Abstract A53: Hypermethylated RASSF1A as circulating tumor marker in pediatric and adolescent solid tumors
Authors: van Zogchel Lieke M. J. , Lobo João , Lak Nathalie , van Wezel Esther M. , van Wijk Jalenka , Stutterheim Janine , Zappeij-Kannegieter Lily , Looijenga Leendert H.J. , van der Schoot Ellen , Tytgat Godelieve A. M. 
Published: June 2020

Abstract
Background: The tumor suppressor gene RASSF1A is hypermethylated in many adult and pediatric cancers. With the absence of recurrent mutations or translocations in various pediatric and young adult (malignant) tumors, detection of hypermethylated RASSF1A (RASSF1A-M) is an attractive biomarker for diagnosis as well as follow-up. Previously, we showed that RASSF1A-M can be used to detect neuroblastoma cells in bone marrow. Here we provide proof of concept of the detection of RASSF1A-M in plasma and serum of patients with neuroblastoma, renal tumors, rhabdomyosarcoma and germ cell tumors.
Method: Plasma was collected at diagnosis from patients with neuroblastoma (n=47), renal tumors (n=13), and rhabdomyosarcoma (n=19) and during treatment and follow-up for neuroblastoma (n=121). Serum was collected from patients with germ cell tumors at diagnosis (n=94). Initially, cell-free DNA (cfDNA) was isolated, bisulfite treated, and tested by qPCR for actin beta (ACTB), unmethylated and hypermethylated RASSF1A (35 diagnostic and 121 follow-up neuroblastoma samples, 19 rhabdomyosarcoma samples). Subsequently, a droplet digital PCR (ddPCR) method for RASSF1A-M and ACTB, including a methylation-specific restriction enzymatic digestion, was applied on the remaining samples to ensure accurate quantification and reducing bisulfite induced cfDNA loss. A threshold for detection of RASSF1A-M was established by testing plasma (73 by qPCR, 25 by ddPCR) and serum samples (21 by ddPCR) from healthy donors.
Results: In plasma samples, the total cfDNA level (ng/mL; median) was significantly higher in patients with renal tumors and neuroblastoma (localized and metastatic disease), but not in rhabdomyosarcoma patients, when compared to healthy donors (66.4; 34.4; 80.9; 3.0 and 2.4, respectively). For diagnostic neuroblastoma samples, RASSF1A-M was detected in all 26 patients with metastatic disease and in 10/21 patients with localized disease (mean 71.4% and 8.4% RASSF1A-M, respectively). RASSF1A-M levels decreased during therapy and re-elevated at relapse. RASSF1A-M was detected in 10/13 renal tumor and 8/21 rhabdomyosarcoma diagnostic samples (18.6% and 19.7% RASSF1A-M, respectively). Total cfDNA levels in healthy donor samples were higher in serum when compared to plasma (39.9 versus 2.4 ng/mL). cfDNA levels were significantly higher in patients with germ cell tumors (230.20 ng/mL) compared to healthy donor serum. RASSF1A-M was detected across all subtypes, including 15/16 embryonal carcinomas, 16/18 seminomas, 2/2 yolk sac tumors, 10/11 teratomas, 1/1 choriocarcinoma, and 42/46 mixed tumors.
Conclusion: Our findings demonstrate the value of RASSF1A-M as a molecular circulating tumor marker in various solid tumors. We developed a ddPCR-based sensitive and quantitative assay for RASSF1A-M detection. Based on the data presented, RASSF1A hypermethylation is an interesting biomarker for detection of minimal residual disease across various solid tumor types, including those lacking recurrent mutations/translocations.
Citation Format: Lieke M. J. van Zogchel, João Lobo, Nathalie Lak, Esther M. van Wezel, Jalenka van Wijk, Janine Stutterheim, Lily Zappeij-Kannegieter, Leendert H.J. Looijenga, Ellen van der Schoot, Godelieve A. M. Tytgat. Hypermethylated RASSF1A as circulating tumor marker in pediatric and adolescent solid tumors [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A53.