Metastatic retinoblastoma exhibit MYCN alterations, recurrent CNAs and hypomethylation as molecular characteristics of aggressive disease

GANIEWICH, DAIANA; ANDREA S. LLERA

Congreso; Annual Meeting of the American Association for Cancer Research; 2022

Retinoblastoma is a rare pediatric malignancy but the most frequent tumor of the eye. Metastatic disease is the most common cause of disease-related death and is rare in high-income countries. Therefore, little is known about the genomics, transcriptomics and methylomics of extraocular retinoblastoma. Here we present a series of extraocular retinoblastoma cases analyzed at DNA, RNA and methylation level to elucidate the landscape of the metastatic disease. These samples were analyzed by OncoScan CNV, CytoScan HD or whole-exome sequencing, depending on sample availability, for CNA (clonal and subclonal) and mutation analysis; RNAseq for fusion and expression data, and Infinium MethylationEPIC assay for methylation examination. Hierarchical clustering and subtype classification was performed using previously published expression and methylation signatures. Cell cultures were successfully established for 4 tumor specimens. In vitro pharmacological sensitivity of those cell cultures was determined by MTT assay. In total, 11 patients and 14 samples were studied (55% male, 64% unilateral). One patient was RB+/+ and harbored a MYCN amplification. Dissemination sites included orbit (n=8), systemic (n=3), and central nervous system (n=5). Median age at diagnosis was 27 months (5-48); 36% were already metastatic. All samples were classified as subtype 2 retinoblastoma according to Liu et. al., 2021, both at the expression and methylation level. Among recurrent CNAs in all samples, common retinoblastoma alterations were observed: 1q (86%), 2p (71%, 29% MYCN amplified), 6p (93%) gains and 16q (64%) loss. Other less frequent alterations such as 17q gain (21%) and 11q (43%) and 19q (29%) loss were found. MYCN pathway alteration was the most outstanding feature, evidenced by frequent gains/amplification on that gene and in gene-expression clustering based on MYCN expression signatures. Global hypomethylation was also observed when comparing metastatic to intraocular samples and it was consistent with the general gene overexpression seen in metastatic samples. Metastasis-derived in vitro cultures treated either with melphalan, topotecan, idarubicin, doxorubicin or carboplatin showed various degrees of sensitivity (i.e. EC50 in the nM to mM range), with no clear association to MYCN overexpression. In conclusion, even considering the caveats regarding our current sample size, we could detect the alteration of the MYCN pathway as a distinct characteristic of extraocular retinoblastoma. Global hypomethylation has been related to the activation of oncogenic drivers and is consistent with metastatic behavior. These results suggest a pattern of molecular features that may have been selected during tumor evolution. Our results do not support a straightforward relationship between MYCN activation and resistance to therapy.