Non-small Cell Lung Cancer Patient-derived Xenograft Model Research Articles

Abstract A30: Predictive and pharmacodynamic biomarkers of vantictumab (OMP-18R5; anti-Frizzled) in non-small cell lung cancer

Abstract Background: Vantictumab is a monoclonal antibody that blocks canonical WNT/β-catenin signaling through binding of five FZD receptors (1, 2, 5, 7, 8). This antibody inhibits the growth of several tumor types, reduces tumor-initiating cell frequency (TIC) and exhibits synergistic activity with standard-of-care (SOC) chemotherapeutic agents (Gurney et al., 2012). To target responsive patients and understand the mechanism of action of the drug, we set out to identify predictive and pharmacodynamic (PD) biomarkers of vantictumab in non-small cell lung cancer (NSCLC). Materials and methods: The response to vanticutmab was established from in vivo efficacy experiments including different treatment groups: control, vantictumab, paclitaxel and vantictumab in combination with paclitaxel. For combination treatment, same day dosing and sequential dosing (paclitaxel dosed 2 days after the antibody) were compared. Samples were collected for PD biomarker analysis. To identify a predictive biomarker for the response to vantictumab in NSCLC patients, gene expression data from 7 NSCLC patient derived xenograft (PDX) models was analyzed. We utilized support vector machine-recursive feature elimination (SVM-RFE, Guyon et al., 2002) to select genes and support vector machine (SVM) for classification. Results: Vantictumab showed significant tumor growth inhibition as a single agent as well as in combination with paclitaxel. The reduction of TIC and the antitumor efficacy of vantictumab were significantly enhanced with sequential dosing compared with same day dosing. These findings suggested that optimal synergy occurs using sequential dosing, likely due to enhanced blockade of cell cycle progression at mitosis. PD biomarker analysis confirmed inhibition of genes in Wnt, Notch, and stem cell pathways by vantictumab both as a single agent and also in combination with paclitaxel. Wnt pathway targets including AXIN2 and LEF1 were down-regulated significantly by vantictumab in both sequential dosing and same day dosing confirming the mechanism of action. From a series of 7 in vivo efficacy PDX experiments, LEF1 was identified as a predictive biomarker of vantictumab response and achieved the best performance with cross-validated positive predictive value (PPV) = negative predictive value (NPV) = sensitivity = specificity = 100%. Strong correlation was also observed between LEF1 gene expression and the ratio of tumor volume. Furthermore, LEF1 was able to successfully predict the response to vantictumab in 2 independent NSCLC PDX models. Prevalence estimation for LEF1 ranged from 35% to 50% based on public microarray datasets. LEF1 was also found to be significantly correlated with the response to vantictumab in combination with paclitaxel in 12 NSCLC PDX models (p = 0.0162), indicating LEF1 as a potential predictive biomarker of the response vantictumab as a single agent and in combination with SOC in NSCLC. Conclusions: A biomarker study for the pharmacodynamics and response to vantictumab was performed using a series of PDX NSCLC models. PD biomarkers were identified which confirmed the mechanism of action of vantictumab. LEF1 was identified as a predictive biomarker and is being evaluated in the Phase 1b study of vantictumab in combination with SOC in previously treated NSCLC: NCT01957007. Comprehensive PD and predictive biomarker data will be presented. Citation Format: CHUN ZHANG, Fiore Cattaruzza, Pete Yeung, Wan-Ching Yen, Marcus Fischer, Alayne Brunner, Min Wang, Belinda Cancilla, Rainer Brachmann, Tim Hoey, John Lewicki, Ann M. Kapoun. Predictive and pharmacodynamic biomarkers of vantictumab (OMP-18R5; anti-Frizzled) in non-small cell lung cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A30.

Association of EGFR Expression Level and Cetuximab Activity in Patient-Derived Xenograft Models of Human Non–Small Cell Lung Cancer

To explore in a panel of patient-derived xenograft models of human non-small cell lung cancer (NSCLC) whether high EGFR expression, was associated with cetuximab activity. NSCLC patient-derived xenograft models (n=45) were implanted subcutaneously into panels of nude mice and randomization cohorts were treated with either cetuximab, cisplatin, cisplatin plus cetuximab, vehicle control, or else were left untreated. Responses according to treatment were assessed at week 3 by analyzing the relative change in tumor volume and an experimental analogue of the Response Evaluation Criteria in Solid Tumors (RECIST) guidelines. An EGFR IHC score was calculated for each patient-derived xenograft model and response was assessed according to EGFR expression level. When tumors were stratified into high and low EGFR expression groups (IHC score threshold 200; scale 0-300), a stronger antitumor activity was seen in the high EGFR expression group compared with the low EGFR expression group in both the cetuximab monotherapy and cisplatin plus cetuximab combination therapy settings. For tumors treated with cisplatin plus cetuximab, the objective response rate was significantly higher in the high EGFR expression group compared with the low EGFR expression group (68% vs. 29%). Objective response rates were similar in high and low expression groups for tumors treated with cisplatin alone (27% vs. 24%, respectively). Cetuximab activity in NSCLC patient-derived xenograft models was demonstrated clearly only in tumors that expressed high levels of EGFR, as defined by an IHC score of ≥200.

HIP1–ALK, A Novel ALK Fusion Variant that Responds to Crizotinib

The aim of this study was to identify anaplastic lymphoma kinase (ALK) rearrangements in lung cancer patient-derived xenograft (PDX) models and to explore their responses to crizotinib. Screening of 99 lung cancer PDX models by the NanoString ALK fusion assay identified two ALK-rearranged non-small-cell lung cancer (NSCLC) tumors, including one harboring a previously known echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion and another containing an unknown ALK fusion variant. Expression array, RNA-Seq, reverse transcription polymerase chain reaction, and direct sequencing were then conducted to confirm the rearrangements and to identify the novel fusion partner in the xenograft and/or the primary patient tumor. Finally, pharmacological studies were performed in PDX models to evaluate their responses to ALK inhibitor crizotinib. Two ALK-rearranged NSCLC PDX models were identified: one carried a well-known EML4-ALK variant 3a/b and the other harbored a novel huntingtin interacting protein 1 (HIP1)-ALK fusion gene. Exon 28 of the HIP1 gene located on chromosome 7 was fused to exon 20 of the ALK gene located on chromosome 2. Both cases were clinically diagnosed as squamous cell carcinoma. Compared with the other lung cancer PDX models, both ALK-rearranged models displayed elevated ALK mRNA expression. Furthermore, in vivo efficacy studies demonstrated that, similar to the EML4-ALK-positive model, the HIP1-ALK-containing PDX model was sensitive to treatment with crizotinib. Discovery of HIP1 as a fusion partner of ALK in NSCLC is a novel finding. In addition, the HIP1-ALK-rearranged tumor is sensitive to treatment with crizotinib in vivo, implicating HIP1-ALKas an oncogenic driver of lung tumorigenesis. Collectively, our results indicate that HIP1-ALK-positive NSCLC may benefit from clinical applications of crizotinib.

Abstract A145: In vivo and in vitro generation and characterization of EGFR TKI resistance in a patient-derived and a cell line-derived xenograft model of NSCLC with activating EGFR mutations.

Abstract Introduction: Most non-small cell lung cancer (NSCLC) patients that have activating mutations in the EGFR gene will respond to treatment with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) e.g. erlotinib and gefitinib. However, in about 50% of these cases a secondary mutation in EGFR (T790M) subsequently occurs, which results in resistance to treatment. Other mechanisms of clinical resistance can also occur such as amplification of c-MET kinase, HER2 and epithelial-mesenchymal transition (EMT) conversion; however, additional routes of resistance are poorly defined. Using a novel Caucasian NSCLC patient-derived xenograft (PDX) model and a cell line derived model, driven by the L858R EGFR mutation, we set out to recapitulate the reported clinical routes of resistance to EGFR inhibitors and to evaluate if additional mechanism could also be identified. Methods: LU6422 is a Caucasian NSCLC adenocarcinoma PDX model with an activating EGFR mutation (L858R) which is maintained subcutaneously in vivo admixed with a human stromal cell component. HCC827 is NSCLC adenocarcinoma cell line with an activating EGFR mutation (del E746-A750). Resistant models of LU6422 and HCC827 were generated in vivo and in vitro (respectively) through repeated dosing or exposure to EGFR TKIs. Resistant tumour material was characterised for further mutations in the EGFR gene by direct sequencing as well as for c-MET, AXL and HER2 over-expression and genomic amplification by quantitative PCR. Results: Naïve LU6422 and HCC827 tissue exhibited exquisite sensitivity to EGFR TKIs (100% & 30% reduction respectively in pre-treatment tumor volume, p<0.0001). Following successive cycles of EGFR-TKI treatment several resistant subtypes were generated, cloned and characterized. Conclusions: EGFR TKI resistant subtypes were generated in vivo and in vitro from a proprietary Caucasian NSCLC PDX model (LU6422) and the HCC827 NSCLC cell line and were characterized for their resistance mechanisms. Models of resistance will be invaluable in assessing novel agents targeting the EGFR pathway and the development of new combination strategies which seek to prevent or overcome resistance to EGFR TKIs. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A145. Citation Format: Andrew J. McKenzie, Aaron N. Cranston, Nektaria Papadopoulou, Simon Jiang, Jane Wrigley, Yinfei Yin, Henry Li, Martin Page, Rajendra Kumari. In vivo and in vitro generation and characterization of EGFR TKI resistance in a patient-derived and a cell line-derived xenograft model of NSCLC with activating EGFR mutations. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A145.

Abstract B23: Selective inhibition of a long non-coding RNA (lncRNA), MALAT1 by antisense oligonucleotides results in significant anti-tumor effects in a variety of preclinical cancer models

Abstract An increasing body of evidence supports the notion that long noncoding RNAs (lncRNAs) play important roles in diseases including cancer. MALAT1 (also called NEAT2) was originally identified in tumors of highly metastatic non-small cell lung cancer (NSCLC), however, the functional role of MALAT1 in carcinogenesis has remained largely unknown. However, MALAT1 is highly expressed in various types of cancer and is implicated in the regulation of alternative splicing. To determine if MALAT1 constitutes an important driver of tumor growth in vivo, we developed potent antisense oligonucleotides (ASOs) that were able to achieve strong inhibition of MALAT1 in the tumor cells of a variety of preclinical models following systemic delivery. MALAT1 ASOs were well tolerated in rodents at doses leading to >95% inhibition of MALAT1 RNA in the liver. Inhibition of MALAT1 expression in tumor and tumor-associated stromal cells was determined by both q-RT-PCR using species-specific probe/primer sets and/or in situ ‘viewRNA’ technology, where target knockdown can be visualized on a cell by cell basis. Systemic administration of mouse MALAT1 ASOs resulted in a decrease in the numbers of polyps and proliferation index (BrdU (+)) in the small intestine of Apcmin mouse model of colon cancer and correlated well with MALAT1 inhibition in the polyps. Mouse MALAT1 ASOs were also effective in a DEN-induced HCC model, where ASO treatment reduced the target RNA ~ 90% in tumor cells with a concomitant decrease in tumor numbers, while control ASO had no effects on either measure. In addition, higher expression of MALAT1 in non-treated tumors compared to adjacent normal hepatocytes was also clearly visualized by the in situ ‘view RNA’ method. Furthermore, MALAT1 ASO significantly delayed tumor growth in C26 colon cancer and reduced tumor size in TRAMP mouse model of prostate cancer, where the target RNA was decreased by 80% in tumor cells. In a human NSCLC patient-derived xenograft model, both significant MALAT1 RNA reduction and a delay in tumor growth were achieved after MALAT1 ASO treatment. The effects of MALAT1 downregulation by ASO were not limited to the inhibition of tumor growth alone. MALAT1 ASO treatment not only inhibited the growth of primary tumors in the EBC-1 human NSCLC xenograft model, but also resulted in a decrease in lung metastasis as measured by micro CT scanning. Furthermore, cross-species MALAT1 ASOs greatly improved the survival of animals bearing Hep3B human hepatocellular carcinoma (HCC) tumor orthotopically (48.5 days with control ASO vs 88 days with MALAT1 ASO, p=0.005). Taken together, these results demonstrate previously undiscovered roles of MALAT1 as an important regulator in vivo tumor growth and metastasis and suggest that selective inhibition of MALAT1 by ASO could have therapeutic value for the cancer treatment. Citation Format: Jeff Hsu, Guobin He, Gourab Bhattacharjee, Tianyuan Zhou, Chris May, Xiaokun Xiao, Gene Hung, Brett P. Monia, A. Robert MacLeod, Youngsoo Kim. Selective inhibition of a long non-coding RNA (lncRNA), MALAT1 by antisense oligonucleotides results in significant anti-tumor effects in a variety of preclinical cancer models [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr B23.