Retinoblastoma (RB) represents the most common intraocular malignancy of infancy and childhood (1). Although the survival rates are over 95% for intraocular forms, in low-income countries the prognosis remains poor, ranging from 20% to 60% (2, 3). Among the dissemination routes, RB may spread to the central nervous system (CNS) through the optic nerve. This occurrence correlates with poor prognosis despite intensive multimodal treatment (4). In particular, in patients with advanced RB, CNS remains the predominant site of relapse. Thus, an early detection of CNS involvement is crucial to set up timely and tailored therapeutic approaches. The conventional diagnosis of CNS involvement relies on magnetic resonance imaging (MRI) and on the detection of tumor cells in CSF. Detection of biomarkers in the CSF could be useful in identifying patients at risk of developing CNS disease, even before the evidence of CNS involvement. In recent years, mass spectrometry has emerged as a sensitive tool in characterizing proteomic profiles of different biofluids, such as CSF, in neurological diseases and brain tumors (5–8). Importantly, the proteomic analysis of CSF may detect the presence of brain tumors before the onset of symptoms or imaging in animal models (9), reveling that CSF may be altered even at the earliest stages of malignancy. In this report, we perform a proteomic study of CSF in a patient affected by stage III (IRSS) RB in order to detect proteins changes occurring during CNS recurrence.

A total of 1029 proteins were identified in all CSF samples, of these 500 proteins were in common in all samples ( Figure 2A ). The Heatmap, resulting from the comparisons among the samples, showed 46 differentially expressed proteins ( Figure 2B , Table 1 ). In all 3 CSF-RB samples we identified seven downregulated and seventeen upregulated proteins compared with the CSF-CTRL ( Figure 2B , Table 1 ). By gene enrichment analysis performed by FunRich software (15), the downregulated proteins were mainly involved in cell growth, metabolism and cell communication, whereas the upregulated ones in cell growth, signal transduction and metabolism ( Figure 2C ). Four proteins were commonly and differentially expressed in CSF-RB2 and CSF-RB3 samples (positive for neoplastic cells). Specifically, 3 proteins were up-regulated [Creatine kinase B-type (CKB), Retinolbinding protein 3 (RBP3), Nucleoside diphosphate kinase A (NME1)] and one down-regulated [RNAbinding protein 44 (RBM44)]. Four proteins were down-regulated in CSF-RB1: Tubulin beta proteins (TUBB4B, TUBB2B, TUBA1B) and Cathepsin B (CTSB). Interestingly, FSTL5 was upregulated only in CSF-RB3, whereas 2 proteins (DOCK10 and ENDOD1) were down regulated in the same sample.

Although RB primarily affects the eyes, advanced disease mostly associated with delayed diagnosis, can lead to CNS involvement, including the brain and CSF, associated with extremely poor prognosis. Therefore, early detection of CNS involvement is crucial for allowing early therapeutic intervention. In RB, different biofluids, such as plasma, aqueous humor or CSF have been studied to detect tumor related biomarkers as prognostic predictors of disease evolution or treatment response (16). CSF samples from patients with non-metastatic RB and high-risk pathologic features showed that, among four cases with CSF relapse, two patients had GD2 synthase positivity in CSF as marker of minimal dissemination disease (MD) (17). In view of these findings, Laurent et al. suggested early and intensified intrathecal treatment for children with MD in CSF in order to improve the outcomes of these patients. Dimaras et al. also demonstrated that MD could be detected in patients with metastatic RB by using RB1 mutational analysis in CSF (18). Proteomic analysis involves the comprehensive study of proteins present in a biological sample. Since CSF is considered a primary route for metastatic dissemination (19), its proteomic analysis has been useful in improving the understanding of CNS tumors (20). Furthermore, proteins that are overexpressed in the CSF may serve as targets for developing new tailored treatment. For the afore mentioned reasons in this study, we have performed a proteomic evaluation of serial CSF samples in a patient affected by stage III RB with the aim to identify molecular changes occurring during treatment and CNS relapse. The differential analysis revealed a specific proteomics signature in CSF-RB samples. Among the upregulated proteins in CSF-RB, several have structural and physiological functions in the eye (COL1A1, COL3A1, TMSB4X, NID1) (21). NID1 has been described to promote tumor cell migration, invasion, epithelial-to-mesenchymal transition (EMT) and chemo-resistance in different tumors (22–24) similarly to MCAM (25). Other upregulated proteins are involved in neuronal/retinal development and cell migration (Fat2, RELN, NEGR1, SEZ6) (26–29). RELN is an extracellular matrix protein expressed during normal retinogenesis and seems to play an important role in regulating stem cell trafficking in neuronal and non-neuronal tissues (30). SEZ6 has been found be elevated in the CSF from patients with multiple sclerosis and inflammatory conditions (31), furthermore, has been shown to promote metastatic spread by affecting cellular adhesion, cytoskeletal and extracellular matrix remodeling and inducing EMT in cancer cells (32). Of note, RNPEP and PON1 progressively increased during treatment. RNPEP is a secreted enzyme belonging to M1 metallopeptidase family (33) and its circulating mRNA levels have been demonstrated to be an independent prognostic factor in different tumors (34). PON1 is an enzyme associated with high-density lipoprotein (HDL) and plays a role in detoxification processes (35); this protein has potential implications in ocular disorders associated with oxidative stress and inflammation (36–38). Similarly, SERPINA1, is an acute inflammatory protein involved in corneal defense and neuroinflammation (39). Among the down-regulated proteins in CSF-RB, we identified proteins involved in glucose transport (SLC5A) (40), synthesis of glycogen and lipids (CA1) (41), and exosomes biogenesis (SDCBP) (42); those proteins have been described to contribute to aggressive behavior of tumor via metabolic reprogramming and cell signaling. Other downregulated proteins were: protease inhibitor (SERPINB4), which may regulate the immune response against tumor cells (43), protein involved in brain development and synaptic plasticity (TNR) (44) and keratin (KRT14), which provides structural support and integrity to epithelial cells and is abnormal expressed in several tumors (45). The CSF samples that were positive for neoplastic cells, CSF-RB2 and -RB3, presented two commonly upregulated proteins involved in visual pathways: RBP3, which is involved in retinol transport (46), and CK-B, an enzyme that provides energy to photoreceptors for the visual cycle particularly expressed in retinal diseases (47, 48). Another protein, NME1, is involved in oxidative stress response (49) and its increased expression correlates with tumor aggressiveness in specific cancer types (melanoma, liver and breast) (50). CSF-RB3 showed two down-regulated proteins (DOCK10 and ENDOD1) and the upregulation of FSTL5. FSTL5 is a secretory glycoprotein expressed in restricted areas of CNS and few studies indicate its role on neo-plastic transformation and metastasis development (51, 52). DOCK 10 is also expressed in the brain and seems to have a role in neuroinflammation and cancer development by regulating cell adhesion and migration (53). ENDOD1 is a member of nucleases, which participate in DNA repair with abnormal expression in different tumors (54). Lastly, in the CSF-RB1 sample, which was negative for neoplastic cells, we found down-regulated three tubulin beta proteins (TUBB4B, TUBB2B, TUBA1B). Tubulins are microtubules subunits involved in cell shape maintenance (55). In particular, TUBB4B expression is correlated whit EMT initiation and affection of cell polarity (56) and TUBB2B is involved in visual development and function (57); mutations of TUBB4B and TUBB2B have been linked to several ocular disorders (57, 58).

Overall, the proteomic analysis of CSF showed in our patient deregulation of proteins mainly involved in inflammation, extracellular matrix remodeling, EMT initiation, migration, invasion and cell metabolism. The CSF samples positive for neoplastic cells revealed proteins with a prominent role in visual pathways, including transport of retinol, photoreceptor’s function and oxidative stress response. The timepoint negative for tumor infiltration revealed several TUBB proteins that are related with cellular shape maintenance, cell polarity and EMT initiation. These results demonstrate how proteomic analysis of CSF can help to improve our understanding in RB. Further studies including large cohorts of patients with RB should be conducted to identify new prognostic biomarkers and potential therapeutic targets in this tumor.