Regional and temporal differences in gene expression of LH(BETA)T(AG) retinoblastoma tumors

Retinoblastoma (RB) is the most common intraocular malignancy in children, affecting approximately 1 in 15,000, for an incidence of 250 to 300 new diagnoses a year in the United States.1–4 As treatment has progressed from external beam radiation therapy (EBRT) to chemoreduction combined with focal therapies, survival rates have climbed to 99% with a large percentage of children maintaining vision.5 Despite the significant advancements in treatment and survival, current chemotherapy regimens and focal therapies may result in complications. Children are subjected to toxic chemotherapeutic drugs for multiple cycles, resulting in considerable risk for systemic toxicities.6–8 Focal consolidation therapies also contribute to morbidity, depending on the intraocular tumor size and location.9 Finally, chemoreduction success varies depending on tumor classification, with more advanced eyes achieving tumor control in 47% to 83% of cases.10,11 As a result, a greater understanding of tumorigenesis is necessary to develop adjuvant therapies to potentially treat tumors that are unresponsive to current treatment protocols and to minimize local and systemic complications of treatment. The genetics of RB development have been studied, beginning with Knudson's “two hit” hypothesis.12 In RB development, mutations or epigenetic changes in both alleles of the RB1 gene lead to loss of retinoblastoma protein (pRB). pRB binds to E2F, which acts as a transcriptional regulator of the cell cycle. Loss of both RB1 alleles leads to susceptibility of retinal cells to formation of RB. It has been proposed that development of RB requires more than the two hits proposed by Knudson, with Corsen and Gallie13 reviewing the literature for evidence of further genetic changes necessary for tumor development. The paradigm of cancer treatment and understanding has shifted from solely targeting hyperproliferative tumor cells and associated oncogenes/tumor suppressor genes to also targeting cancer stromal tissue, which consists of complex multicellular interactions, termed the tumor microenvironment.14,15 This environment consists of a plethora of cell types, including endothelial cells, fibroblasts, and inflammatory cells,16 that, along with numerous growth factors and signaling molecules, contribute to tumorigenesis. Hypoxia has been strongly correlated with tumor growth, progression, resistance to therapy, and metastasis.17 It has been shown that through O2-sensitive pathways, hypoxia alters tumor cell behavior, resulting in an integrated response of tumor cells to the tumor microenvironment, leading to altered gene expression and tumor adaptation and survival. Alterations to hypoxia include signaling through the mammalian target of rapamycin (mTOR), hypoxia inducible factor (HIF), and the unfolded protein response (UPR). These responses lead to altered cellular metabolism, angiogenesis, and other cell survival mechanisms.18 The genetic changes associated with a tumor's adaptation to the microenvironment are prospective avenues for more specific and targeted therapy. In addition, an understanding of the timing of gene expression is fundamental in the optimal use of novel, multimodal adjuvant treatments. Hypoxia and angiogenesis have been shown to be significantly associated with tumor proliferation and metastasis. Our previous studies with the LHBETATAG mouse model of RB have shown that 20% to 26% of tumoral areas are hypoxic and that there is a spatial difference, with hypoxia primarily in the central and basal regions of the tumor.19 In addition, we have shown that there is a spatial distribution of blood vessel maturation, with mature blood vessels concentrated centrally, while immature neovessels radiate peripherally. As a result, we have proposed that RB tumors grow radially from the center, with the apex, anterior, and posterior margins serving as the leading edges.20 We hypothesize that there are regional and temporal differences in gene expression of murine RB tumors corresponding to areas of tumor heterogeneity and variations in tumor microenvironment. These variations may provide valuable information regarding specific genes and pathways that facilitate tumor growth, progression, and resistance to treatment. The purpose of our study was to evaluate these regional and temporal differences by using mRNA microarray analysis in a transgenic model of RB.