Gene expression analysis of retinoblastoma tissues with clinico-histopathologic correlation

Abstract

Purpose: Retinoblastoma (Rb) is the most common intraocular malignant tumor, which is not only unique but also has unraveled many novel aspects of tumor-suppressor genes. Genetic mutations of Rb, loss of phosphorylation, and many other factors resulted in uncontrolled cell division of the retinal cells resulting in tumor progression. In this study, we have analyzed the gene expression patterns of unilateral tumors (n = 11) in comparison to the normal-appearing retina (n = 2) from Rb patients who underwent enucleation for advanced Rb. With recent advances in the knowledge of the role of stem cells in these tumors, it is important to evaluate and understand the self-renewal signaling involved in these tumors. Here, in this study, we particularly aimed at evaluating the aberrant self-renewal signaling pathways in human Rb tumors and genes which show differential expression in cases with and without histologic risk factors (HRF). Materials and Methods: Freshly unfixed eyeballs (n = 11) were obtained. Normal-appearing retinas were pooled together (n = 2) and used as a control for microarray experiments. Total RNA was isolated from tumors and control tissues, and expression of genes was evaluated by hybridizing to expression arrays. Using real-time polymerase chain reaction (PCR), the results, thus, obtained were validated (for expression of N-Myc, HMGA2, LIN-28b, and Activin receptor 1C [ACVR1C]) in tissues compared to two control retinas latter obtained from enucleated Rb eyeballs without tumor. Furthermore, immunohistochemistry (IHC) was done on retrospective (n = 19) cases to confirm the expression of ACVR1C. Results: In Rb tumors, 5593 genes were upregulated and 4864 genes were downregulated (P ≤ 0.05 and fold change ≥1.5 folds). Changes in N-Myc, HMGA2, LIN28b, and ACVR1C expression detected by microarray were validated by real-time PCR. The analysis shows significant up-regulation of HMGA2 and its downstream regulator LIN-28b, which is involved in self-renewal pathway of fetal neural stem cells. ACVR1C is one of the markers, which shows differential expression between histological subtypes of tumors as evident in IHC. CBLB (P ≤ 0.05) and MAPK 8 (P ≤ 0.05) were shown to be highly upregulated in tumors without HRF compared to cases with HRF. Conclusions: This study showed up-regulation of genes involved in neural stem cell self-renewal and marginally in notch signaling. While other stem cell pathways such as Wnt and sonic hedgehog (SHH) pathways were upregulated in these tumors. Targeting these self-renewal pathways would aid in eliminating the resistant cells in this tumor and thus may help in treating the recurrence. Genes such as CBLB, MAPK 8, and ACVR1C can be used as potential biomarkers in this tumor to prognosticate cases with or without HRFs and differentiation of the tumors.