Supplementary Material for: Diffuse Infiltrating Retinoblastoma with Anterior Chamber Involvement: Conservative Management and Identification of <i>RB1</i> Alterations in Aqueous Humor

<b><i>Introduction:</i></b> The aim of the study was to describe the successful conservative management of diffuse infiltrating retinoblastoma (DIR). Identification of <i>RB1</i> pathogenic variant was done after cell-free DNA (cfDNA) analysis in aqueous humor. <b><i>Case presentation:</i></b> Herein, we report 2 patients with unilateral, non-familial DIR with anterior and posterior involvement. Both patients underwent liquid biopsy for tumor cfDNA analysis in aqueous humor. Treatment consisted of a combination of systemic and intra-arterial chemotherapy, with consecutive intracameral and intravitreal injections of melphalan. One patient also required iodine-125 brachytherapy. In both cases, tumor cfDNA analysis revealed biallelic somatic alterations of the <i>RB1</i> gene. These alterations were not found in germline DNA. Both patients retained their eyes and had a useful vision after a follow-up of 2 years. <b><i>Conclusion:</i></b> In selected cases, conservative management of DIR is safe and effective. Tumor cfDNA analysis in aqueous humor is an effective technique to disclose <i>RB1</i> somatic alterations that guide the germline molecular explorations and improve genetic counseling after conservative treatment.