Simultaneous Detection of Single Nucleotide Variants and Expression of RNA and Protein in Multiple Myeloma Bone Marrow Aspirates

Introduction: Multiple myeloma (MM) is generally an incurable plasma cell neoplasm that evolves from a premalignant state known as monoclonal gammopathy of undetermined significance (MGUS). Primary genomic events leading to the development of MGUS include hyperdiploidy, translocations involving the immunoglobulin heavy chain genes, and copy number variations. Single-nucleotide variants (SNVs) as well as insertions and deletions are secondary events that trigger progression to active MM. Identifying nucleotide variants that lead to changes in RNA and protein expression is critically important to understanding the biology of the cancer and ultimately identifying therapeutic targets. Analysis of DNA, RNA, and protein has traditionally involved integration of three separate assays and is frequently limited by the availability of sufficient material from MM patient specimens. We used a more efficient approach that simultaneously detects DNA variants, RNA and protein expression in a single sample from as little as 5 ng DNA, 25 ng RNA, and 250 ng protein. We evaluated the NanoString nCounter® Vantage 3D™ DNA:RNA:Protein Heme Assay performed on 1 x 10⁶cells from bone marrow biopsies obtained from patients with MM.

Methods: Bone marrow aspirates from 15 patients diagnosed with MM were obtained through an Institutional Review Board approved protocol. Seven patients had newly diagnosed MM and 8 had relapsed disease. DNA, RNA, and protein were extracted from unsorted, cryopreserved, ficoll-hypaque separated bone marrow mononuclear cells and hybridized with optical barcodes designed to detect 124 DNA somatic variants common to hematologic malignancies as well as 180 mRNA transcripts and 38 total and phospho-proteins. We also used the nCounter PanCancer Pathways Panel Assay to measure gene expression of 770 additional genes from 13 cancer-associated canonical pathways. The hybridized probes in each sample were counted using the nCounter Digital Analyzer and statistical analysis was performed using nSolver™ v4.0 alpha software.

Results: The percentage of CD138+ bone marrow plasma cells measured by immunohistochemistry at the time of biopsy varied from 44 to 100% in each sample. One or more DNA variants were detected in 3 pre-treatment and 4 relapse samples. Mutations in KRAS, NRAS, and BRAF were most common and 2 patients were found to have variants in both KRAS and NRAS or KRAS and BRAF . Additionally, variants in DNMT3A and IDH2 were detected in 1 patient each. Since KRAS, NRAS, and BRAF are all members of the mitogen-activated protein kinase (MAPK) signaling pathway, a differential gene expression analysis of both RNA and protein was performed comparing samples with and without mutations in these genes. Findings confirmed that a higher level of expression was detectable in MAPK pathway genes, although this result did not reach statistical significance following correction for replicate testing, potentially the consequence of a small sample size.

Conclusions: The Vantage 3D DNA:RNA:Protein Heme Assay is a rapid, sensitive and resource sparing platform for the simultaneous detection of DNA variants, RNA transcripts, and protein expression. We show that this method can feasibly be performed on as few as 1 x 10⁶ cryopreserved bone marrow cells in patients with MM and our findings confirm detection of 3 of the most common MM variants. This technology should have broad application in other hematological malignancies and further evaluation of blood and lymph node biopsies from fresh, frozen and formalin-fixed paraffin-embedded (FFPE) samples is warranted.