title: Methylated RASSF1a is the first specific DNA marker for minimal residual disease testing in neuroblastoma.
abstract: OBJECTIVE
  PCR-based detection of minimal residual disease (MRD) in neuroblastoma (NB) is presently based on NB-specific transcripts. However, the expression of these targets varies between patients and upon treatment, and only PHOX2B is truly specific. RASSF1a is methylated (RASSF1a(M)) in NB, and we investigated whether it can serve as a specific and stable DNA MRD marker.
  
  
  METHODS
  The RASSF1a(M)-specific quantitative real-time PCR was tested on control bone marrow (BM; n = 50), on 71 NB tumors, and on 159 clinical BM samples at diagnosis and at follow-up of 77 patients. Results were compared with a panel of RNA markers and correlated with prognosis.
  
  
  RESULTS
  RASSF1a(M) was present in all stage 4 and 4s tumors (n = 50) and in 86% stages 1 to 3 tumors (n = 21). The level of methylation in stage 4 NB was correlated with overall survival (P = 0.02). RASSF1a(M)-PCR was highly specific (only 1 amplification in 50 control samples tested in triplicate) and had a similar sensitivity as the RNA-based PCRs, as shown on clinical samples. Moreover, RASSF1a(M) enabled accurate quantification without need for the original tumor.
  
  
  CONCLUSIONS
  RASSF1a(M) is a novel, highly specific DNA marker for MRD detection in NB, equal to PHOX2B in specificity and sensitivity, and better suitable for MRD quantification. We propose to include RASSF1a(M) in further prospective MRD studies in NB alongside RNA MRD markers. In addition, this assay might also be applicable for detection of circulating tumor cells in patients with other cancers withRASSF1a(M) such as breast or lung cancer.
authors.affiliation: Department of Pediatric Oncology, Emma Children's Hospital, Amsterdam, the Netherlands.
authors.forename: Janine
authors.initials: J
authors.lastname: Stutterheim
authors.forename: Fatima Ait
authors.initials: FA
authors.lastname: Ichou
authors.forename: Emmy
authors.initials: E
authors.lastname: den Ouden
authors.forename: Rogier
authors.initials: R
authors.lastname: Versteeg
authors.forename: Huib N
authors.initials: HN
authors.lastname: Caron
authors.forename: Godelieve A M
authors.initials: GA
authors.lastname: Tytgat
authors.forename: C Ellen
authors.initials: CE
authors.lastname: van der Schoot
mesh_terms: D014408:Biomarkers, Tumor / Q000235:genetics*; D019175:DNA Methylation; D005819:Genetic Markers; D006801:Humans; D053208:Kaplan-Meier Estimate; D009367:Neoplasm Staging; D018365:Neoplasm, Residual / Q000175:diagnosis / Q000235:genetics* / Q000401:mortality; D009447:Neuroblastoma / Q000175:diagnosis / Q000235:genetics* / Q000401:mortality; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D025521:Tumor Suppressor Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism
chemical_list: D014408:Biomarkers, Tumor; D005819:Genetic Markers; C411762:RASSF1 protein, human; D025521:Tumor Suppressor Proteins