different tumor indications resulted in an overall response (OR) of 29% (47/162). Although DR4 (TNFSFR10A) and/or DR5 (TNFSFR10B) expression levels did not predict the level of response to ABBV-621 activity in vivo, KRAS mutations were associated with elevated TNFSFR10A and TNFSFR10B and were enriched in ABBV-621-responsive colorectal carcinoma PDX models. To build upon the OR of ABBV-621 monotherapy in colorectal cancer (45%; 10/22) and pancreatic cancer (35%; 7/20), we subsequently demonstrated that inherent resistance to ABBV-621 treatment could be overcome in combination with chemotherapeutics or with selective inhibitors of BCL-XL. In summary, these data provide a preclinical rationale for the ongoing phase 1 clinical trial (NCT03082209) evaluating the activity of ABBV-621 in patients with cancer. SIGNIFICANCE: This study describes the activity of a hexavalent TRAIL-receptor agonistic fusion protein in preclinical models of solid tumors that mechanistically distinguishes this molecular entity from other TRAIL-based therapeutics.

Abstract 2692: Novel chemotherapy stable subpopulations are conserved across multiple small cell lung carcinoma patient derived xenograft models

Abstract Introduction: Small cell lung cancer (SCLC) is an aggressive neuroendocrine carcinoma of the lung responsible for up to 25% of lung cancer deaths and the 6th leading cause of cancer death. SCLC initially responds well to chemotherapy, but inevitably recurs even after initial complete responses. The etiology of this relapse is likely secondary to tumor heterogeneity and/or chemotherapy resistance subpopulations reconstituting tumor. Characterization of these resistant subpopulations could yield novel therapeutic targets for SCLC treatment. Mass cytometry uses metal labeled antibodies to profile expression and phosphorylation of multiple proteins in a single cell and offers the opportunity to identify new subpopulations as targets for novel therapies in SCLC. Methods: Nude mice with SCLC patient derived xenografts (PDXs) were treated with a single cycle of carboplatin/etoposide or saline injection. PDX samples were stained with a 26-35 marker panel and an intercalator dye to identify nucleated cells. This panel measured phospho-signaling, neuroendocrine, immune, and mesenchymal cell markers, and functional markers including ki67 and cleaved caspase 3. Mouse cells, including leukocytes, were excluded using mouse MHC1 gating and Histone H3 was used to identify nucleated cells Single cell protein expression and phosphorylation was analyzed using viSNE, manual gating, as well as unsupervised clustering approaches with SPADE to identified multiple subpopulations with neuroendocrine and non-neuroendocrine features. Subpopulations were compared across multiple patient derived xenograft (PDX) models with and without chemotherapy treatment. Results: Patient derived Xenograft (PDX) tumors across 3 distinct models including with and without a single cycle of chemotherapy treatment released viable tumor and stromal cells suitable for cryopreservation and mass cytometry. Chemotherapy treated tumors had dramatic changes in subpopulation distribution compared to matched mock treated tumor. This included enrichment in EPCAM+, CD24+, CD44- progenitor like subpopulations. Similar patterns of population shift were observed in multiple models. Of note, chemotherapy stable subpopulations were conserved across 5 different PDX models including SOX2+ and Oct ¾+ tumor populations. These subpopulations sorted similarly in multidimensional space in multiple PDX models suggesting conserved origins. Conclusions: Mass cytometry was able to identify multiple NE and non-neuroendocrine cell populations from SCLC PDXs and characterize their signaling include rare subpopulations with stem like signaling factors of interest. Chemotherapy treated PDX had differential subpopulation distribution with enrichment of progenitor like cells with chemotherapy treatment similar to previous work in mouse genetic SCLC. However, rare conserved chemotherapy stable subpopulations enriched in stem-like signaling factors were identified across 5 PDX models including those with and without chemotherapy treatment. This work raises the possibility that chemotherapy stable subpopulations may contribute to progenitor populations which lead to relapse. Future work in this lab will focus on characterizing the stem like features of these subpopulations and their phenotypic dynamics and signaling. Citation Format: Jonathan M. Lehman, Jeremy Staub, Yong Zou, Jonathan Irish, Pierre Massion. Novel chemotherapy stable subpopulations are conserved across multiple small cell lung carcinoma patient derived xenograft models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2692.

Immuno-PET Detects Changes in Multi-RTK Tumor Cell Expression Levels in Response to Targeted Kinase Inhibition.

Receptor tyrosine kinase (RTK) coexpression facilitates tumor resistance due to redundancies in the phosphatidylinositol-3'-kinase/protein kinase B and KRAS/extracellular-signal-regulated kinase signaling pathways, among others. Crosstalk between the oncogenic RTK hepatocyte growth factor receptor (MET), epidermal growth factor receptor (EGFR), and human epidermal growth factor receptor 2 (HER2) are involved in tumor resistance to RTK-targeted therapies. Methods: In a relevant renal cell carcinoma patient-derived xenograft model, we use the 89Zr-labeled anti-RTK antibodies (immuno-PET) onartuzumab, panitumumab, and trastuzumab to monitor MET, EGFR, and HER2 protein levels, respectively, during treatment with agents to which the model was resistant (cetuximab) or sensitive (INC280 and trametinib). Results: Cetuximab treatment resulted in continued tumor growth, as well as an increase in all RTK protein levels at the tumor in vivo on immuno-PET and ex vivo at the cellular level. Conversely, after dual MET/mitogen-activated protein kinase inhibition, tumor growth was significantly blunted and corresponded to a decrease in RTK levels. Conclusion: These data show the utility of RTK-targeted immuno-PET to annotate RTK changes in protein expression and inform tumor response to targeted therapies.

Near-Infrared Fluorescent Imaging for Monitoring of Treatment Response in Endometrial Carcinoma Patient-Derived Xenograft Models.

Imaging of clinically relevant preclinical animal models is critical to the development of personalized therapeutic strategies for endometrial carcinoma. Although orthotopic patient-derived xenografts (PDXs) reflecting heterogeneous molecular subtypes are considered the most relevant preclinical models, their use in therapeutic development is limited by the lack of appropriate imaging modalities. Here, we describe molecular imaging of a near-infrared fluorescently labeled monoclonal antibody targeting epithelial cell adhesion molecule (EpCAM) as an in vivo imaging modality for visualization of orthotopic endometrial carcinoma PDX. Application of this near-infrared probe (EpCAM-AF680) enabled both spatio-temporal visualization of development and longitudinal therapy monitoring of orthotopic PDX. Notably, EpCAM-AF680 facilitated imaging of multiple PDX models representing different subtypes of the disease. Thus, the combined implementation of EpCAM-AF680 and orthotopic PDX models creates a state-of-the-art preclinical platform for identification and validation of new targeted therapies and corresponding response predicting markers for endometrial carcinoma.

Impact of Tumour Hypoxia on Evofosfamide Sensitivity in Head and Neck Squamous Cell Carcinoma Patient-Derived Xenograft Models.

Tumour hypoxia is a marker of poor prognosis and failure of chemoradiotherapy in head and neck squamous cell carcinoma (HNSCC), providing a strategy for therapeutic intervention in this setting. To evaluate the utility of the hypoxia-activated prodrug evofosfamide (TH-302) in HNSCC, we established ten early passage patient-derived xenograft (PDX) models of HNSCC that were characterised by their histopathology, hypoxia status, gene expression, and sensitivity to evofosfamide. All PDX models closely resembled the histology of the patient tumours they were derived from. Pimonidazole-positive tumour hypoxic fractions ranged from 1.7–7.9% in line with reported HNSCC clinical values, while mRNA expression of the Toustrup hypoxia gene signature showed close correlations between PDX and matched patient tumours, together suggesting the PDX models may accurately model clinical tumour hypoxia. Evofosfamide as a single agent (50 mg/kg IP, qd × 5 for three weeks) demonstrated antitumour efficacy that was variable across the PDX models, ranging from complete regressions in one p16-positive PDX model to lack of significant activity in the three most resistant models. Despite all PDX models showing evidence of tumour hypoxia, and hypoxia being essential for activation of evofosfamide, the antitumour activity of evofosfamide only weakly correlated with tumour hypoxia status determined by pimonidazole immunohistochemistry. Other candidate evofosfamide sensitivity genes—MKI67, POR, and SLFN11—did not strongly influence evofosfamide sensitivity in univariate analyses, although a weak significant relationship with MKI67 was observed, while SLFN11 expression was lost in PDX tumours. Overall, these data confirm that evofosfamide has antitumour activity in clinically-relevant PDX tumour models of HNSCC and support further clinical evaluation of this drug in HNSCC patients. Further research is required to identify those factors that, alongside hypoxia, can influence sensitivity to evofosfamide and could act as predictive biomarkers to support its use in precision medicine therapy of HNSCC.

677 - Ultrasound molecular imaging to predict response to sunitinib in a renal carcinoma patient derived xenograft model

677 - Ultrasound molecular imaging to predict response to sunitinib in a renal carcinoma patient derived xenograft model

Abstract 3992: Varlitinib demonstrates tumor regression and vessel normalization in ErbB-dependent and mutated beta-catenin hepatocellular carcinoma patient-derived xenograft model

Abstract Hepatocellular carcinoma (HCC) is the most common form of liver cancer and current therapies such as sorafenib, regorafenib and lenvatinib, prolong median overall survival by less than a year. Hence, there is an urgent need for additional HCC therapies. Varlitinib is a potent, reversible, small molecule pan – HER inhibitor that selectively binds to EGFR, HER2 and HER4. Varlitinib is currently being clinically tested across multiple indications including biliary tract cancer and gastric cancer. In HCC patient – derived xenograft (PDX) models, varlitinib has been shown to mediate potent anti – tumour activities (ASCO 2016). To understand the molecular profiles of HCC PDX sensitive to varlitinib, we studied the ErbB family signalling in 56 HCC PDX models and varlitinib was found to be efficacious in HCC PDX with activated ErbB2 / 3 as well as mutated beta – catenin. In HCC PDX models with activated ErbB2 / 3 , treatment with varlitinib inhibited multiple proliferation and anti – apoptosis pathways including AKT, PI3K and Survivin pathways. In addition, normalisation of blood vessels by varlitinib in the PDX models, as assessed by staining with biotinylated lectin and anti – CD31, also reduced hypoxia in the tumour microenvironment as well as enhanced the infiltration of immune cells. Activating mutations in the Wnt / beta – catenin pathway lead to tumour development in HCC and occur in a subset of HCC tumours. HCC PDX models with beta – catenin mutation were found to be sensitive to varlitinib. Varlitinib led to down – regulation of mutated beta – catenin signalling, and enhanced staining of beta – catenin at the membrane instead of in the nucleus. Since activating mutations in the Wnt / beta – catenin pathway are detected in 26 – 40 % of HCC samples, our data shows that varlitinib may represent an alternative treatment for a significant subset of HCC which are ErbB / beta – catenin – dependent. Citation Format: Wai Ho Shuen, Richard Ong, Chloe Yeo, Rebecca Banu, Lip Seng Koh, Chit Lai Chee, Qihui Seet, Lisa Ooi, Mark McHale, Bertil Lindmark, Han Chong Toh, Huynh Hung. Varlitinib demonstrates tumor regression and vessel normalization in ErbB-dependent and mutated beta-catenin hepatocellular carcinoma patient-derived xenograft model [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 3992.

MEK inhibition abrogates sunitinib resistance in a renal cell carcinoma patient-derived xenograft model

Background:Renal cell carcinoma (RCC) patients treated with tyrosine kinase inhibitors (TKI) typically respond initially, but usually develop resistance to therapy. We utilised transcriptome analysis to identify gene expression changes during development of sunitinib resistance in a RCC patient-derived xenograft (PDX) model.Methods:RCC tumours were harvested during pre-treatment, response and escape phases. Direct anti-proliferative effects of sunitinib plus MEK inhibitor were assessed. Activation status (phosphorylation) of MEK1/2 and ERK1/2 was determined, myeloid-derived suppressor cells (MDSC) sub-fractions were quantitated and G-CSF was measured by ELISA.Results:During the response phase, tumours exhibited 91% reduction in volume, characterised by decreased expression of cell survival genes. After 4-week treatment, tumours developed resistance to sunitinib, associated with increased expression of pro-angiogenic and cell survival genes. During tumour escape, cellular movement, inflammatory response and immune cell trafficking genes were induced, along with intra-tumoural accumulation of MDSC. In this PDX model, either continuous treatment with sunitinib plus MEK inhibitor PD-0325901, or switching from sunitinib to PD-0325901 was effective. The combination of PD-0325901 with TKI suppressed intra-tumoural phospho-MEK1/2, phospho-ERK1/2 and MDSC.Conclusions:Continuous treatment with sunitinib alone did not maintain anti-tumour response; addition of MEK inhibitor abrogated resistance, leading to improved anti-tumour efficacy.

Preclinical Evaluation of AMG 337, a Highly Selective Small Molecule MET Inhibitor, in Hepatocellular Carcinoma.

Aberrant hepatocyte growth factor (HGF)/MET signaling has been implicated in hepatocarcinogenesis, suggesting that MET may serve as an attractive therapeutic target in hepatocellular carcinoma. We sought to investigate the in vitro and in vivo antitumor activity of AMG 337, a potent and highly selective small molecule MET kinase inhibitor, in preclinical models of hepatocellular carcinoma. The antiproliferative activity of AMG 337 was evaluated across a panel of hepatocellular carcinoma cell lines in a viability assay. Daily oral administration was used to evaluate the in vivo antitumor activity of AMG 337 in two patient-derived xenograft (PDX) models of hepatocellular carcinoma (LI0612 and LI1078). AMG 337 exerted potent antiproliferative activity against 2 of 40 hepatocellular carcinoma cell lines, namely, MHCC97H (IC50, 0.015 μmol/L) and HCCLM3 (IC50, 0.025 μmol/L). Both sensitive cell lines showed MET amplification (MET/CEN-7 >2.0) assessed by FISH, and high MET expression (3+ IHC) assessed by IHC. AMG 337 potently inhibited p-MET in all cell lines with detectable levels of total MET. However, the dose-dependent inhibition of downstream effectors of HGF/MET signaling, including p-GAB1, p-AKT, and p-ERK, was limited to those cell lines sensitive to AMG 337 in a viability assay (MHCC97H and HCCLM3). AMG 337 significantly inhibited tumor growth at all doses tested in the MET-amplified and MET-high-expressing hepatocellular carcinoma PDX model LI0612 and had no effect on tumor growth in the non-MET-amplified and MET-low-expressing hepatocellular carcinoma PDX model LI1078. AMG 337 represents a promising and novel therapeutic strategy for targeting hepatocellular carcinomas with a dependence on HGF/MET signaling. Mol Cancer Ther; 15(6); 1227-37. ©2016 AACR.

Tumor Take Rate Optimization for Colorectal Carcinoma Patient-Derived Xenograft Models.

Background. For development of individualized treatment on a routine basis, transfer of patients' tumor tissue in a xenograft model (i.e., generation of patient-derived xenografts (PDX)) is desirable for molecular, biochemical, or functional analyses. Drawbacks are dissatisfactory tumor take rates, the necessity of fast tumor tissue processing, and extensive logistics demanding teamwork of surgeons, pathologists, and laboratory researchers. Methods. The take rates of ten colorectal cancer (CRC) tissue samples in immunodeficient mice were compared after direct cryopreservation and after a 24 h cooling period at 4°C prior to cryopreservation. Additionally, the effect of simultaneous Matrigel application on the take rates was investigated. Beside take rates, tumor growth characteristics and cell culture success were analyzed. Results. Tumor takes of CRC tissue samples were significantly improved after Matrigel application (8 versus 15 takes, p = 0.04). As expected, they diminished furthermore after 24 h cooling. Application of Matrigel could counteract this decrease significantly (2 versus 7 takes, p = 0.03). Cumulative take rate after cryopreservation was satisfactory (70%). Conclusion. Matrigel application after 24 h delay in tissue processing facilitates CRC PDX model development. These data help developing strategies for individualized tumor therapies in the context of multicenter clinical studies and for basic research on primary patient tumors.

Abstract 1477: Pharmacodynamic response and anti-tumor activity of the MET inhibitor AZD6094 in papillary renal cell carcinoma patient derived xenograft models

Abstract Papillary renal cell carcinoma (PRCC) is the second most common cancer of the kidney and carries a poor prognosis for patients with non-localized disease. The central role of the hepatocyte growth factor (HGF) receptor MET in PRCC has been explored, demonstrating that MET aberrations, either through mutation, copy number gain, or trisomy of chromosome 7 (the location of MET and HGF) occur in the majority of PRCC cases. We sought to evaluate AZD6094 (HMPL-504), a potent and selective small molecule MET kinase inhibitor, in this disease setting. However, the development of effective therapies targeting MET and other targets in PRCC has been hampered in part by a lack of available preclinical models to test novel targeted therapies. Here we describe for the first time the pharmacodynamic (PD) response and anti-tumor activity of the selective MET inhibitor AZD6094 in two preclinical patient derived xenograft (PDX) models of PRCC (RCC-43b and RCC-47). Both PDX models have increased MET copy number of 8 and 9 copies by FISH in RCC-43b, and RCC-47 respectively, and robust MET protein staining by IHC. AZD6094 treatment resulted in dose dependent anti-tumor responses reaching ∼85% tumor growth inhibition (TGI) when dosed at 2.5 mg/kg daily (qd), stasis when dosed 10 mg/kg qd, and ∼20% regression when dosed at 25 mg/kg qd in the RCC-43b model and ∼63% TGI, ∼89% TGI, ∼64% regression, and ∼96% regression in the RCC-47 model when dosed 0.5, 2.5, 10, and 25 mg/kg qd respectively. The standard of care for RCC, sunitinib, showed no activity in RCC-43b when dosed at 10 mg/kg qd (∼10% TGI, p>0.05 vs vehicle) and ∼60% TGI when dosed at 80 mg/kg qd. Pharmacodynamic analysis of RCC-47 tumors revealed that two hours after an acute dose of AZD6094 pMET levels were reduced >95% at all dose levels tested (0.5 - 25 mg/kg). Eight hours after dosing, pMET levels returned to ∼50% in the 0.5 and 2.5 mg/kg dose groups whereas pMET was still inhibited >90% in the 10 and 25 mg/kg dose groups indicating that the duration of target inhibition was dose related. AZD6094 inhibited multiple signaling nodes including MAPK, PI3K, and EGFR. Finally, at doses that induced tumor regression, AZD6094 resulted in a dose and time dependent induction of cleaved PARP, a marker of cell death. The finding that lower, sub-optimal doses of AZD6094 showed return of pMET 8 hours after a single administration, raised the question whether splitting the dose over a longer duration would increase anti-tumor activity. Indeed, twice a day dosing (bid, 8;16 hours) of AZD6094 at 1.25 mg/kg was more efficacious than daily administration of 2.5 mg/kg resulting in 8% regression compared to 89% TGI (p<0.05). Taken together, these data provide the first report testing therapeutics in preclinical in vivo models of PRCC and support the clinical development of AZD6094 in this indication. Citation Format: Alwin Schuller, Evan Barry, Rhys Jones, Melanie Frigault, Garry Beran, Ryan Henry, David Linsenmayer, Maureen Hattersley, Aaron Smith, Joanne Wilson, Ammar Adam, Michael Zinda, Corinne Reimer, Stephen Fawell, Edwin Clark, Celina D'Cruz. Pharmacodynamic response and anti-tumor activity of the MET inhibitor AZD6094 in papillary renal cell carcinoma patient derived xenograft models. [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 1477. doi:10.1158/1538-7445.AM2015-1477

Abstract 342: In vivo response and molecular characterisation of a NSCLC squamous cell carcinoma PDX model exhibiting reproducible sensitivity to FGFR inhibitors.

Abstract BACKGROUND: Non-small cell lung cancer (NSCLC) accounts for 85% of lung cancer cases, carries a poor prognosis and remains an area of high unmet need. Despite recent advances in treating NSCLC adenocarcinomas with targeted agents such as Erlotinib (Tarceva©) and Crizotinib (Xalkori©) few advances have been made in the treatment of squamous cell carcinoma (SCC), which accounts for 25% of all NSCLC. Although the incidence of fibroblast growth factor receptor (FGFR) mutations in SCC is modest, amplification of FGFR is well documented and there are several first generation FGFR inhibitors currently in clinical trial. It is therefore essential to develop relevant in vivo patient-derived xenograft (PDX) models for the development and characterisation of new FGFR agents and/or combination strategies which may prolong benefit and delay the emergence of resistance. METHODS: LION137 is a novel NSCLC SCC patient-derived xenograft (PDX) model which is maintained subcutaneously in vivo admixed with a human stromal cell component. Cohorts of mice were dosed with AZD4547 alone or in combination with chemotherapy (CTx; parallel or serial dosing regimens). The extent of inhibition was measured over several in vivo passages and following cryopreservation/resuscitation. Response to treatment was evaluated by tumour growth inhibition and waterfall plots. Tumour material was characterised for 50 key oncogenes and mutations by Ion Torrent sequencing and FGFR1-4 expression and genomic amplification by quantitative PCR. Immunohistochemistry (IHC) was used to confirm SCC subtype, EMT status and FGFR expression. RESULTS: LION137 PDX tumours (SCC confirmed by IHC) exhibited reproducible sensitivity to AZD4547 alone (P<0.001) which was maintained both in continuous serial passage and following cryopreservation (at -196°C). FGFR expression and amplification and mutational analysis were assessed in naïve tumour material, following 10 weeks of continuous dosing and following outgrowth from CTX treatment and correlated with treatment response. CONCLUSIONS: LION137 is a novel patient-derived NSCLC SCC xenograft model with reproducible FGFRi-sensitivity which is directly relevant to the clinical setting. This model could be invaluable in assessing novel agents targeting the FGFR pathway and in the development of new combination strategies which may prevent or overcome resistance. Citation Format: Andrew Mckenzie, Aaron Cranston, Phil Mallinder, Nektaria Papadopoulou, Simon Jiang, Kerry Moakes, Yinfei Yin, David Onion, Anna Grabowska, Martin Page, Rajendra Kumari. In vivo response and molecular characterisation of a NSCLC squamous cell carcinoma PDX model exhibiting reproducible sensitivity to FGFR inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 342. doi:10.1158/1538-7445.AM2013-342