Lung Cancer Patient-derived Xenograft Research Articles

FGFR1 and FGFR4 oncogenicity depends on n-cadherin and their co-expression may predict FGFR-targeted therapy efficacy.

BackgroundFibroblast growth factor receptor (FGFR)1 and FGFR4 have been associated with tumorigenesis in a variety of tumour types. As a therapeutic approach, their inhibition has been attempted in different types of malignancies, including lung cancer, and was initially focused on FGFR1-amplified tumours, though with limited success.MethodsIn vitro and in vivo functional assessments of the oncogenic potential of downregulated/overexpressed genes in isogenic cell lines were performed, as well as inhibitor efficacy tests in vitro and in vivo in patient-derived xenografts (PDXs). mRNA was extracted from FFPE non-small cell lung cancer samples to determine the prognostic potential of the genes under study.FindingsWe provide in vitro and in vivo evidence showing that expression of the adhesion molecule N-cadherin is key for the oncogenic role of FGFR1/4 in non-small cell lung cancer. According to this, assessment of the expression of genes in different lung cancer patient cohorts showed that FGFR1 or FGFR4 expression alone showed no prognostic potential, and that only co-expression of FGFR1 and/or FGFR4 with N-cadherin inferred a poorer outcome. Treatment of high-FGFR1 and/or FGFR4-expressing lung cancer cell lines and patient-derived xenografts with selective FGFR inhibitors showed high efficacy, but only in models with high FGFR1/4 and N-cadherin expression.InterpretationOur data show that the determination of the expression of FGFR1 or FGFR4 alone is not sufficient to predict anti-FGFR therapy efficacy; complementary determination of N-cadherin expression may further optimise patient selection for this therapeutic strategy.

New Preclinical Development of a c-Met Inhibitor and Its Combined Anti-Tumor Effect in c-Met-Amplified NSCLC.

c-Met is a receptor tyrosine kinase with no commercially available product despite being a pivotal target in cancer progression. Unlike other c-Met inhibitors that fail clinically, ABN401 is a newly synthesized c-Met inhibitor that is not potentially degraded by aldehyde oxidase (AO) in human liver cytosol. This study aimed to determine the physicochemical stability, pharmacokinetics in beagle dogs, and therapeutic effect of ABN401 in a c-Met-amplified non-small-cell lung cancer (NSCLC) patient-derived xenograft (PDX) model. ABN401 was found to be a weak basic compound, with pKa and log P values of 7.49 and 2.46, respectively. It is poorly water-soluble but soluble at acidic pH. The accelerated storage stability is dependent on temperature, but the purity remains at over 97% after 6 months. The bioavailability is approximately 30% in dogs and it is highly efficient in the PDX model, achieving around 90% tumor growth inhibition in combination with erlotinib. These observations indicate that the compound is acceptable for the next phase of trials.

An improved method to build lung cancer PDX models by surgical resection samples and its association with B7-H3 expression.

BackgroundPatient-derived xenograft (PDX) models are biologically stable and can accurately reflect the primary tumor in histopathology and genetic expression in many cancers. In lung cancer, the models’ engraftment rate by endobronchial ultrasound transbronchial needle aspiration (EBUS-TBNA) and computed tomography (CT)-guided biopsy is exceptionally low, and this limits the development of PDX models in lung cancer research. In this study, we aimed to improve the traditional method, and explore the feasibility and convenience of establishing lung cancer PDX models using the samples directly from surgical resection. We also endeavored to explore the correlation between PDX formation and primary tumor B7-H3 protein expression.MethodsFrom September 2017 to July 2018, 24 patients were enrolled in this study. The pathological diagnoses were as follows: 15 adenocarcinomas, 7 squamous cell carcinomas, 1 large cell carcinoma, and 1 small cell carcinoma. The tumor tissues were cut into sizes of 2×2×2 mm3/fragment and inoculated subcutaneously into immunodeficiency mice with a trocar needle. The engrafted tumors were passaged at least 3 generations, and B7-H3 IHC staining was performed on primary tumors. To explore the correlation between PDX formation and B7-H3 protein expression, we also performed H&E staining to evaluate whether the established PDX models could retain the histological features of the primary tumor.ResultsXenograft formation success was achieved in 19 out of 24 cases (79.2%). The time between implantation to tumor formation was an average of 81.5 days (27–154 days) in P1, an average of 44.4 days (14–122 days) in P2, and an average of 26.9 days (12–75 days) in P3. The diameter of the tumor was associated with tumor engraftment: the longer the diameter, the easier engraftment formation was (P=0.0342). The data also suggests that lung cancers with B7-H3 expression greatly induced PDX formation (P=0.0375). The histological characters of each passage resembled the primary tumors.ConclusionsThis study shows that the samples from carefully selected lung cancer by surgical resection can be used for the establishment of PDX models with a high success rate; this improved method is closer to actual clinical work. Compared with the same kind of research, the success rate of the xenograft indicates significant improvement, so this improved method is suitable for promotion and application. The results of H&E staining showed that the histological characters of each passage resembled the primary tumors. Furthermore, the diameter of the tumor was associated with tumor engraftment, and higher B7-H3 expression demonstrated a statistically significant difference compared with the PDX model formation.

1199P - MG1124, a novel CEACAM1-targeted monoclonal antibody, has therapeutic potential as a combination partner of PD-1 inhibitors in NSCLC patients

BackgroundCEACAM1 is a member of the CEA family expressed in cell membranes of T cells and NK cells as well as tumor cells. CEACAM1 overexpressed in tumor cells interacts with CEACAM1 expressed in tumor infiltrating lymphocytes (TIL) and suppresses TIL activity by sending an inhibitory signal to immune cells, which is an immune escape mechanism in cancer. Therefore, we developed MG1124, a monoclonal antibody that specifically binds to CEACAM1 but not to other CEA family members, thereby blocking tumor growth by triggering immune response. MethodsCEACAM1 specific binding of MG1124 was tested in CEACAM1 or other CEA family over-expressing cells. MG1124 was examined by in vitro tumor cell lysis assays using NK92, a human NK cell line. The anti-tumor efficacy of MG1124 alone or in combination with a PD-1 inhibitor was studied in a humanized mouse model engrafted with non-small cell lung cancer (NSCLC) patient-derived tumor xenografts (NSCLC hu-PDX models). ResultsAn anti-CEACAM1 antibody, MG1124 binds to CEACAM1 overexpressing Jurkat cells but not to other CEA family members and blocks ligands interaction in a dose-dependent manner. MG1124 increased NK92-mediated tumor cell lysis against cancer cells with strong CEACAM1 expression, but not to those with no or weak CEACAM1 expression. In a single mouse trial, MG1124 (tumor growth inhibition (TGI) 55%) was efficacious in the high CEACAM1 expressing NSCLC hu-PDX model as monotherapy. In addition, the combination therapy (TGI 42%) with a PD-1 inhibitor displayed enhanced antitumor activity than the anti-CEACAM1 monotherapy (TGI 17%) in a CEACAM1-low expression model as well as in a CEACAM1-high expression model (combination therapy TGI 63% vs monotherapy TGI 46%). Moreover, tumor tissues (n=150) of NSCLC patients showed extensive expression of CEACAM1, suggesting a potential therapeutic use of MG1124 in NSCLC patients. ConclusionsIn this study, the anti-CEACAM1 antibody MG1124 showed antitumor efficacy in human CEACAM1-expressing cancer cell lines and NSCLC hu-PDX xenograft models, and demonstrated potential as a targeted therapy for NSCLC, both as a single agent and in combination with a PD-1 inhibitor. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

Patient-derived xenograft models of non-small cell lung cancer for evaluating targeted drug sensitivity and resistance.

Patient‐derived xenograft (PDX) models are a useful tool in cancer biology research. However, the number of lung cancer PDX is limited. In the present study, we successfully established 10 PDX, including three adenocarcinoma (AD), six squamous cell carcinoma (SQ) and one large cell carcinoma (LA), from 30 patients with non‐small cell lung cancer (NSCLC) (18 AD, 10 SQ, and 2 LA), mainly in SCID hairless outbred (SHO) mice (Crlj:SHO‐PrkdcscidHrhr). Histology of SQ, advanced clinical stage (III‐IV), status of lymph node metastasis (N2‐3), and maximum standardized uptake value ≥10 when evaluated using a delayed 18F‐fluoro‐2‐deoxy‐d‐glucose positron emission tomography (FDG‐PET) scan was associated with successful PDX establishment. Histological analyses showed that PDX had histology similar to that of patients’ surgically resected tumors (SRT), whereas components of the microenvironment were replaced with murine cells after several passages. Next‐generation sequencing analyses showed that after two to six passages, PDX preserved the majority of the somatic mutations and mRNA expressions of the corresponding SRT. Two out of three PDX with AD histology had epidermal growth factor receptor (EGFR) mutations (L858R or exon 19 deletion) and were sensitive to EGFR tyrosine kinase inhibitors (EGFR‐TKI), such as gefitinib and osimertinib. Furthermore, in one of the two PDX with an EGFR mutation, osimertinib resistance was induced that was associated with epithelial‐to‐mesenchymal transition. This study presented 10 serially transplantable PDX of NSCLC in SHO mice and showed the use of PDX with an EGFR mutation for analyses of EGFR‐TKI resistance.

An Improved Patient-Derived Xenograft Humanized Mouse Model for Evaluation of Lung Cancer Immune Responses.

Human tumor xenograft models do not replicate the human immune system and tumor microenvironment. We developed an improved humanized mouse model, derived from fresh cord blood CD34+ stem cells (CD34+ HSC), and combined it with lung cancer cell line-derived human xenografts or patient-derived xenografts (Hu-PDX). Fresh CD34+ HSCs could reconstitute detectable mature human leukocytes (hCD45+) in mice at four weeks without the onset of graft-versus-host disease (GVHD). Repopulated human T cells, B cells, natural killer (NK) cells, dendritic cells (DC), and myeloid-derived suppressor cells (MDSC) increased in peripheral blood, spleen, and bone marrow over time. Although cultured CD34+ HSCs labeled with luciferase could be detected in mice, the cultured HSCs did not develop into mature human immune cells by four weeks, unlike fresh CD34+ HSCs. Ex vivo, reconstituted T cells, obtained from the tumor-bearing humanized mice, secreted IFNγ upon treatment with phorbol myristate acetate (PMA) or exposure to human A549 lung tumor cells and mediated antigen-specific CTL responses, indicating functional activity. Growth of engrafted PDXs and tumor xenografts was not dependent on the human leukocyte antigen status of the donor. Treatment with the anti-PD-1 checkpoint inhibitors pembrolizumab or nivolumab inhibited tumor growth in humanized mice significantly, and correlated with an increased number of CTLs and decreased MDSCs, regardless of the donor HLA type. In conclusion, fresh CD34+HSCs are more effective than their expanded counterparts in humanizing mice, and do so in a shorter time. The Hu-PDX model provides an improved platform for evaluation of immunotherapy.

Abstract 4736: Dose optimization of olaparib plus temozolomide in small cell lung cancer (SCLC) patient-derived xenograft (PDX) models for clinical translation

Abstract Introduction: SCLC is an aggressive high-grade neuroendocrine malignancy in which targeting DNA-damage repair pathways has shown efficacy in pre-clinical and clinical contexts. We previously conducted a phase I/II trial combining the PARP inhibitor olaparib (O) tablets with the alkylating agent temozolomide (T) in patients with relapsed SCLC, in which O and T were both given days (d) 1-7 of each 21 d cycle. The recommended phase 2 dose (RP2D) was O 200 mg PO BID and T 75 mg/m2 PO daily, and the response rate among the first 29 evaluable patients was 41.4%, with the primary toxicities being hematologic (Farago et al., ASCO 2018). Continuous PARP inhibition with O may improve efficacy, though it is unknown how best to incorporate T onto this backbone. Simultaneous clinical testing of multiple dosing schedules for concurrent OT would be impractical. Here we present an optimization of the OT regimen using PDX models generated from patients treated with OT. Methods: SCLC PDX models derived from two patients prior to their durable responses to OT, MGH1518-1B and MGH1528-1, were used for dose optimization. Based on a human-murine comparison of serum Cmax for OT, the estimated murine-equivalents for the RP2D doses were O 25 mg/kg BID and T 6.25 mg/kg/d. The RP2D regimen was then modulated in both dose intensity and duration in a 9-arm PDX trial. The arms included 3 schedule categories, with doses varied within each category: discontinuous regimens (OT d 1-7); continuous O regimens (O d1-21 + T d 1-7); and rapid- or slow-alternating regimens administering O and T on non-overlapping days. Tumor bearing mice were treated when subcutaneous tumors reached a volume of 400-800 cc, enabling measurement of tumor regression (PDX response) and time to volume doubling (PDX TTP). Findings: The RP2D regimen resulted in near-complete response in both models. This degree of regression was recapitulated with nearly all dosing schedules, and was not improved by increasing T to 2x-RP2D. However, the PDX TTP varied. In both models, the longest TTP was seen with continuous O at full- or half-RP2D and d 1-7 T at half-RP2D. This regimen prolonged TTP by 12% in MGH1518-1B and 20% in MGH1528-1, versus discontinuous OT at RP2D. Based on these results, we initiated clinical treatment in a new cohort of patients on OT, with a phase 1 dose escalation starting with O 50 mg BID d 1-21 and T 50 mg/m2 d 1-7 of each 21 d cycle (NCT02446704). At this first dose level, a partial response by RECIST 1.1 criteria was observed in a heavily pre-treated patient, indicating clinical activity of this combination with this new dosing strategy. Conclusions: In PDX models sensitive to OT at our previously determined RP2D, we find that continuous O with intermittent T permits dose reduction of both O and T without loss of efficacy. A clinical trial is ongoing to determine whether this strategy may enable longer-term dosing and improve efficacy in patients. Citation Format: Benjamin J. Drapkin, Sarah Phat, David T. Myers, Jun Zhong, Beow Y. Yeap, Elisabetta Leo, Anna Stansiszewska, Aaron Smith, Elaine Cadogan, Maria C. Pietanza, Nicholas J. Dyson, Anna Farago. Dose optimization of olaparib plus temozolomide in small cell lung cancer (SCLC) patient-derived xenograft (PDX) models for clinical translation [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 4736.

Abstract 1059: A case study: OncoRat is a viable patient avatar for a NSCLC patient with a Y1248H Met activating mutation

Abstract Human cancer xenografts in rodents can provide predictive data on the success of candidate drugs in clinical trials and have been a pivotal tool in moving new drugs from the bench to the clinic. However, currently available immunodeficient mouse models have shown some limitation and variability in tumor take rates and growth kinetics in cancer cell lines. In addition, commercially available human cancer cell lines aren’t representative of the genomic and molecular diversity of cancers found in patients. Patient Derived Xenograft (PDX), in which tumor tissue is transplanted directly into rodents after biopsy from the patient, better represents that molecular signature, heterogeneity, and pathology of the original tumor. Therefore, in vivo efficacy studies with PDX models could be highly predictive for treatment sensitivity. Despite the many advantages of PDXs for preclinical research, PDX mouse models are hindered by low engraftment rates and slow tumor growth kinetics. The loss of patient tumor heterogeneity and stromal cells as the PDX is passaged multiple times to generate sufficient tumor tissue to inoculate a cohort of animals for efficacy studies is also a disadvantage in the immunodeficient mouse models. To address these limitations, we have introduced the OncoRat®; built on the SRGTM Platform, a Sprague-Dawley Rag2/Il2rg double knockout rat that lacks mature B cells, T cells, and circulating NK cells. We have demonstrated that the OncoRat has improved tumor take rate and growth kinetics for non-small cell lung cancer (NSCLC) PDXs. The NSLSC PDXs in the OncoRat have a much larger tumor volume, over 20,000 mm3 in the first passage (P0) in the rat, which provides an ample source of tissue for characterization and/or subsequent passage (P1) into OncoRat for drug efficacy studies. This leads to fewer animals used for study and faster timelines to drug efficacy data, resulting in a reduction in cost. In addition, we have used genomic analysis for guidance in planning in vivo efficacy studies. One of our NSCLC PDX models harbors a novel mutation in the MET pathway, suggesting this tumor would not be responsive to standard of care treatment. An efficacy study we performed in the OncoRat suggests that this particular tumor would respond well to Type II MET inhibitors, such as Cabonzantinib. This proof of concept study demonstrates that genomic and molecular analysis can provide insight into treatment outcomes and that PDX models in the OncoRat could serve as patient avatars for predicting treatment outcomes. Citation Format: Fallon K. Noto, Bisoye Towobola Adedeji, Sam Moody, Chris Brenzel, Jack Crawford, Goutham Narla, Tseten Yeshi Jamling. A case study: OncoRat is a viable patient avatar for a NSCLC patient with a Y1248H Met activating mutation [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 1059.

Abstract 4407: Next generation CDK2/9 inhibitor CYC065 triggers anaphase catastrophe in diverse aneuploid cancers and markedly inhibits growth and metastasis

Abstract Genetically-unstable cancer cells have supernumerary centrosomes and aneuploidy (a hallmark of cancer). CDK2 antagonism inhibits clustering of excessive centrosomes at mitosis leading to multipolar cell division and apoptotic death. This process is called anaphase catastrophe. Anaphase catastrophe affects preferentially aneuploid cancer cells while sparing normal cells with two centrosomes. CYC065 (Cyclacel) is a next-generation CDK2/9 inhibitor that is undergoing clinical trials. Here, we explored CYC065 activity against aneuploid lung and other cancers. CYC065 substantially inhibited growth, triggered apoptosis, and induced anaphase catastrophe in murine (ED1, LKR13, and 393P) and human (Hop62, A549, and H1299) lung cancer cells. In marked contrast, these effects were largely unseen in bipolar immortalized pulmonary epithelial (murine C10 and human BEAS-2B) cells. Notably, anaphase catastrophe was induced by CYC065-treatment even in pancreatic and colon cancers that are often driven by the KRAS oncoprotein. This suggests that engaging anaphase catastrophe is a general antineoplastic mechanism. Comprehensive expression analysis of nearly 300 growth-regulatory proteins was performed after CYC065 treatment in lung cancer cells using Reverse Phase Protein Arrays (RPPAs). In addition to known CDK targets (such as phosphorylated retinoblastoma protein), FAK phosphorylation and SAC phosphorylation that are known to regulate metastasis were unexpectedly down-regulated. Consistent with this finding, in vitro migration and invasion CYC065 treatment assays markedly inhibited migration and invasion of lung cancer cells. CYC065 anti-neoplastic effects were interrogated in mice. CYC065 significantly inhibited tumor growth in several lung cancer syngeneic xenografts and patient-derived xenografts (PDXs) and reduced metastasis of 344SQ lung cancer cells in a syngeneic tail-vein injection model. Immunohistochemistry (IHC) studies of resected PDX tumors revealed downregulation of p-CDK2 (Thr160) and MCL1 expression after CYC065 treatment which is consistent with CDK2/9 antagonism, respectively. In summary, CYC065 engages anaphase catastrophe and elicits marked antineoplastic effects in diverse cancers. These effects are due to mechanisms that confer after CDK2/9 inhibition, anaphase catastrophe, MCL1 down-regulation and suppression of proteins that regulate metastasis. Taken together, findings reveal that anaphase catastrophe is a general antineoplastic mechanism engaged in aneuploid cancers. Intriguingly, this mechanism is activated after CDK2/9 inhibition of lung and other cancers, including those driven by the KRAS oncoprotein. Citation Format: Masanori Kawakami, Lisa Maria Mustachio, Yulong Chen, Zibo Chen, Jason Roszik, Xi Liu, Ethan Dmitrovsky. Next generation CDK2/9 inhibitor CYC065 triggers anaphase catastrophe in diverse aneuploid cancers and markedly inhibits growth and metastasis [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 4407.

Antibody-Fc/FcR Interaction on Macrophages as a Mechanism for Hyperprogressive Disease in Non-small Cell Lung Cancer Subsequent to PD-1/PD-L1 Blockade.

Hyperprogression (HP), a paradoxical boost in tumor growth, was described in a subset of patients treated with immune checkpoint inhibitors (ICI). Neither clinicopathologic features nor biological mechanisms associated with HP have been identified. Among 187 patients with non-small cell lung cancer (NSCLC) treated with ICI at our institute, cases with HP were identified according to clinical and radiologic criteria. Baseline histologic samples from patients treated with ICI were evaluated by IHC for myeloid and lymphoid markers. T-cell-deficient mice, injected with human lung cancer cells and patient-derived xenografts (PDX) belonging to specific mutational subsets, were assessed for tumor growth after treatment with antibodies against mouse and human programmed death receptor-1 (PD-1). The immune microenvironment was evaluated by flow cytometry and IHC. Among 187 patients, 152 were evaluable for clinical response. We identified four categories: 32 cases were defined as responders (21%), 42 patients with stable disease (27.7%), 39 cases were defined as progressors (25.7%), and 39 patients with HP (25.7%). Pretreatment tissue samples from all patients with HP showed tumor infiltration by M2-like CD163+CD33+PD-L1+ clustered epithelioid macrophages. Enrichment by tumor-associated macrophages (TAM) was observed, even in tumor nodules from immunodeficient mice injected with human lung cancer cells and with PDXs. In these models, tumor growth was enhanced by treatment with anti-PD-1 but not anti-PD-1 F(ab)2 fragments. These results suggest a crucial role of TAM reprogramming, upon Fc receptor engagement by ICI, eventually inducing HP and provide clues on a distinctive immunophenotype potentially able to predict HP.See related commentary by Knorr and Ravetch, p. 904.

Establishment of a patient derived xenograft mouse model of lung cancer

Objective To establish a patient-derived xenograft (PDX) model library of lung cancer, and to compare the biological consistency between primary tumors and passaged tumors, at the same time to find the key factors affecting the formation of PDX tumor. Methods Fresh surgical or biopsy samples from lung cancer patients were collected and subcutaneously transplanted into immunodeficient mouse model (NOD-Prkdcnull-Il2rgnull) to establish the PDX model and then passaged with 3-5 generation. Structure and cell morphology, and biomarkers of passages of PDX tumor tissue and primary tumor tissue were compared. Combined with patients’ clinical data, key factors affecting the tumor take rate were analyzed. Results a total of 195 lung cancer samples were collected and 59 cases of lung cancer PDX were successfully established. The rate of PDX tumor formation was 29.65%. The pathological analysis results of the tumor samples from all the tumor models of PDX were consistent with the corresponding primary tumors. The key factors affecting the take rate and tumor formation time were pathological types of primary tumors. The take rate of adenocarcinoma was 14.75% whereas the take rate of squamous cell carcinoma was 56.60%. Conclusion The library of lung cancer PDX models we have established retained the key features of primary cancers, providing an effective resource for research and development of drug efficacy evaluation in pre-clinical trial and identification of biomarkers for drug responsiveness. Key words: Lung cancer; Patient derived xenograft; Mouse model; Take rate

Patient-derived tumor immune microenvironments in patient-derived xenografts of lung cancer

BackgroundBecause patient-derived xenografts (PDXs) are grown in immunodeficient mouse strains, PDXs are regarded as lacking an immune microenvironment. However, whether patients’ immune cells co-exist in PDXs remains uncharacterized.MethodsWe cultured small pieces of lung PDX tissue in media containing human interleukin-2 and characterized the proliferated lymphocytes by flow cytometric assays with antibodies specific for human immune cell surface markers. Presence of immune cells in PDXs was also determined by immunohistochemical staining.ResultsHuman tumor-infiltrating lymphocytes (TILs) were cultured from nine of 25 PDX samples (36%). The mean time of PDX growth in immunodeficient mice before obtaining TILs in culture was 113 days (range 63–292 days). The TILs detected in PDXs were predominantly human CD8+ T cells, CD4+ T cells, or CD19+ B cells, depending on cases. DNA fingerprint analysis showed that the TILs originated from the same patients as the PDXs. Further analysis of two PDX-derived CD8+ T cells showed that they were PD-1−, CD45RO+, and either CD62L+ or CD62L−, suggesting they were likely memory T cells. Immunohistochemical staining showed that human T cells (CD8+ or CD4+), B cells (CD19+), and macrophages (CD68+) were present in stroma or intraepithelial cancer structures and that human PD-L1 was expressed in stromal cells. Moreover, the patient-derived immune cells in PDX can be passaged to the F2 generation and may migrate to spleens of PDX-bearing mice.ConclusionsPatient-derived immune cells co-exist in early passages of PDXs in some lung cancer PDX models. The CD8+ cells from PDXs were likely memory T cells. These results suggest that PDXs can be used for evaluating the functionality of immune components in tumor microenvironments.

DNAX-activating protein 10 co-stimulation enhances the anti-tumor efficacy of chimeric antigen receptor T cells

ABSTRACT Chimeric antigen receptor (CAR) T cell immunotherapies have shown remarkable efficacy in treating multiple types of hematological malignancies but are not sufficiently effective at treating solid tumors. NKG2D is a strong activating receptor for NK cells and a co-stimulatory receptor for T cells. NKG2D signal transduction depends on DNAX-activating protein 10 (DAP10). Here, we introduced the cytoplasmic domain of DAP10 into the second-generation CARs M28z and G28z to generate M28z10 and G28z10, which target mesothelin (MSLN) and glypican 3 (GPC3), respectively. T cells expressing M28z10 or G28z10 showed enhanced and prolonged effector function against MSLN+ lung cancer or GPC3+ hepatocellular carcinoma cell lines in culture and secreted elevated levels of cytokines, including IL-2, IFN-γ, granzyme B, and GM-CSF. In addition, M28z10 CAR-T cells showed greater anti-tumor activity than those expressing M28z in both A549 cell line xenografts and human lung cancer patient-derived xenografts (PDX). Similarly, G28z10 exhibited higher efficacy in causing tumor regression than did G28z in hepatocellular carcinoma PDX. Therefore, our results show that DAP10 signaling contributes to the function of CAR-T cells in both lung cancer and hepatocellular carcinoma and can enhance the efficacy of CAR-T cells.

PARP trapping by Talazoparib is a Potent Mechanism of Radiosensitization in Small Cell Lung Cancer Cell Lines and Patient-Derived Xenografts

Small cell lung cancer (SCLC) is an aggressive malignancy with a critical need for novel molecular therapies. We hypothesized that poly(ADP)-ribose polymerase (PARP) inhibitors could effectively sensitize SCLC cells to ionizing radiation (IR). We further hypothesized that PARP trapping, a cytotoxic mechanism of action distinct from inhibition of the PARP enzyme and the DNA damage response, contributes to radiosensitization by PARP inhibitors. Short-term viability assays and clonogenic survival assays (CSA) were used to assess radiosensitization in six SCLC cell lines. PARP trapping and PARP enzymatic inhibition were analyzed by quantifying the chromatin-bound fraction of PARP1 and the total cell PAR polymer, respectively, using western blot. Doses of veliparib, a weak PARP trapper, and talazoparib, a potent PARP trapper, that had equivalent enzymatic inhibitory activity but differing PARP trapping activity were identified and compared in CSAs. The number of g-H2AX foci induced by veliparib and talazoparib were compared with and without IR using immunofluorescence assays. Talazoparib, IR, and their combination were tested in three patient-derived xenograft (PDX) models, and time for the tumors to grow to 1000 mm3 was compared using the log-rank test. Radiosensitization by talazoparib was seen in 5 of 6 cell lines in short term viability assays: one cell line at 0.2 nM (Dose Modification Factor [DMF] 1.56), two cell lines at 2 nM (DMF 1.34 – 1.86), and four cell lines at 20 nM (DMF 1.61 – 2.88). Using CSAs, sensitization to IR was seen in 3 of 3 cell lines, with two cell lines sensitized at 20 nM (DMF 1.40-2.13) and one at 200 nM (DMF 2.20). Doses of 200 nM talazoparib and 1600 nM veliparib similarly inhibited PAR polymerization; however, as expected, talazoparib exhibited greater PARP trapping activity. This dose of veliparib was insufficient for radiosensitization, while the greater PARP trapping observed with 200 nM talazoparib was associated with significant radiosensitization (DMF 3.3). This further correlated with more double-stranded DNA breaks (g-H2AX foci) seen with talazoparib compared to veliparib both in the absence of IR (mean foci per cell: 8.4 DMSO, 19.3 veliparib, 58.7 talazoparib) and with IR (22.4 DMSO, 32.8 veliparib, 75.6 talazoparib). Finally, a dose of 0.2 mg/kg talazoparib in vivo led to statistically significant tumor growth inhibition in combination with IR but not as a single agent in three SCLC PDX models. PARP inhibition effectively sensitizes SCLC cell lines and PDXs to IR, and PARP trapping correlates with radiosensitization. PARP inhibitors, especially those with high PARP trapping activity, may provide a powerful tool to improve the efficacy of radiation therapy in SCLC.

MS32.02 Patient Derived Models (PDX and CTC-Derived)

Patient-derived xenograft (PDX) models are a powerful preclinical tool for studying small cell lung cancer (SCLC) biology and therapeutic vulnerabilities. SCLC PDX models can be generated from biopsies or circulating tumor cells (CTCs), though these approaches can be limited by scarcity of tissue and low efficiency of tumor growth. Applying an established clinical-translational pipeline for tissue collection and an automated microfluidic platform for CTC-enrichment, we have generated over 40 PDX models. In a 24-month period, we initiated 17 biopsy-derived PDXs and 17 CTC-derived PDXs, at 89% and 38% efficiency, respectively (Drapkin et al., Cancer Discovery 2018). Whole exome sequencing showed that somatic alterations are stably maintained between patient tumors and PDXs. Early-passage PDXs maintain the genomic and transcriptional profiles of the founder PDX. We have standardized quantitative measurements of efficacy of in vivo treatments, utilizing both depth of tumor shrinkage (response) and time to tumor volume doubling (time to progression). With these approaches, we have assessed responses to both standard of care and experimental therapies across a large panel of mice. We find that sensitivity to etoposide and platinum (EP) corresponds with the treatment history of patients, and that resistance to EP corresponded to increased expression of a MYC gene signature. Similarly, responses of the PDXs to an experimental combination, olaparib and temozolomide, mirrors the responses seen in patients treated on a clinical trial (Farago et al., ASCO 2018). Biomarker analysis in this panel reveals that basal PARylation is a highly sensitive and specific predictor of response to olaparib and temozolomide. Collectively, this experience highlights the utility of PDX models in SCLC for biomarker discovery and assessment of novel therapeutic strategies. 1. Drapkin BJ, George J, Christensen CL, Mino-Kenudson M, Dries R, Sundaresan T, Phat S, Myers DT, Zong J, Igo P, Hazar-Rethinam MH, Licausi JA, Gomez-Caraballo M, Kem M, Jani KN, Azimi R, Abedpour N, Menon R, Lakis S, Heist RS, Buttner R, Haas S, Sequist LV, Shaw AT, Wong K-K, Hata A, Toner M, Maheswaran S, Haber DA, Peifer M, Dyson N, Thomas RK, Farago AF. Genomic and functional fidelity of small cell lung cancer patient-derived xenografts. Cancer Discovery 2018 May 8(5):600-615. PMID: 29483136 2. Farago AF, Drapkin BJ, Charles A, Yeap B, Heist RS, Azzoli CG, Jackman D, Marcoux P, Barbie D, Myers DT, Phat S, Zhong J, Grinnell JB, Sequist LV, Mino-Kenudson M, Maheswaran S, Haber D, Hata A, Dyson N, Shaw AT. Safety and efficacy of combination olaparib (O) and temozolomide (T) in small cell lung cancer (SCLC). American Society of Clinical Oncology Annual Meeting, June 1-5 2018. Poster presentation

Establishment of a platform of non-small-cell lung cancer patient-derived xenografts with clinical and genomic annotation

BackgroundPreclinical models that can better predict therapeutic activity in clinical trials are needed in this era of personalized cancer treatment. Herein, we established genomically and clinically annotated patient-derived xenografts (PDXs) from non-small-cell lung cancer (NSCLC) patients and investigated whether these PDXs would faithfully recapitulate patient responses to targeted therapy. MethodsPatient-derived tumors were implanted in immunodeficient mice and subsequently expanded via re-implantation. Established PDXs were examined by light microscopy, genomic profiling, and in vivo drug testing, and the successful engraft rate was analyzed with the mutation profile, histology, or acquisition method. Finally, the drug responses of PDXs were compared with the clinical responses of the respective patients. ResultsUsing samples from 122 patients, we established 41 NSCLC PDXs [30 adenocarcinoma (AD), 11 squamous cell carcinoma (SQ)], among which the following driver mutation were observed: 13 EGFR-mutant, 4 ALK-rearrangement, 1 ROS1-rearrangement, 1 PIK3CA-mutant, 1 FGFR1-amplification, and 2 KRAS-mutant. We rigorously characterized the relationship of clinical features to engraftment rate and latency rates. The engraft rates were comparable across histologic type. The AD engraft rate tended to be higher for surgically resected tissues relative to biopsies, whereas similar engraft rates was observed for SQ, irrespective of the acquisition method. Notably, EGFR-mutants demonstrated significantly longer latency time than EGFR-WT (86 vs. 37days, P = 0.007). The clinical responses were recapitulated by PDXs harboring driver gene alteration (EGFR, ALK, ROS1, or FGFR1) which regressed to their target inhibitors, suggesting that established PDXs comprise a clinically relevant platform. ConclusionThe establishment of genetically and clinically annotated NSCLC PDXs can yield a robust preclinical tool for biomarker, therapeutic target, and drug discovery.

Abstract 2589: Novel predictor of FGFR1 inhibition efficacy in non-small cell lung cancer

Abstract Fibroblast growth factor receptor (FGFR)1 has been associated with tumorigenesis in a variety of tumors, including lung cancer. FGFR1 amplification is detected in up to 20% of lung squamous cell carcinomas, and was initially related to tumorigenesis and to response to FGFR inhibitors in preclinical models of this pathology. These data encouraged the establishment of FGFR1 amplification as the main eligibility criterion for inclusion of patients in FGFR inhibitors clinical trials. However, the use of FGFR inhibitors in FGFR1-amplified tumors has shown limited success, with only 5-10% of selected patients showing partial response, suggesting that this genomic aberration may not be a good predictive biomarker for the efficacy of these inhibitors. In this work, we provide in vitro and in vivo data showing that the oncogenic effects of FGFR1 expression depend on the expression of the adhesion molecule N-cadherin in non-small cell lung cancer (NSCLC). In line with these results, FGFR1 expression correlates with poorer prognosis only in tumors with high N-cadherin levels in two independent NSCLC patient cohorts. We observed that high FGFR1 expression alone is not sufficient to predict FGFR inhibition efficacy in lung cancer cell lines and patient-derived xenografts, with only high FGFR1- and high N-cadherin-expressing models responding to selective FGFR inhibitors. Altogether, our data show that the determination of the expression of FGFR1 alone is not sufficient to predict FGFR inhibition efficacy. The co-determination of N-cadherin expression may optimize patient selection for this therapeutic strategy. Citation Format: Álvaro Quintanal-Villalonga, Irene Ferrer, Ángela Marrugal, Laura Ojeda-Márquez, Jon Zugazagoita, Laura García-Redondo, Fernando López-Ríos, Luis Montuenga, Sonia Molina-Pinelo, Sonia Molina-Pinelo, Amancio Carnero, Luis Paz-Ares. Novel predictor of FGFR1 inhibition efficacy in non-small cell 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 2589.

Abstract 1616: Novel caged hapten proximity assay platform enables detection of oncogenic signaling-associated complexes in the clinical pathology laboratory setting

Abstract Assays measuring protein complexes represent a novel class of protein-based biomarkers. Using proximity-based readouts, we and others have previously shown utility in monitoring signaling-associated complexes to predict response to targeted therapeutics in lung cancer. However, fluorescence-based proximity assay readouts are difficult to implement in a clinical setting and are not conducive to typical pathology laboratory workflow. Here, we provide evidence of a novel proximity-based technology platform that utilizes a “caged hapten” proximity readout followed by chromogenic immunohistochemistry (IHC) detection. We have synthesized haptens modified with a phosphorylated quinone methide precursor. This enzyme- labile caging group abrogates antibody binding; however, upon exposure to alkaline phosphatase (AP) the native hapten is regenerated. These caged haptens may be applied in a proximity assay format by the use of a first antibody labeled with caged haptens that can be uncaged by AP conjugated to the second antibody. Only when the two epitopes of interest are in close proximity to one another will the AP interact with the caged hapten and uncage it. The native hapten, which represents the protein complex population, can then be visualized by an anti-hapten antibody conjugated to horseradish peroxidase (HRP) followed by 3,3'-diaminobenzidine (DAB) detection. These reagents and assays are fully automated on the VENTANA DISCOVERY ULTRA platform. We demonstrate detection of β-Catenin:E-Cadherin, EGFR:GRB2 and MET:GRB2 complexes in formalin-fixed paraffin embedded (FFPE) lung cancer cell lines, patient-derived xenografts (PDX) and non-small cell lung cancer (NSCLC) patient specimens. Assay specificity was confirmed through technical controls involving reagent omission experiments, and biologically by treatment with small molecule kinase inhibitors that disrupt kinase:adaptor interactions. We revealed heterogeneous EGFR and MET signaling in MET-amplified PDX specimens, replicating previously-published findings. For EGFR:GRB2, we developed a pathologist-guided ordinal scoring system and evaluated inter-rater concordance using a tissue microarray containing 120 NSCLC cores. Kappa statistics and inter-rater correlations reveal excellent correlation among multiple raters.Collectively, these data support the adaptation of proximity-based approaches in clinical pathology settings. The use of caged hapten proximity assays on VENTANA automated slide stainers provides a novel and robust platform to evaluate protein complexes as potential biomarkers that may be incorporated as molecular correlates in ongoing early phase clinical trials. This approach also facilitates analysis of a large number of slides in a reproducible assay format enabling retrospective evaluation of tumor specimens from completed trials. Citation Format: Brian D. Kelly, Nathan W. Polaske, Yuri Belosludtsev, Matthew A. Smith, Theresa Boyle, Y. Ann Chen, Eric B. Haura. Novel caged hapten proximity assay platform enables detection of oncogenic signaling-associated complexes in the clinical pathology laboratory setting [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 1616.

Abstract 988: Short-term culture of organotypic tumor spheroids derived from patient xenografts in a novel 3D microfluidic chip predicts in vivo response of targeted therapies

Abstract Introduction: Patient-derived xenografts (PDX) are increasingly adopted to evaluate the efficacy of new treatment approaches, including the development of combination therapies and testing of predictive biomarkers hypotheses. However, PDXs can take months to generate and are impractical for screening large sets of drug combinations. Here we report on a novel 3D microfluidic platform that allows for evaluating ex vivo responses to targeted therapies from genotyped defined patient-derived tumor spheroids (XDOTS) (1). Methods: Non-small cell lung cancer (NSCLC) PDXs with known genotype, including EGFR ex19 del, HER-2 ins, and BRAF V600E, were generated from either core needle biopsies or pleural effusions under an IRB-approved protocol. XDOTS (<100 μm and >40μm) were grown from PDX tumors using DAX-1 3D cell culture chips (AIM, Singapore) and treated with genotype matched inhibitors that are either approved (e.g., erlotinib for EGFR mut) or are being tested in the clinic (neratinib for HER-2 ins) at known peak plasma concentrations for 3 days. Live/death quantification was performed by dual labeling fluorescence microscopy using acridine orange for live and propidium iodide for dead cells. Cell type characterization was performed by immunofluorescence in the devices. Results: We first characterized our XDOT system on models for which we had orthogonal validation. We show by dual labeling fluorescence microscopy that PC9GR4 (EGFR Del19/T790M) cells are sensitive to osimertinib (IC50 = 12nM), but not gefitinib (IC50 >10uM). We further show that HCC827GR6 (Del 19/MET Amp) and DFCI32 (EML4-ALK/EGFR/HER2) cells are sensitive to crizotinib/gefitinib combo, but not to each of the single agents. Results were validated using the IncuCyte Live Cell analysis. We then went on to show that genotype-specific responses can be recapitulated in tumor spheroids generated from PDXs. In DFCI 359, a PDX derived from a HER2 mutant (755_757 LRdelinsPR), we show the antitumor efficacy of afatinib and confirm that effect is specific to tumor cells and not stromal. We observe significant drug efficacy only on cells growing in our microfluidic devices and not on spheroids growing 3D in 384 well plates. Similar results were obtained for additional PDXs with known NSCLC driver mutations. Finally, we evaluate our system on a small cohort of direct patient specimens. Conclusions: Taken together, our data show that short culture experiments using XDOTS are feasible and recapitulate key futures of in vivo response and resistance ex vivo. Reference: 1. Jenkins RW, et al. Cancer Discov DOI:10.1158/2159-8290. Citation Format: Amir Aref, Elena Ivanova, Emily Chambers, Andrew Portell, Russell Jenkins, Mika Lin, Man Xu, Jacob Haworth, Magda Bahcall, Cloud Paweletz, Pasi Janne. Short-term culture of organotypic tumor spheroids derived from patient xenografts in a novel 3D microfluidic chip predicts in vivo response of targeted therapies [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 988.

Abstract 5778: Preclinical evaluation of TQB3616, a highly potent and selective small-molecule CDK4/6 inhibitor

Abstract Objective: The G1 restriction point is critical for regulating the cell cycle and is controlled by the retinoblastoma protein (Rb) pathway (CDK4/6-cyclin D1-Rb-p16/ink4a). Selective CDK4/6 inhibitors, such as palbociclib and ribociclib have been approved by US FDA in combination with letrozole to treat patients with HR+/Her2- breast cancer, and selective CDK4/6 inhibitor LY2835219 is in Phase 3 trials for the same and related indications. Here, we disclose TQB3616 for the first time, a novel selective, potent, oral inhibitor of CDK4/6. Method: The anti-proliferative activity of TQB3616 was evaluated in a panel of 18 Rb-proficient cell lines in a 72-hour viability assay. Daily oral administration was used to evaluate in vivo antitumor activity of TQB3616 in the MCF-7 cell-derived xenograft (CDX) breast cancer model. Effect on CDK4/6-cyclin D1-Rb signaling was assessed by western blot analysis of downstream effector protein p-Rb. In vivo antitumor activity of TQB3616 was also tested in a patient-derived xenograft (PDX) lung cancer model LU-01-0393. Result: TQB3616 displayed potent anti-proliferative activity in 8 of 18 Rb-proficient cancer cell lines (>50% inhibition at 0.3 µM). Inhibition of p-Rb in the tumor samples was verified by western blot analysis. TQB3616 showed in vivo antitumor activity in the MCF-7 CDX model with TGI of 60% @7.5 mpk and 93% @15 mpk, better than palbociclib (TGI = 52% @20 mpk, 80% @40 mpk) and LY2835219 (TGI = 45% @7.5 mpk, 76% @15 mpk). TQB3616 also showed good in vivo antitumor activity in the LU-01-0393 lung cancer PDX model with 65% of TGI @35 mpk. Conclusion: TQB3616 is a potent and selective CDK4/6 inhibitor and shows excellent antitumor activity in preclinical tumor models. These results warrant TQB3616 going to the clinic for exploration of its potential for treating HR+/Her2-/Rb+ breast cancer as well as other Rb+ cancer types. Citation Format: Xu Zhaobing, Hu Lihong, Liu Yingchun, Charles Z. Ding, Xiaoyu Zhu, Chen Chen, Yusong Zhu, Jiahu Wu, Dongdong Wu, Shuhui Chen, Xiquan Zhang, Ling Yang, Xin Tian. Preclinical evaluation of TQB3616, a highly potent and selective small-molecule CDK4/6 inhibitor [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 5778.