Abstract 5563: 3D Biology™ view of cancer: Simultaneous detection of somatic DNA mutations and expression profiling of genes and signaling proteins from melanoma tumor FFPE samples
Authors: Lee Jinho , Vellano Christopher P. , Meredith Gavin , Mckay-Fleisch Jill , Ross P. Martin , Tetzlaff Michael , Reuben Alexandre , Hudgens Courtney , Wargo Jennifer , Garber Jessica , White Andrew , Pan Joseph , Krouse Mike , Pansalawatta Mekala , Dennis Lucas , Kharkia Anisha , Piazza Erin , Mashadi-Hossein Afshin , Boykin Rich , Elliott Nathan , Filanoski Brian , Demirkan Gokhan , Warren Sarah , Geiss Gary , Kim Dae , Beechem Joseph , Mills Gordon B. 
Published: July 2017

Abstract
Prognosis is favorable in patients with primary localized melanoma but poor in patients with metastatic disease. With more than 76,000 cases expected to be diagnosed in 2016, more precise prognostic technologies and new therapies are needed. Although targeted treatment regimens have been approved in recent years, resistance has emerged in large part due to adaptive response mechanisms and intratumoral heterogeneity. Analysis of tumor samples across multiple molecular platforms will help elucidate the complexities within and across tumors which may underlie response to therapy as well as assist in identifying predictive biomarkers; however, these approaches require significant amounts of sample, time, and resources.
In order to integrate the strengths of analyzing different molecular analytes, we have modularized Nanostring Technologies' molecular barcoding technology to permit simultaneous digital measurement of cancer-associated DNA mutation variants, mRNA expression, and protein expression in one assay from the same sample (3D Biology). Novel nucleotide variant probes enable sensitive and specific identification of DNA mutant allele sequences down to a level of detection of ≤ 5% from 5 ng of FFPE-extracted genomic DNA. Gene expression is measured via unique digital barcoding technology to measure mRNA transcripts, and protein expression and activity (via phosphorylation) is measured by DNA-labeled antibodies. The multi-omic workflow requires only two 5-10 micron sections of FFPE tissue, whereby DNA and RNA are extracted from one section and multiplex digital protein profiling is conducted on the second.
As proof of concept demonstrating the utility of this 3D Biology platform, we have simultaneously analyzed DNA variants, RNA expression, and protein expression using NanoString’s nCounter Vantage 3D™ Solid Tumor Panel on 12 FFPE melanoma tumor samples and one normal tissue from six patients. This sample set included two metastatic tumors from each patient, and in one instance multiple regions from each metastatic site (7 and 2 regions) in order to assess intratumoral heterogeneity. Importantly, this sample set has associated mRNA expression measured using the nCounter® PanCancer Pathways for Human panel as well as whole exome sequencing (WES) data. Somatic variants seen in this latter dataset were compared with the results from the DNA SNV Solid Tumor Panel. Samples with variants that were detected in the nCounter assay but not WES were subjected to deep sequencing for validation. Overall, we show that this multiplex and multi-omic platform has the potential for rapid and sensitive assessment of patient samples that will impact clinical care.
Citation Format: Jinho Lee, Christopher P. Vellano, Gavin Meredith, Jill Mckay-Fleisch, P. Martin Ross, Michael Tetzlaff, Alexandre Reuben, Courtney Hudgens, Jennifer Wargo, Jessica Garber, Andrew White, Joseph Pan, Mike Krouse, Mekala Pansalawatta, Lucas Dennis, Anisha Kharkia, Erin Piazza, Afshin Mashadi-Hossein, Rich Boykin, Nathan Elliott, Brian Filanoski, Gokhan Demirkan, Sarah Warren, Gary Geiss, Dae Kim, Joseph Beechem, Gordon B. Mills. 3D Biology™ view of cancer: Simultaneous detection of somatic DNA mutations and expression profiling of genes and signaling proteins from melanoma tumor FFPE samples [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 5563. doi:10.1158/1538-7445.AM2017-5563