Abstract A026: A novel ddPCR detects hypermethylated RASSF1A in plasma of patients with pediatric Wilms tumor
Authors: Sprokkerieft Julia , Lak Nathalie S.M. , van Gestel-Fadaie Zeinab , van den Heuvel-Eibrink Marry M. , Tytgat Godelieve A.M. 
Published: November 2024

Abstract
Background: Wilms tumor is the most common pediatric renal cancer. Within the SIOP-Renal Tumor Study Group (SIOP-RTSG), diagnosis is based on imaging and patients receive neo- adjuvant treatment. Final diagnosis is established via histopathological analysis at the timepoint of surgery, which also determines the intensity of adjuvant treatment. Overall, patients with Wilms tumor have a 90% survival rate, but one in five patients will suffer from a relapse, which requires intensive treatment and induces long-term toxicity. It is challenging to identify patients at risk of suffering from relapse at initial diagnosis. Hence the need for additional biomarkers that will optimize risk stratification and minimal residual disease monitoring. RASSF1A is a tumor suppressor gene that is silenced via hypermethylation of its promotor region in multiple adult and pediatric tumors and is reported to represent an aggressive phenotype. We have previously reported on thirteen patients with Wilms tumor where increased hypermethylation of RASSF1A (RASSF1A-M) was observed in plasma cell-free DNA (cfDNA). The aim of this study was to further investigate the levels of RASSF1A-M in a larger cohort. Methods: The presence of RASSF1A-M in plasma cfDNA of patients with Wilms tumor was quantified using a multiplexed droplet digital PCR comprising methylation-specific restriction enzymes. Diagnosis and pre- surgery samples were included to evaluate RASSF1A-M levels during treatment. Results: A total of 49 patients were evaluated. At diagnosis, 37/46 (80.4%) samples were positive for RASSF1A-M. Negative samples were from patients with localized disease (N=6), regressive type regionally advanced Wilms (N=1) or mixed type metastatic Wilms (N=2). RASSF1A-M levels were significantly elevated in regionally advanced and metastatic disease compared to localized disease. Of the 37 positive diagnosis samples, 23 pre-surgical follow-up samples were available. In 7/23, RASSF1A-M was still detectable. These belong to patients with stromal-, regressive- or diffuse anaplastic predominant tumors. Interestingly, an increasing methylation level was seen in one patient with a regressive tumor. Conclusion: RASSF1A-M is a potential biomarker for patients with Wilms tumor, since it was detected in the majority of patients. Furthermore, significantly increased levels were observed in regionally advanced and metastatic disease. In the small number of patients with persisting levels of RASSF1A-M, we did not observe an association with risk groups. In collaboration with SIOP-RTSG, additional follow-up samples will be analyzed to explore the potential of RASSF1A-M as biomarker for relapse detection.
Citation Format: Julia Sprokkerieft, Nathalie S.M. Lak, Zeinab van Gestel-Fadaie, Marry M. van den Heuvel-Eibrink, Godelieve A.M. Tytgat. A novel ddPCR detects hypermethylated RASSF1A in plasma of patients with pediatric Wilms tumor [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A026.