An Assay for Simultaneous Profiling of Gene Expression, Phospho- and Total Protein Abundance, and Somatic DNA Mutations for Hematology-Oncology Research

Introduction:

Despite the great diversity in cell of origin and other clinical features, hematological malignancies share a core set of pathways and processes that drive tumorigenesis. Pathways critical to the normal growth and development of the hematopoietic compartment, such as MAPK, MYC signaling, NF-kB, PI3K-AKT, B and T cell receptor signaling are frequently mutated and disrupted. Additionally, dysregulation of cytokines, apoptosis, the DNA damage response, and epigenetic factors are frequently seen in gene expression as well as mutational profiles across a variety of these cancers.

To address the need to profile such pathways across multiple classes of biological macromolecules from small amounts of sample, NanoString has developed 3D Biology™ Technology, which enables DNA, RNA, and protein to be simultaneously detected from a single sample utilizing digital molecular barcoding technology on the nCounter® system. For hematology-oncology specifically, the nCounter® Vantage 3D™ DNA:RNA:Protein Heme Assay embodies a collection of probes, molecular barcodes, and workflows to measure 180 mRNA targets, more than 30 protein targets (total and phosphorylated forms including JAK-STAT, Src, Syk, Ik-Ba, PI3K-Akt), and more than 120 DNA mutations for the conserved and crucial pathways underlying hematological malignancies.

Methods and Results:

Development of 3D Biology™ Technology is based on Nanostring's proven digital molecular barcoding technology. Probe-reporter complexes are formed through highly specific and predictable nucleic acid hybridization events allowing for parallel design and development across multiple analytes. The RNA portion is composed of probes designed using a well-established pipeline and are verified against synthetic oligonucleotide targets and Universal Human Reference total RNA (Agilent). The protein portion utilizes a mixture of antibodies each of which has been chemically labeled with a target-specific 'alien' synthetic oligonucleotide. The capacity for each antibody within the mix to specifically detect its target is tested against lysates and formalin-fixed, paraffin-embedded (FFPE) tissue samples. Finally, the DNA panel is composed of novel 'SNV' probes that can discriminate between targets that differ by a single base. These probes are designed and screened against synthetic mutant and reference (hg19) allele targets for specificity and sensitivity to ensure detection down to 5% allele frequency with >95% sensitivity. Standard genomic DNA samples from NIST (NA12878) and Horizon Discovery are also used to verify assay performance.

Workflows have been established and validated for DNA:RNA:Protein profiling for fresh/frozen or FFPE-preserved samples. For fresh/frozen samples, the assay enables simultaneous DNA:RNA:Protein profiling from as little as 5 ng DNA, 25 ng RNA, and 250 ng protein. This is achieved by extracting genomic DNA and creating a lysate for RNA and protein analysis. The purified DNA is pre-amplified before overnight hybridization with SNV probes and reporters and the lysate is processed to permit a combined overnight hybridization reaction with both RNA- and protein-specific probes and reporters. For FFPE samples, equivalent profiling can be obtained from two 100 mm², 5-micron thick FFPE sections; one section is incubated with antibodies and the second section is used for extraction of RNA and DNA using a standard kit. After hybridization, processed DNA, RNA, and protein are pooled and co-analyzed using an nCounter system and nSolver™ software (alpha version 4.0). Concordant results were obtained from both workflows for two heme-derived cell lines, CCRF-CEM and Hut78, showing reproducibility between fresh/frozen and FFPE sample types. Additionally, all known DNA mutations were detected and Hut78 showed higher and concomitant RNA and protein expression of NF-kB p65 (RELA), BCL-xL (BCL2L1), Stat3 (STAT3), and IkBa ( IKBA ) than occurs in CCRF-CEM cell lines. Results from human archived tissues will also be shown.

Conclusion:

Combined analysis of SNV, mRNA, and protein expression in a 3D Biology experiment promises broad and unique utility for hematology-oncology research, particularly for the evaluation of blood, lymph node, and bone marrow biopsies.