Abstract 4280: Paralleled workflow for expansion of limited patient material using CRC for in vitro/PDX/biological/genetic studies of a low-grade mucoepidermoid carcinoma
Authors: Alamri Ahmad M. , Liu Xuefeng , Wang Weisheng , Zhong Xiaogang , Kallakury Bhaskar , Davidson Bruce , Furth Priscilla A. 
Published: July 2016

Abstract
Acquiring live cancer cells preserving original characteristics is a critical step for precision medicine. Here we illustrate a paralleled workflow using a low-grade sublingual salivary mucoepidermoid carcinoma (MEC) as an example. Methods: Working under IRB approval, tissue from two distinct regions of a MEC were obtained and primary epithelial cell cultures established using CRC (F medium with Rho Kinase (ROCK) inhibitor Y-27632 with irradiated Swiss 3T3-J2 mouse fibroblast feeder cells). Epithelial cells were separated from feeders to test under non-CRC conditions including 2D (MammoCult™), colony formation in Matrigel (F medium, Y-27632), patient derived xenograft (PDX) formation (10*6 cells/50:50 matrigel/PBS/mammary fatpad), and RNA and DNA extracted for RNAseq, exome sequencing and PCR/RT-PCR for a CRTC1-MAML2 fusion gene (reported in MEC). Sequences were analyzed for relative transcript abundance, differentially expressed genes (DEGs), and single nucleotide polymorphisms (SNPs) (TopHat/CuffLinks, SAMTOOLS, variant database: dbSNP & 1000G, UCSC hg19) between the two MEC regions and fusion genes (FusionCatcher). Potentially pathophysiological SNPs were identified (OMIM and SNPedia). Gene ontology was performed on transcriptome data (PARTEK Genomic Suite, Pathway Studio). Immunohistochemistry (IHC) was used to compare protein expression of selected DEG and downstream effectors in original cancer and surrounding normal tissue, cell pellets, Matrigel colonies and PDX (confirmed as human using MAB1273). Results: CRC cultures established from both sites grew similarly under all in vitro and in vivo conditions. PDX showed well-differentiated histology comparable to original cancer. One of the 11 DEG was amphiregulin but protein expression was equivalent in the two cell cultures. Amphiregulin, EGFR, p-EGFR, p-AKT, and mTOR were expressed in vitro (CRC and non-CRC), in vivo (PDX) and in original cancer, where they were up-regulated as compared to surrounding normal tissue. Five potentially pathophysiologically significant SNPs were detected (BRCA2 (rs144848), TP53 (rs1042522), AURKA (rs2273535), DBYD (rs1801265), and (SOD2 rs4880)) but no CRCTC1-MAML2 fusion gene. FusionCatcher detected a possible novel fusion product currently under investigation. Conclusion: The paralleled approach provided sufficient material to identify amphiregulin as an upregulated growth factor pathway in a CRTC1-MAML2 fusion gene negative MEC. Previously, amphiregulin upregulation was pathophysiologically linked to the CRTC1-MAML2 fusion gene. Results from the two MEC sites were biologically concordant and genetically similar. In summary, CRC can be used to expand limited patient derived material for more extensive studies under non-CRC conditions. Support: R56DE023259 (PAF)
Citation Format: Ahmad M. Alamri, Xuefeng Liu, Weisheng Wang, Xiaogang Zhong, Bhaskar Kallakury, Bruce Davidson, Priscilla A. Furth. Paralleled workflow for expansion of limited patient material using CRC for in vitro/PDX/biological/genetic studies of a low-grade mucoepidermoid carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4280.