NanoString Digital Molecular Profiling of Protein and microRNA in Rhabdomyosarcoma.

Abstract

Simple SummaryNanoString digital profiling methods are novel techniques to identify biologic markers from human formalin-fixed, paraffin-embedded cancer tissue. We have applied NanoString Digital spatial profiling and microRNA profiling methods in non-alveolar rhabdomyosarcoma, a common soft tissue tumor in young adults and children with variable prognosis. Our results have highlighted aberrant miRNA expression and over-expression of several members of PI3-AKT, MAPK and apoptosis signaling pathways in fusion-negative rhabdomyosarcoma, particularly in tumors with unfavorable prognosis. INPP4B, an entry molecule in the PI3/AKT pathway, was significantly over-expressed in tumors with poor prognosis, confirmed by traditional immunohistochemistry. Several microRNAs had increased expression in association with poor patient prognosis. These results highlight the utility of NanoString digital profiling as a screening method to identify prognostic biomarkers of interest in rhabdomyosarcoma from formalin-fixed paraffin-embedded tissue.Purpose: Rhabdomyosarcoma (RMS) exhibits a complex prognostic algorithm based on histologic, biologic and clinical parameters. The embryonal (ERMS) and spindle cell-sclerosing RMS (SRMS) histologic subtypes warrant further studies due to their heterogenous genetic background and variable clinical behavior. NanoString digital profiling methods have been previously highlighted as robust novel methods to detect protein and microRNA expression in several cancers but not in RMS. Methods/Patients: To identify prognostic biomarkers, we categorized 12 ERMS and SRMS tumor cases into adverse (n = 5) or favorable (n = 7) prognosis groups and analyzed their signaling pathways and microRNA profiles. The digital spatial profiling of protein and microRNA analysis was performed on formalin-fixed, paraffin-embedded (FFPE) tumor tissue using NanoString technology. Results: The detectable expression of several component members of the PI3K/AKT, MAPK and apoptosis signaling pathways was highlighted in RMS, including INPP4B, Pan-AKT, MET, Pan-RAS, EGFR, phospho-p90 RSK, p44/42 ERK1/2, BAD, BCL-XL, cleaved caspase-9, NF1, PARP and p53. Compared to cases with favorable prognosis, the adverse-prognosis tumor samples had significantly increased expression of INPP4B, which was confirmed with traditional immunohistochemistry. The analysis of microRNA profiles revealed that, out of 798 microRNAs assessed, 228 were overexpressed and 134 downregulated in the adverse prognosis group. Significant over-expression of oncogenic/tumor suppressor miR-3144-3p, miR-612, miR-302d-3p, miR-421, miR-548ar-5p and miR-548y (p < 0.05) was noted in the adverse prognosis group. Conclusion: This study highlights the utility of NanoString digital profiling methods in RMS, where it can detect distinct molecular signatures with the expression of signaling pathways and microRNAs from FFPE tumor tissue that may help identify prognostic biomarkers of interest. The overexpression of INPP4B and miR-3144-3p, miR-612, miR-302d-3p, miR-421, miR-548y and miR-548ar-5p may be associated with worse overall survival in ERMS and SRMS.