Mesenchymal Neuroblastoma Cells Are Undetected by Current mRNA Marker Panels: The Development of a Specific Neuroblastoma Mesenchymal Minimal Residual Disease Panel

Abstract

PURPOSE Patients with neuroblastoma in molecular remission remain at considerable risk for disease recurrence. Studies have found that neuroblastoma tissue contains adrenergic (ADRN) and mesenchymal (MES) cells; the latter express low levels of commonly used markers for minimal residual disease (MRD). We identified MES-specific MRD markers and studied the dynamics of these markers during treatment. PATIENTS AND METHODS Microarray data were used to identify genes differentially expressed between ADRN and MES cell lines. Candidate genes were then studied using real-time quantitative polymerase chain reaction in cell lines and control bone marrow and peripheral blood samples. After selecting a panel of markers, serial bone marrow, peripheral blood, and peripheral blood stem cell samples were obtained from patients with high-risk neuroblastoma and tested for marker expression; survival analyses were also performed. RESULTS PRRX1, POSTN, and FMO3 mRNAs were used as a panel for specifically detecting MES mRNA in patient samples. MES mRNA was detected only rarely in peripheral blood; moreover, the presence of MES mRNA in peripheral blood stem cell samples was associated with low event-free survival and overall survival. Of note, during treatment, serial bone marrow samples obtained from 29 patients revealed a difference in dynamics between MES mRNA markers and ADRN mRNA markers. Furthermore, MES mRNA was detected in a higher percentage of patients with recurrent disease than in those who remained disease free (53% v 32%, respectively; P = .03). CONCLUSION We propose that the markers POSTN and PRRX1, in combination with FMO3, be used for real-time quantitative polymerase chain reaction–based detection of MES neuroblastoma mRNA in patient samples because these markers have a unique pattern during treatment and are more prevalent in patients with poor outcome. Together with existing markers of MRD, these new markers should be investigated further in large prospective studies.

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

“…The number of control samples with expression of our markers was slightly increased, possibly due to the more efficient cDNA synthesis. However, ΔCt results were comparable to earlier established thresholds ( Table S3 ), with the exception of FMO3 in BM, which showed an expression of 2.2 Ct lower than previously published data [ 9 , 13 ]. Of note, FMO3 amplifications were not detected in unconverted RNA, excluding the possibility that detection of gDNA resulted in lower Ct values.…”

“…In the current protocols, 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 ]. Multiple studies showed that detection of MRD with various mRNA markers throughout and post-induction therapy could be prognostic of outcome [ 3 , 4 , 11 , 12 , 13 ].…”

“…Several patients scoring negative for MRD detection with the above-mentioned adrenergic (ADRN) marker panels still experience relapse [ 4 , 13 ]. Epithelial to mesenchymal transition (EMT) is a process that plays a role in tumorigenic progression, resulting in an increased motility of cancer cells.…”

“…Together with flavin containing monooxygenase 3 ( FMO3 ), which is not expressed in NBL cells but is expressed in mesenchymal stromal cells and used to differentiate these from MES, these markers form the MES marker panel. High expression of POSTN and PRRX1 and low/no expression of FMO3 can identify MES NBL cells in BM and PB [ 13 ].…”

11q Deletion or ALK Activity Curbs DLG2 Expression to Maintain an Undifferentiated State in Neuroblastoma

“…In contrast, ADRN-type cells represent a more differentiated state, with similarities to the ADRN lineage as in chromaffin cells (van Groningen et al, 2017). ADRN mRNAs decrease during the treatment of NB patients, which may indicate the depletion of ADRN cells in the tumor (Wezel et al, 2019). An ADRN gene enrichment was observed for genes responding to DLG2 Article ll OPEN ACCESS overexpression (2.6 times enriched, p = 4.3eÀ5, Fisher's exact test), but not for genes responding to RA treatment (p = 0.46; Figure 3D).…”