Clinicohistopathological implications of MMP/VEGF expression in retinoblastoma: a combined meta-analysis and bioinformatics analysis

Retinoblastoma is an extremely rare cancer that rapidly develops from immature retina cells, the light-detecting portion of the eye [1]. It is the most common pediatric malignant cancer of the eye and can easily lead to the loss of either sight or the eyeball and even death during early childhood [2]. The incidence and degree of malignancy of retinoblastoma are higher in developing countries than those in developed countries; moreover, cure rates remain unsatisfactory in developing countries, with relatively high morbidity and mortality, lower rates of eye salvage and higher rates of extraocular dissemination [3‐5]. Survival rates in high-income countries can be greater than 95%, while the global survival rate is less than 30% [2]. Poor outcomes in retinoblastoma patients from developing countries are a concern worldwide.

The therapeutic options for retinoblastoma have undergone sweeping changes over the years; these mainly include chemotherapy, focal or consolidation therapy (e.g., laser photocoagulation, cryotherapy, thermotherapy and plaque brachytherapy), external beam radiotherapy and enucleation [6, 7]. While enucleation is progressively being supplanted by globe-sparing treatments to save the eyeball, this strategy may lead to compromised survival rates, as conservative treatments may not be as effective as enucleation for preventing tumor metastasis for potentially high-risk eyes. Therefore, the choice of conservative treatments with fewer side effects or aggressive therapies with greater survival rates is highly dependent on the assessment of the extent of intraocular disease and the extraocular tumor spread [7, 8]. However, in-depth systematic evidence available for estimating outcomes and eligibility for subsequent treatments is limited in retinoblastoma. Under these circumstances, it is crucial to identify biomarkers that predict retinoblastoma development, progression and clinical outcomes and can help determine the optimal treatment in the delicate balance between preserving the eyeball, vision and life.

Multiple markers have been suggested to be associated with the clinical features of retinoblastoma [9]. However, our preliminary search suggested that matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF) have been studied most. MMPs and VEGF regulate extracellular matrix (ECM) remodeling and microvascular permeability during angiogenesis [10‐12]. The MMP family, a zinc-dependent endopeptidase family, regulates the degradation of the ECM and basal membrane [13]. Additionally, clinical studies have identified MMPs as prognostic markers and therapeutic targets in multiple types of cancer [14, 15]. VEGF is the most thoroughly and widely explored proangiogenic factor in tumors, including retinoblastoma, a heavily vascularized tumor [16‐18]. Activated MMPs and VEGF are suggested to facilitate malignant cell growth, invasion, metastasis, angiogenesis and even chemoresistance in retinoblastoma [18‐21]. The importance of these genes in retinoblastoma highlights their importance as candidates in our research. Moreover, MMPs and VEGF are closely related in oncogenesis and tumor progression, which increases our interest in their correlation [22‐24]. For example, MMP-9 and MMP-2 inhibition may reduce VEGF expression and, thus, angiogenesis in retinoblastoma cell lines [25, 26]. However, whether MMPs and VEGF are coexpressed in retinoblastoma how they interact in pathways related to tumorigenesis and metastatic spread remain unknown.

Although remarkable progress in retinoblastoma therapies has been achieved, identification of systematic evidence for basing therapy recommendations or consensus has not progressed to the same extent. To the best of our knowledge, no systematic reviews or meta-analyses have investigated this issue. A scattering of single-center experimental studies exists, but the inadequate sample sizes of single studies reduce their credibility. Therefore, we conducted a meta-analysis to evaluate the significance of the MMPs/VEGF in predicting clinicopathological characteristics, thus providing comprehensive evidence for a better understanding of retinoblastoma biology, predicting prognosis and developing novel targeted therapies.

Retinoblastoma is a curable intraocular tumor of the pediatric population [1, 54]. However, due to relatively late detection and primitive medical care, retinoblastoma appears to be a serious vision- and life-threatening disease in developing countries. Management of retinoblastoma depends on cooperative, multidisciplinary efforts and comprehensive surveillance and evaluation of the tumor [6, 55]. Unfortunately, we have lagged behind in providing systematic evidence on which to base the choice of treatment; nevertheless, great progress has been achieved in therapeutic methods for retinoblastoma. Thus, our systematic review and meta-analysis sought to contribute missing data examining tumor invasion, differentiation and clinical stages in the hope of more precisely predicting the long-term outcomes and determining individualized therapies for retinoblastoma.

In this meta-analysis, we assessed the clinicopathological significance of MMPs and VEGF in retinoblastoma and detected a potential relationship between MMP-9 and VEGF expression. The results confirm that MMP-1, MMP-2, MMP-9, and VEGF overexpression is highly related to poor retinoblastoma differentiation and tumor invasion. Moreover, MMP-9 overexpression is associated with tumor stage, regardless of the gender of the patients. Except for MMP-1, the lower confidence intervals (LCIs) of the ORs are all greater than 3, which implied intensely significant correlations. The findings suggest that these genes participate in tumorigenesis and metastasis, which is in agreement with previous experimental studies. However, according to the study by Nermeen, overexpression of VEGF may be irrelevant to poor differentiation [9], whereas our study of a Chinese population reported the opposite result. This contradiction implies that VEGF may play distinct roles in retinoblastoma oncogenesis among different races.

Multiple measures, including quality assessment, public bias evaluation, and sensitivity and heterogeneity analyses have been adopted to control potential biases while exploring the main outcomes. The quality of studies included in this analysis was acceptable, as their NOS scores were all greater than 6. Regarding publication bias, the funnel plots display asymmetry between certain groups, probably due to language and ethic confinement in enrolled studies. Additionally, it is possible that MMP/VEGF expression in progressed retinoblastoma has been previously reported in other studies, leading more researchers to validate the findings in their specific patient populations. The limited number of trials deters us from further investigation. Aside from conventional meta-analysis, our study employs a novel QE model in addition to the FE and RE models to pool the effects. The QE model is an updated estimator for meta-analyses and has the advantage of a decreased mean squared error and reduced observed variance compared with the RE model. Regardless of the level of heterogeneity, the QE model maintains the correct coverage probability of the confidence interval [56]. With three different models to weight the average, our results should be more convincing. Certain included studies introduced a high level of heterogeneity across studies, such as Nermeen’s study as shown in Additional file 2: Figure S2D, Mohan’s study as shown in Additional file 3: Figure S3B and Lin Zhou’s study as shown in Additional file 3: Figure S3C, but their removal did not lead to changed conclusions, implying that the results are relatively stable.

Although prognostic markers are widely investigated and applied in other malignant tumors, such as lung cancer, they have remained undetermined in retinoblastoma [57, 58]. Possible reasons include an insufficient number of samples due to the rarity of the disease and cautious application of biopsy. Fearing tumor dissemination and extraocular metastasis development, the diagnosis and grading of retinoblastoma using biopsy are usually discouraged [59]. However, several biopsy techniques, such as fine needle aspiration cytology and lipid biopsy (using the aqueous humor as a surrogate biopsy material for retinoblastoma DNA), have emerged as safe and effective methods [59, 60]. Nevertheless, long-term follow-up in a larger cohort of patients is needed to validate the results and provide safety data. To the best of our knowledge, our paper is the first meta-analysis to provide a series of markers for predicting retinoblastoma prognosis. These oncogenes contribute to a more accurate estimation of clinical outcomes and provide early indications for possible retinoblastoma metastasis. Patients with high protein expression are considered to be at high-risk and potential candidates for aggressive treatment. In addition, these proteins may help monitor patients’ responses to therapy and facilitate further treatments.

Major advances have been made in small molecule therapeutics. Targets such as inhibitors of the MDMX-p53 response, histone deacetylase inhibitors, and spleen tyrosine kinase inhibitors have shown promise in the treatment of retinoblastoma, with minimal off-target effects in animal models or clinic trials [61, 62]. Our study supplements MMPs and VEGF as promising druggable molecular targets for retinoblastoma treatment. Retinoblastoma exhibits invasive behavior to adjacent tissues and the blood stream at the early stage of the tumor, which emphasizes the urgency of preventing retinoblastoma metastasis at the early stage [4, 63]. However, although current therapy focuses on blocking tumor cell division and tumor growth, specific treatments targeted to prevent retinoblastoma metastasis and invasion are unavailable. Our results may pioneer the first indication for targets that interfere with tumor spread. Additionally, molecular suppression or silencing of these targets has the advantage of superior selectivity and lower systemic toxicities compared to chemotherapeutic agents. The use of an anti-VEGF antibody in combination with chemotherapy may enhance the efficacy of chemotherapy toward retinoblastoma [64, 65], despite a distinct toxicity profile in use for clinical trials. Whether a VEGF inhibitor will work effectively in humans and not only in nonhuman or human tumor sample trials needs to be validated as well.

We have demonstrated prognostic concordance among targeted gene expression signatures. Next, we investigated how the various genes are related to one another. Therefore, we further investigated the associations among gene targets. Regrettably, due to a lack of original studies, we were only able to show that MMP-9 and VEGF are coexpressed. Nonetheless, this represents a novel piece of system-level evidence elucidating the cellular pathways and biological processes regulating retinoblastoma phenotype, progression and prognostic performance. The identification of a synchronized overexpression pattern between MMP-9 and VEGF may generate many insights into tumor biology and pathogenesis. Notably, the degree of overlap among regulatory programs greatly influences the degree of coexpression [66]. According to previous studies, MMP-9 and VEGF are indeed closely related on the cellular level. MMP-9 plays an essential role in the acquisition of an angiogenic phenotype by assisting in VEGF liberation from the ECM and is involved in VEGF–VEGF-receptor interactions [23, 67, 68]. VEGF can also induce MMP-9 expression through pathways such as the Ras-activated extracellular signal-regulated kinase (ERK) pathway [24, 69]. In addition to positive feedback regulation, MMP-9 and VEGF also share a generous number of upstream pathways. Although we confirmed that MMP-9 and VEGF are coexpressed in retinoblastoma, further exploration is needed to identify the biological role of their interaction.

Last but not least, the GO functional annotation and KEGG pathway analysis of the four target genes provides additional information about their roles in retinoblastoma, indicating that they play important roles in multiple cellular components, thus promoting tumorigenesis and metastasis, which prompt potential directions for further investigations.

This study has some inherent limitations that should be highlighted. First, although all studies utilized IHC, the antibodies used are different, and the threshold value is inconsistent among trials. Thus, despite the implementation of a standard regimen to avoid divergence, heterogeneity from these factors is inevitable. Ethnic groups also vary, but subgroup analysis of different ethnic groups is not feasible due to the limited number of trails. Second, prognostic indexes, such as overall survival and disease-free survival, were not end points for most included studies, which restricts our study to an analysis of clinicohistopathological characteristics. Life-long follow-up should be performed in subsequent studies, as retinoblastoma survivors are at high risk of developing secondary cancers [70, 71]. Third, our search was restricted to studies published in English or Chinese, which may be responsible for the publication bias. Our results are more applicable to certain ethnic groups; as most relevant reports were conducted in Chinese populations. Outcomes in developing countries are relatively unsatisfactory [2], so these studies may add additional bias. Fourth, tumor invasion data in most of the included studies were related to optic nerve invasion except Mohan’s report, which included the choroid, optic nerve and orbit [41]. Optic nerve invasion is emphasized mainly because it is the most common route for retinoblastoma to progress into the brain and then further metastasize [52]. However, excluding other types of invasion may reduce the available information in the early detection of retinoblastoma progression. Finally, analyses of MMP-1 and MMP-2 expression are based on only 3 studies. Corresponding results based on small samples should be interpreted with caution.

We concluded that the overexpression of MMP-1, MMP-2, MMP-9 and VEGF is highly related to poor retinoblastoma differentiation, tumor invasion and advanced clinical stage and, thus, have a role in predicting the prognosis of retinoblastoma patients. Meanwhile, MMP-9 and VEGF proteins exhibit a system-level coexpression pattern in retinoblastoma, indicating their potentially important biological relationships in tumor development and progression.