Imaging, Diagnosis, Prognosis Methylated RASSF1a Is the First Specific DNA Marker for Minimal Residual Disease Testing in Neuroblastoma

Purpose: PCR-based detection of minimal residual disease (MRD) in neuroblastoma (NB) is presently based onNB-specific transcripts. However, the expression of these targets varies between patients and upon treatment, and only PHOX2B is truly specific. RASSF1a is methylated (RASSF1aM) in NB, and we investigated whether it can serve as a specific and stable DNA MRD marker. Patients and Methods: The RASSF1aM-specific quantitative real-time PCR was tested on control bone marrow (BM; n1⁄4 50), on 71NB 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: RASSF1aM was present in all stage 4 and 4s tumors (n 1⁄4 50) and in 86% stages 1 to 3 tumors (n1⁄4 21). The level of methylation in stage 4 NBwas correlated with overall survival (P1⁄4 0.02). RASSF1aM– 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, RASSF1aM enabled accurate quantification without need for the original tumor. Conclusions: RASSF1aM 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 RASSF1aM in further prospective MRD studies in NB alongside RNA MRD markers. In addition, this assay might also be applicable for detectionof circulating tumor cells inpatientswithother cancerswithRASSF1aM such as breast or lung cancer. Clin Cancer Res; 18(3); 808–14. 2011 AACR.