Vasculogenic Mimicry And Its Clinical Significance In Malignant Tumors Of Neuroepithelial Tissue

Neuroepithelial tumors are the most common intracranial tumor, generally account for more than half of the incidence of intracranial tumor. According to the2007WHO classification of tumors of the central nervous system, neuroepithelial tumors include astrocytic tumors, oligodendroglial tumors, mixed gliomas, ependymal tumors, choroid plexus tumors, glial tumors of uncertain origin, neuronal and mixed neuronal-glial tumors, neuroblastic tumors, pineal parenchymal tumors, embryonal tumors. The most important treatment of these tumors is to achieve maximal surgical resection and further supplemented by postoperative radiotherapy and chemotherapy and other comprehensive treatments. Our research targets in this thesis are two of the most malignant neuroepithelial tumors in central nervous system-medulloblastoma and glioblastoma.Glioblastoma is characterized by a high rate of incidence, difficult to total resection and high postoperative recurrence, short median survival time, high mortality rate. In recent decades, the diagnosis and treatment of malignant tumor has made significant progress, however, the treatment of patients with glioblastoma yield little effect, due to the rapid growth rate, extensive invasion and high heterogeneity nature of this tumor. Until now, patients prognosis is still very poor. The median survival time is only12to15months, with most cases die within two years and less than5%patients survive more than five years. Compared with glioblastoma, a few patients with medulloblastoma might get longer postoperative survival after comprehensive treatments, however, these treatments also resulted in chronic suffering for patients. Cognitive dysfunction and neurological problems were two of the most common complications. How to develop safe, effective and less side effects treatment, which prolong survival time while at the same time can improve the quality of survival, remains a huge challenge.As early as1971, Folkman put forward the theory that the growth and metastasis of tumor depends on angiogenesis. Angiogenesis has been shown to be chemotactically-driven. In response to an angiogenic stimulus, the endothelial cells will sprout from the existing vessels, grow towards the source of stimulus and thus forming new blood vessels. Angiogenesis has been considered to be the most important way for tumors to sustain growth. Without angiogenesis, tumors cannot grow beyond2-3mm3. Folkman further proposed that since tumor growth is dependent on sufficient blood supply, and the blood vessels are lined by endothelial cells, thus the specific killing tumor vascular endothelial cells might be able to deprive the blood supply of tumor and bear the chance of curing cancer. Based on this assumption, Researchers developed antitumor drugs targeted at tumor vascular endothelial cells, such as bevacizumab and sunitib et al. But, the efficacy of these drugs was far from people’s expectations. Researchers have been probing into this question, but there has been no satisfactory answer. Until the introduction of vasculogenic mimicry by Maniotis et al, that provided a reasonable explanation for this question. In1999, Maniotis et al defined the conception of vasculogenic mimicry for the first time. Based on the in-depth research on the vascular morphology of melanoma, they confirmed that highly malignant melanoma cells are genetically dysregulated and can be able to express the phenotypes of epithelial, endothelial and stem cells, and form the capillary-like structure which is independent of endothelail cells. Functionally, vasculogenic mimicry just as blood vessel, can transport blood, oxygen and nutrients to tumor cells, but morphologically, it has distinct difference to endothelial cell blood vessels, in that the cells lining in the inner wall of vasculogenic mimicry are tumor cells not endothelial cells. Vasculogenic mimicry enables the direct contact between tumor cells and blood circulation, through which a proportion of tumor cell can enter the blood circulation and metastasize to distant organs. It is known that anti-angiogenic drugs can destroy tumor blood vessels, but these drugs have no effect on vasculogenic mimicry. There is report revealed that treatment with some anti-angiogenic agents, which inhibited tumor perfusion and increase intratumoral hypoxia, had demonstrated increasing metastasis and inducing vasculogenic mimicry.In2004, Maniots and Folberg reviewed the research papers on vasculogenic mimicry and put forward two distinctive types of vasculogenic mimicry in malignant tumors. Vasculogenic mimicry of the patterned matrix type in no way resembles blood vessels morphologically:the channels are composed of a basement membrane, lined by tumor cells in their external superficies and no endothelial cells are found on their inner wall despite blood plasma and red blood cells flowing through the channels. In contrast, vasculogeni mimicry of the tubular type may be confused morphologically with endothelial cell-lined blood vessels. The inner surface of tubular type of vasculogenic mimicry is lined by tumor cells not endothelial cells, and endothelial cells were not identified within channels by light microscopy, by transmission electron microscopy, or by using an immunohistochemical panel of endothelial cell markers (Factor Ⅷ-related antigen, Ulex, CD31, CD34, and KDR [Flk-1]). Matrix proteins such as laminin, heparan sulfate proteoglycan, and collagens IV and VI have been identified in vasculogenic mimicry. The patterned matrix anastomoses with blood vessels, and systemically injected tracers co-localize to these patterns. Vasculogenic mimicry was based on the studies of melanoma, since then, it has been seen in several malignant tumor types such as breast cancer, liver cancer, glioma, ovarian cancer, melanoma, prostate cancer, and bidirectional differentiated malignant tumors. Studies in most malignant tumors have revealed that tumor cells with the capability of vasculogenic mimicry associate with high invasiveness and metastatic potential, and tumors with vasculogenic mimicry is a prognostic factor for poorer clinical outcome.In studies of vasculogenic mimicry in central nervous system tumor, there are reports revealed that vasculogenic mimicry exists in glioblastoma. In2005, Yue et al examined45astrocytomas by CD34-PAS dual staining, and confirmed patterned matrix type of vasculogenic mimicry in high grade astrocytoma. In2010, El Hallani et al analysed human glioblastoma tissues and found non-endothelial cell-lined blood vessels that were formed by tumor cells (vasculogenic mimicry of the tubular type). The clinical significance of vasculogenic mimicry in glioma has been studied in detail by Liu et al. After evaluating vasculogenic mimicry in gliomas of different grades, they concluded that gliomas with vasculogenic mimicry may identify patients with poor prognoses. However, it is particularly noteworthy that there was no significant difference in survival time between patients with vasculogenic mimicry-positive and Vasculogenic mimicry-negative glioblastomas. Their conclusions might be unconvincing because their study based on a group of21glioblastoma patients with only eight vasculogenic mimicry-positive cases. The association between vasculogenic mimicry and a poor prognosis in glioblastoma needs to be studied further in larger number of patients. In addition, there is no report about vasculogenic mimicry in medulloblastoma which is the most common pediatric malignant brain tumor.Based on the above research background, our research is about vasculogenic mimicry and its clinical significance in medulloblastoma and in glioblastoma. CD34-PAS dual staining was applied to identify vasculogenic mimicry in medulloblastoma and glioblastoma. Univariate analyses were performed to identify the prognostic variables for overall survival. Multivariate analyses were carried out to identify the independent prognostic factors for survival using the Cox regression hazard model. The survival analysis was accomplished using the Kaplan-Meier method and the differences between groups were compared using the log-rank test. Our results contain two relatively independent parts which share similar experimental methods.Chapter Ⅰ. Vasculogenic mimicry and its clinical significance in medulloblastomaObjective:To explore the existence of vasculogenic mimicry and its clinical significance in medulloblastoma.Method:A total of62paraffin-embedded medulloblastoma tissues between1999and2010were obtained from the Department of Pathology at Zhujiang Hospital of Southern Medical University. After exclusion, we enrolled41patients in this study. Detailed clinical and pathological data were collected for all samples. CD34-PAS dual staining, immunohistochemistry and microvessel density counting were performed. Statistical analyses were performed using SPSS13.0for Windows (SPSS Inc.). A p value of less than0.05was defined as statistically significant. The differential expression levels of VEGF, MMP-2, MMP-14, laminin5y2, EphA2in the vasculogenic mimicry-positive and vasculogenic mimicry-negative groups were determined by the Mann-Whitney test. Microvessel density counts in the vasculogenic mimicry-positive and vasculogenic mimicry-negative groups were also compared by the student’s t test. Associations between vasculogenic mimicry and clinical and pathological data were determined by the χ2test. The survival analysis was calculated by the Kaplan-Meier method and the differences between groups were compared using the log-rank test. Univariate analysis was performed to identify prognostic variables for overall survival. Multivariate analysis was performed to identify the independent prognostic factors for survival using the Cox regression hazard model. Data on surviving patients and patients without follow-up information were considered censored data in the analysis.Result:Vasculogenic mimicry was identified in9out of41(22%) medulloblastoma tissues. Immunohistochemical studies revealed that the presence of vasculogenic mimicry was associated with the expression of MMP-2(P=0.001), MMP-14(P=0.039), EphA2(P=0.036) and laminin5γ2(P=0.005). Tumor tissues with vasculogenic mimicry were associated with lower microvessel density (P=0.000), which was indirect evidence of the blood supply function of vasculogenic mimicry. Survival analysis and log-rank tests showed that patients with vasculogenic mimicry had shorter overall survival time than those without vasculogenic mimicry (P=0.000). Univariate analysis revealed that vasculogenic mimicry (P=0.001), metastasis (P=0.044) and KPS score (P=0.010) are prognostic variables. Multivariate analysis and the Cox proportional hazards model identified vasculogenic mimicry (P=0.000) and metastasis (P=0.019) as independent prognostic factors for overall survival.Conclusion:Our results confirmed the existence of vasculogenic mimicry in medulloblastoma for the first time and revealed that vasculogenic mimicry is a strong independent prognostic factor for survival in patients with medulloblastoma. Chapter II. Vasculogenic mimicry is a prognostic factor for postoperative survival in patients with glioblastomaObjective:To reevaluate the clinical significance of vasculogenic mimicry in glioblastoma.Method:A total of112paraffin-embedded glioblastoma tissues that were collected between2002and2009from the Department of Pathology at Zhujiang Hospital of Southern Medical University. Eighty-six eligiable patients were enrolled into this study. Patients were classified as vasculogenic mimicry-positive or vasculogenic mimicry-negative according to CD34-PAS staining. The associations between vasculogenic mimicry and the clinical characteristics of the patients were analyzed. Univariate and multivariate analyses were carried out to identify the independent prognostic factors for overall survival using the Cox regression hazard model. Survival times were estimated using the Kaplan-Meier method and compared using the log-rank test.Result:Of all86glioblastomas,23(26.7%) were found to have vasculogenic mimicry. The presence of vasculogenic mimicry in glioblastoma was not associated with gender (P=0.587), age (P=0.445), KPS score (P=0.681), hydrocephalus (P=0.908), tumor burden (P=0.223), microvessel density (P=0.808), tumor relapse(P=0.138), or the extent of tumor resection (P=0.241). The univariate and multivariate analyses revealed that vasculogenic mimicry is an independent prognostic factor for overall survival. The median survival time for patients with vasculogenic mimicry was11.17months compared with16.10months for those without vasculogenic mimicry (P=0.017). In addition to vasculogenic mimicry, an age of65years or older (P=0.000), a KPS of60or less (P=0.004), a large tumor burden (P=0.045) are significant prognostic factors for patient survival.Conclusion:Our data suggest that vasculogenic mimicry is an independent adverse prognostic factor in newly diagnosed glioblastoma, further prospective studies are needed to test this conclusion.