Analysis of CRX e RBP3 gene expression for the detection of minimally disseminated disease (DMD) in patients with retinoblastoma by digital PCR

Abstract

Successful management of Retinoblastoma (RB) depends on the ability to detect the disease while still being intraocular and non-disseminated. The dissemination of RB may occur to the central nervous system and/or through hematologic dissemination, but imaging and cytology studies are not able to detect very low levels of disease. Molecular identification of specific tumor markers may increase the standard sensitivity and potentially enable minimally disseminated disease (DMD) analysis. The information about molecular targets available in RB is still limited, which justifies the search for more sensitive methodologies for the study of DMD. The expression of the cone-rod homeobox (CRX) and retinol-3 binding protein (RBP3) genes, a transcription factor and a photoreceptor, respectively, are critical for differentiation and maintenance of normal retinal development and have been proposed as potential markers for RB. The objective of this study is to detect DMD in patients with high risk RB by detection of the expression of CRX and RBP3 genes in cerebrospinal fluid (CSF), bilateral bone marrow (BM), peripheral blood (SP) and plasma (PL). LCR, MO, SP and PL samples. 20 patients with high-risk RB and 20 control patients, with diagnosis of ALL (acute lymphocytic leukemia) and AML (acute myeloid leukemia) in remission, will be analyzed. RB samples will be collected at three different times of treatment: diagnosis, during and at the end of treatment. Patients and controls will not undergo any additional procedure beyond those already in their treatment. After extraction of mRNA and complementary DNA synthesis (cDNA) the targets will be detected through droplet digital PCR (ddPCR). The prognostic role of the potential molecular markers investigated will be determined by correlating the levels of gene expression in the target tissues evaluated, compared to the control tissues and with the clinical-histopathological aspects of the patients. RB is the major cause of death associated with this tumor. Due to socioeconomic conditions and the health system, the delay in the diagnosis of RB is still a common scenario in developing countries. Besides the low frequency of extraocular dissemination in developed countries, the lack of specific tumor markers for RB makes difficult to establish protocols for detecting DMD and has received little attention from the scientific literature. Thus, the development of a molecular protocol for detecting DMD through potential molecular targets, such as CRX and RBP3, would increase sensitivity to identify high-risk patients with very low levels of metastatic disease who require therapeutic intensification. (AU)