In the present study, we validated the ability of 14 commonly used real-time RT-PCR markers to detect MRD based on their expression in neuroblastoma TICs, and we developed a novel MRD detection protocol, which scored the samples as MRD-positive when the expression of one of the 11 real-time RT-PCR markers (CHRNA3, CRMP1, DBH, DCX, DDC, GABRB3, GAP43, ISL1, KIF1A, PHOX2B and TH) exceeded the normal range.
Heterozygous germline mutations and deletions in PHOX2B, a key regulator of autonomic neuron development, predispose to neuroblastoma, a tumor of the peripheral sympathetic nervous system.
Universal mass screening for neuroblastoma is not indicated but targeted screening of infants at risk of hereditary neuroblastoma with germline ALK or PHOX2B mutations is appropriate.
For instance, discoveries in familial NBL have identified genetic aberrations in Phox2b and Alk that predispose to NBL, while advances in epigenetics and MYCN regulation have also offered insight into NBL pathogenesis and future treatment.
In neuroblastoma (NB) patients, minimal residual disease (MRD) can be detected by real-time quantitative PCR (qPCR) using NB-specific target genes, such as PHOX2B and TH.
We undertook mutational analysis of the genes known to predispose to non-syndromic familial Wilms tumor (WT1) or neuroblastoma (PHOX2B, ALK) which excluded these as the underlying predisposition genes in the nine families.
Following this possibility, we first confirmed a striking correlation between the transcription levels of ALK, PHOX2B and its direct target PHOX2A in a panel of NB cell lines.
Consistent with its role as an important neurodevelopmental gene, forced overexpression of wild-type PHOX2B in neuroblastoma cell lines suppressed cell proliferation and synergized with all-trans retinoic acid to promote differentiation.
The PHOX2B gene is implicated in the development of the autonomic nervous system and has been found to be infrequently mutated in sporadic neuroblastoma tumours and in some patients with hereditary neuroblastoma.
These experiments describe for the first time regulation of the Delta-Notch pathway by MSX1, and connect these genes to the PHOX2B oncogene, indicative of a role in neuroblastoma biology.
Transient transfections and electrophoretic-mobility-shift assays suggested that PHOX2B is able to bind the cell-specific element in the 5' regulatory region of the TLX2 gene, determining its transactivation in neuroblastoma cells.
Nevertheless, as only a few NB families but not others have been shown to carry PHOX2B mutations, the role of this gene in NB predisposition has still to be clarified.
Recent studies have shown that 1) PHOX2B is the main disease-causing gene for congenital central hypoventilation syndrome, an autosomal dominant disorder with incomplete penetrance; 2) PHOX2B is the first gene for which germline mutations have been demonstrated to predispose to neuroblastoma; and 3) Hirschsprung disease was associated with an intronic single-nucleotide polymorphism of the PHOX2B gene in a case-control study.
We also report a germline PHOX2B mutation in one patient treated for Hirschsprung's disease who subsequently developed a multifocal neuroblastoma in infancy.
We have subsequently shown that heterozygous mutations of PHOX2B may account for several combined or isolated disorders of autonomic nervous-system development--namely, tumors of the sympathetic nervous system (TSNS), such as neuroblastoma and late-onset central hypoventilation syndrome.