CirculatingMethylated-DCR 2 Gene in Serum as an Indicator of Prognosis andTherapeutic Efficacy in Patients with MYCN

Background: MYCN amplification (MNA) in neuroblastoma is a strong indicator of poor prognosis. However, some MYCN nonamplified (non-MNA) cases show poor outcomes, and examining the status of the gene requires an operation, which may have surgical complications. Therefore, a newmarker is needed to identify cases of non-MNAneuroblastomaswithpoorprognoses using less risky procedures. Aberrant hypermethylationof theDCR2 promoter has recently been associated with rapidly progressing neuroblastoma.We aimed to develop a noninvasive DCR2 methylation assay for patients with neuroblastomausing serumDNA, whichpredominantly originates from tumor-released DNA. Methods: Using DNA-based real-time PCR, we simultaneously quantified a methylated-DCR2 specific sequence (M) and a reference sequence (R) located in the promoter region in serum DNA, and evaluatedDCR2 methylation status as M/R ratios in 86 patients with neuroblastoma. Results: Serum DCR2 M/R ratios were strongly correlated with those in the tumor (r = 0.67; P =0.002). DCR2methylationwas associatedwith stage both in thewhole neuroblastomagroup and in the non-MNA group (P < 0.001), and DCR2-methylated patients showed significantly poorer 5-year event-free survival in the whole neuroblastoma group (43% versus 84%; P < 0.001), especially in the non-MNA group (12% versus 96%;P < 0.001). Among five DCR2methylated patients whose clinical courses were followed, serum M/R ratios were close to 0 in the patients in remission, whereas the ratios increased in patients who relapsed. Conclusions:Detectionof methylated-DCR2 in serumDNAhas promise as a noninvasive assay for predictingprognosis and therapeutic efficacy inneuroblastoma, especially in non-MNA cases. Furthermore, it might be a sensitive marker of tumor recurrence inDCR2-methylated cases. Neuroblastoma is the most common extracranial solid tumor in children and is characterized by a wide range of clinical behaviors, from spontaneous regression to rapid progression with a fatal outcome (1). The current risk classification of patients with neuroblastoma is based on the age of onset, the extent of disease at the time of diagnosis as defined by the International Neuroblastoma Staging System, and evaluation of genetic aberrations, such as MYCN amplification (MNA; ref. 2). MNA is considered the strongest prognostic factor and is routinely assessed for therapy stratification (2–4), but it is a big concern that some cases without MNA also have a poor prognosis (5). Hero and colleagues recently reported that spontaneous regression is often seen in localized infantile neuroblastoma without MNA, and they suggested that a waitand-see strategy avoiding chemotherapy and surgical procedure was justified in such patients (6). Nevertheless, some patients in their cohort showed local progression, progression to stage IVS, or progression to stage IV. Thus, it is important to have additional biomarkers with prognostic value for the management of non-MNA cases of neuroblastoma. We screened many biological markers for neuroblastoma, such as Trk A expression (7) and chromosome allelic loss (8–10), but none of them were found to be useful for risk classification among non-MNA