Specific and Sensitive Detection of Neuroblastoma mRNA Markers by Multiplex RT-qPCR.

Lieke M J Van Zogchel,Nathalie S M Lak,Marjolein Lugtigheid,Danny A Zwijnenburg,Jan Koster,Godelieve A M Tytgat,Ahmad Javadi,C Ellen Van Der Schoot,Nina U Gelineau,Janine Stutterheim,Lily Zappeij-Kannegieter

doi:10.3390/cancers13010150

Abstract

Simple SummarySensitive detection of minimal residual disease by RT-qPCR in patients with neuroblastoma is shown to be predictive of outcome, but has not yet been introduced into clinical practice. A panel of multiple mRNA markers increases the sensitivity of minimal residual disease detection, since neuroblastoma tumors are heterogeneous tumors. Recent studies have identified two distinct phenotypes, an adrenergic and mesenchymal phenotype, that can be identified by using different mRNA markers. As generally only small volumes of bone marrow or blood are available in young neuroblastoma patients, we have developed a multiplex RT-qPCR to be able to test seven different mRNA markers, while we reduce the sample volume needed. Comparison between the multiplex RT-qPCR and RT-qPCR for the single markers showed a comparable sensitivity. This reduction in required sample volume, while saving time and resources, can assist in the introduction of minimal residual disease detection by RT-qPCR into clinical practice.mRNA RT-qPCR is shown to be a very sensitive technique to detect minimal residual disease (MRD) in patients with neuroblastoma. Multiple mRNA markers are known to detect heterogeneous neuroblastoma cells in bone marrow (BM) or blood from patients. However, the limited volumes of BM and blood available can hamper the detection of multiple markers. To make optimal use of these samples, we developed a multiplex RT-qPCR for the detection of MRD in neuroblastoma. GUSB and PHOX2B were tested as single markers. The adrenergic markers TH, GAP43, CHRNA3 and DBH and mesenchymal markers POSTN, PRRX1 and FMO3 were tested in multiplex. Using control blood and BM, we established new thresholds for positivity. Comparison of multiplex and singleplex RT-qPCR results from 21 blood and 24 BM samples from neuroblastoma patients demonstrated a comparable sensitivity. With this multiplex RT-qPCR, we are able to test seven different neuroblastoma mRNA markers, which overcomes tumor heterogeneity and improves sensitivity of MRD detection, even in those samples of low RNA quantity. With resources and time being saved, reduction in sample volume and consumables can assist in the introduction of MRD by RT-qPCR into clinical practice.

Highlights

Disseminated disease to the bone marrow (BM) is present at diagnosis in more than half of the children with neuroblastoma (NBL) [1,2]
We have previously described paired-like homeobox 2b (PHOX2B) as a sensitive and NBL specific mRNA marker for minimal residual disease (MRD) detection by reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR), with high expression in NBL tumors and no expression in normal BM and peripheral blood (PB) [8]
We observed a disturbance of reverse transcriptase (RT)-qPCR amplification plots through fluorescence quenching, which was caused by interaction between Mustang Purple and dithiothreitol (DTT) [16] from the reverse transcription mix Cancers 2021, 13, 150

Summary

Introduction

Disseminated disease to the bone marrow (BM) is present at diagnosis in more than half of the children with neuroblastoma (NBL) [1,2]. BM infiltration at diagnosis and during treatment is assessed by histology or (immuno)cytology, and more sensitive detection of tumor cells by reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) is under investigation [8,9,10]. We have previously described paired-like homeobox 2b (PHOX2B) as a sensitive and NBL specific mRNA marker for minimal residual disease (MRD) detection by RT-qPCR, with high expression in NBL tumors and no expression in normal BM and peripheral blood (PB) [8]. Based on high expression in NBL tumors and low/no expression in normal BM or PB, we optimized two mRNA marker panels, one specific for BM, the other specific for PB. PHOX2B, TH, CHRNA3 and dopamine beta hydroxylase (DBH) form the PB panel [8,9]

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