Abstract 5095: Development of a pharmacodynamic biomarker assay for AZD4785, an antisense oligonucleotide targeting KRAS
Authors: Claire Rooney, Sarah Ross, Anna Staniszewska, Sabine Lennarz, Elaine Kilgour, Alexey S. Revenko, A. Robert Macleod, Andrew Pierce, Paul D. Lyne, J. Carl Barrett, Elizabeth A. Harrington
Journal: Cancer Research (2017)

Abstract AZD4785 is a potent and selective high affinity 2’-4’ constrained ethyl residues (cEt) containing therapeutic antisense oligonucleotide (ASO) targeting KRAS. In a patient-derived explant model of non-small cell lung cancer (NSCLC) carrying a KRAS G12C mutation systemic dosing of AZD4785 resulted in tumor regression. To determine the level of KRAS mRNA knockdown achieved by AZD4785 at a dose which causes regression (250 mg/kg/wk), tumor samples were collected and analyzed by RT-PCR. Mean levels of KRAS mRNA knockdown relative to the PBS control group were determined to be 61% and 78% from studies dosed for 2 and 4 weeks, respectively. However, by RT-PCR there was significant variability in KRAS mRNA levels within the PBS control group, and level of knockdown varied with different housekeeper normalization genes. In early clinical studies it will be essential to measure KRAS expression in tumor samples as a pharmacodynamic marker for AZD4785, hence we sought to develop a more robust assay compatible with FFPE clinical samples with low RNA yields. KRAS mRNA levels were quantified by the nanoString platform, using the panCancer pathways 770 gene codeset. This assay generated robust data from RNA extracted from 2 x 5µm sections of FFPE material from each sample, despite very limited yields of RNA. AZD4785 treatment resulted in comparable levels of KRAS knockdown to the RT-PCR assay, but assay variability was greatly reduced, from stdev &amp;gt;50% (RT-PCR) to 5% (nanoString) for PBS control group samples. Using the nanoString assay, mean KRAS mRNA knockdown was 52% and 68% (p&amp;lt;0.005). We also analyzed the effect of AZD4785 treatment on the wider 770 gene panel. Further evidence of KRAS mRNA knockdown was observed through significant downregulation of transcriptional targets of KRAS signalling (FOSL1, DUSP5, DUSP6) relative to the PBS control group. Neither HRAS nor NRAS levels were affected by AZD4785 treatment. This nanoString assay was applied to a panel of 50 clinical FFPE NSCLC samples and KRAS mRNA was detected above baseline in all samples. Furthermore the intrapatient variability of the KRAS mRNA signal was tested in sections &amp;gt;50μm apart within four NSCLC tissues and was found to be ≤21%. Taken together, these data validate the use of the KRAS nanoString assay for use in clinical FFPE tissues, and demonstrate the level of knockdown of KRAS mRNA to be &amp;gt;50% at a dose of AZD4785 that causes regression in a preclinical PDX model. Citation Format: Claire Rooney, Sarah Ross, Anna Staniszewska, Sabine Lennarz, Elaine Kilgour, Alexey S. Revenko, A. Robert Macleod, Andrew Pierce, Paul D. Lyne, J. Carl Barrett, Elizabeth A. Harrington. Development of a pharmacodynamic biomarker assay for AZD4785, an antisense oligonucleotide targeting KRAS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5095. doi:10.1158/1538-7445.AM2017-5095