Research Of Biomarkers Guiding Anti-angiogenic Therapy Of High-grade Gliomas

BACKGROUNDGlioma has been regarded as the most common,highly proliferative and invasive brain tumour,accounting for about 60%of the tumors in the central nervous system.High-grade gliomas have the characteristics of refractory,easy local dissemination and easy recurrence,with poor prognosis and short survival time.For example,the median survival time of glioblastomas is(GBM,WHO IV)is 14.6-16 months,estimated 5-year survival rate is only 3.4%.The treatment of gliomas requires a combination of surgical excision,chemotherapy,radiotherapy and targeted therapy based on tumor histological and molecular characteristics.Molecular targeted therapy for gliomas is based on the design of molecular genetic signal transduction system for the occurrence and growth of gliomas.Target-specific blocking drugs can significantly improve the tumor-free survival of patients.Angiogenesis is a prominent feature of high-grade gliomas.Vascular endothelial growth factor(VEGF)is an attractive target of antiangiogenic therapy in glioblastomas.High levels of vascular endothelial growth factor A(VEGF-A)are in and around the tumor.High grade gliomas express large quantities of VEGF-A,which,through paracrine mechanism,promotes endothelial cell proliferation,survival and migration.And this leads to a propagation of disorganized vasculature with leaky blood brain barrier.The degree of VEGF-A overexpression was directly proportional to blood vessel density,tumor grade and overall outcome.Inhibition of VEGF of Bevacizumab prunes abnormal blood vessels and "normalizes" vasculature,which is proposed to improve tissue oxygenation and drug delivery.The re-establishment of blood-brain barrier induced by Bevacizumab also decreases vasogenic edema surrounding the tumor,often improving symptoms related to edema and mass effects,as well as reductions in corticosteroid use.In addition,shown in animal models,Bevacizumab is also thought to have direct anti-tumor activity and can alter glioblastoma cell migration against gliomas that express VEGF-A.Bevacizumab may also increase cell sensitization to cytotoxic agents,making it effective in combination with concurrent chemotherapy or radiation.Bevacizumab received approval from the US Food and Drug Adminsitration in 2009 based on promising trial results demonstrating improved response rates and 6-month progression-free survival.Bevacizumab treatment may be a salvage option for patients with diffusely infiltrating cerebellar gliomas exhibits rapid progression during standard treatment.Bevacizumab has increasingly been used in the treatment of recurrent glioblastoma.However,the effects of Bevacizumab are transient,and most patients'tumors progress after 6 months.An increased incidence of distant or diffuse disease upon recurrence,and suggestive of a transition to a more aggressive phenotype were noted.The mechanisms of Bevacizumab failure are likely complex.And glioblastoma is a highly infiltrative tumor and almost impossible to resect in its entirety,the majority of recurrences occur locally around the resection cavity.Tumors demonstrate adaptive upregulation of alternative angiogenic pathways.Future therapeutic approaches should target different angiogenic pathways,block tumor invasiveness.Preoperative differential diagnosis of gliomas(such as distinguishing between primary intracranial lymphoma)is a difficult clinical problem.Accurate diagnosis plays a decisive role in the formulation of treatment protocols and can prolong the survival time of patients.The "golden standard" of diagnosis is still histopathological biopsy,but there is a great obstacle in obtaining pathological specimens.It is particularly important to identify high sensitivity and specificity biomarkers for diagnosis.Exosomes are vesicles secreted by cells with bilayer lipid membranes.They are an important tool for the exchange of living substances between cells.They contain proteins and nucleic acids(mainly microRNAs),which can regulate physiological and pathological processes by altering gene regulatory networks or epigenetic recombination.MicroRNA is involved in many important biological processes,such as cell growth,differentiation,proliferation and apoptosis,through sequence-specific inhibition of negative regulation of gene expression by translating or degrading mRNA.MicroRNA is involved in the angiogenesis pathway,and its role in controlling angiogenesis is recognized as a promising therapeutic target for cancer and cardiovascular diseases.High throughput sequencing was used to screen circulating nucleic acid molecules in serum exosomes associated with tumor genesis and development,and to find circulating nucleic acid markers for diagnosis,treatment and prognosis prediction of intracranial malignancies.Part One:The new treatment target of VEGF signaling pathway in anti-angiogenesis and anti-lymphangiogenesis of gliomaObjective:Bevacizumab is primarily targeted at vascular endothelial growth factor-A(VEGF-A),and evaluation of validated biomarkers and combined treatment may make the patients benefit from treatment with VEGF inhibitors.In the first part of our study,the expression of angiogenesis and lymphangiogenesis related markers of tumor tissues from patients with brain malignant glioma,including VEGF-C,VEGF-D and VEGFR-3,were studied by histochemical staining.We want to provide a new treatment target for anti-angiogenesis and anti-lymphangiogenesis therapy of glioma.Methods:From June 2011 to February 2015,121 patients with gliomas were hospitalized in the Department of Neurosurgery,the Second Hospital of Shandong University and Qilu Hospital of Shandong University.The expression of angiogenesis and lymphangiogenesis related markers,such as VEGF-C,VEGF-D,VEGFR-3 and D2-40,were studied by histochemical staining in glioma tissues and normal controls.Nonparametric Spearman correlation analysis was used to analyze the relationship between pathological grade and the expression levels of D2-40,VEGF-C,VEGF-D,VEGFR-3,p53 and Ki67.Nonparametric Spearman correlation analysis assessed the relationship between disease-free survival time and the expression of different markers after the first operation.The expression levels of newly diagnosed and relapsed markers were analyzed by Wilcoxon matched-pairs signed rank.Cox proportional hazards regression model was used to analyze the prognostic factors of glioma.Results:Firstly,101 patients with glioma were studied.The median age was 40.5 years.The most common clinical symptoms were headache,vomiting,limb paralysis,epilepsy and coma.The average size of the tumor was 4.1 ±1.6cm.Of the 101 patients,there were 18 cases of WHO grade I,25 cases of II grade,24 cases of III grade and 34 cases of IV grade.P53 was significantly associated with Ki67(P=0.0005).The age of different WHO grades patients was also significantly different(P=0.0005).D2-40-positive staining was observed in the cytoplasm,cytoplasmic expression of D2-40 was observed in 28 of 101 glioma(27.7%).VEGF-C and-D expression was found on endothelial cells as well as on numerous cells distant from any vessels.VEGF-C-positive staining was mostly observed in the cytoplasm or/and weakly in the nucleus,expression of VEGF-C was observed in 35 of 101 glioma(34.7%).VEGF-D expression was observed in the cytoplasm or/and in the nucleus,expression of VEGF-D was observed in 88 of 101 glioma(87.1%).And VEGFR-3-positive tumor cells were observed in the cytoplasm and nucleus,expression of VEGFR-3 was observed in 25 of 101 glioma(24.7%).Expression of D2-40 and VEGFR3 significantly correlated with glioma tumor grade,with grade IV glioma showing higher expression of D2-40(P =0.047)and VEGFR3(P =0.040).However,tendencies for increase with tumor grade were not observed for VEGF-C and VEGF-D(P=0.3784,P?0.08096).There were significantly differences among different grades in P53 and Ki67 expression in glioma(P<0.0001,P<0.0001).A total of 40 surgical specimens(20 primary and 20 recurrent specimens)from 20 recurrent glioma patients(8 males and 12 females)were also studied.The median age of patients at diagnosis was 43.5 years old,and the mean tumor-free survival was 1.65±1.34 years.The most common clinical presentation was headache,limb paralysis,and epilepsy.The mean tumor size was 4.3±1.7cm.There were 1 case of grade I,6 cases of grade II,9 cases of grade III,and 4 cases of grade IV at diagnosis but 2 cases of grade II,2 cases of grade III,and 16 cases of grade IV at relapse.At relapse malignant transformation rate was 65%(13/20 cases).IHC showed that D2-40,VEGF-C,VEGF-D and VEGFR-3 was expressed in 20%,30%,60%and 20%of primary and 45%,30%,75%and 35%of recurrent glioma tumors(P<0.01,P=1.00,P=0.03,P=0.03).The first disease free survival time was not related to the expressions of D2-40(P=0.78),VEGF-C(P=0.64),VEGF-D(P=0.11),VEGFR-3(P=0.31),Ki67(P=0.69)and p53(P=0.35).There were 6 cases with negative staining for all lymphangiogenesis markers(all of D2-40,VEGF-C,VEGF-D and VEGFR-3).There was no significant difference of the first disease free survival time between them and others(P=1.665).Moreover,the pathological grade was significantly related to the positive stainings for D2-40(P=0.0060),VEGF-D(P=0.017),and VEGFR-3(P=0.032),and to the scores of Ki67(P<0.001)and p53(P=0.003),while was not significantly related to VEGF-C expression(P = 0.059).In 13 cases with increased malignancy,the expression levels of Ki67,p53,D2-40,VEGF-C,VEGF-D and VEGFR-3 were increased at relapse.Ki67 and p53 scores were significantly higher at relapse compared with primary tumor(P=0.001,10(5?27.5)%VS 40(35?55)%for Ki67;P=0.045,10(0?25)%VS 25(17.5?37.5)%for p53).There was significant difference in the D2-40 and VEGF-D expression between recurrent and primary tumors(P = 0.031,P = 0.047),but there is no significant difference in the VEGF-C and VEGFR-3 expression between recurrent and primary tumors(P = 0.094 and P = 0.25).A multivariate survival analysis,including expression of D2-40,VEGF-C,VEGF-D,VEGFR-3,Ki67,p53,primary tumor grade,and relapse tumor grade,showed that VEGF-D was an independent prognostic factor for malignant transformation(HR=0.376,P=0.045).Conclusion:The expression of D2-40,VEGD-D and VEGFR3 is high in malignant gliomas.The recurrence of gliomas is often accompanied by the progression of WHO grade.VEGF-D is an independent prognostic factor for gliomas.It is expected to become a new target for the treatment of glioma.Part Two:Diagnostic markers and therapeutic targets for intracranial lymphoma and glioma from serum exosomes based on high-throughput sequencingObjective:At present,there is no effective and noninvasive method for differential diagnosis between malignant glioma and intracranial lymphoma.In this study,we intend to screen microRNA markers in serum exosomes by high-throughput sequencing technique and want to find differential diagnosis biomarkers of distinguishing malignant glioma and intracranial lymphoma.And we also want to find new targets for angiogenesis therapy of glioma and intracranial lymphoma.Methods:The HiSeq/MiSeq high-throughput sequencing technique was used to determine the expression profiles of microRNA in serum exosomes of patients with intracranial lymphoma,malignant glioma and healthy controls.Serum exosomes were isolated from 12 patients with intracranial lymphoma,32 patients with malignant glioma and 20 healthy controls.The expression of candidate microRNA was verified by RT-q PCR.Small sample size was used to evaluate the diagnostic value of candidate microRNA for intracranial lymphoma and malignant glioma.Then,the candidate microRNAs involved in angiogenesis and lymphangiogenesis were selected from the serum exosome microRNA expression profiles of patients with intracranial lymphoma and malignant glioma,and the expression of the candidate microRNAs was verified by RT-qPCR.Kolmogorov-Smirnov was used to detect the distribution of data,and Mann-Whitney U test or Kruskal-Wallis test was used to detect the difference of microRNA expression between groups.The diagnostic value was analyzed by using the worker's curve.Results:There were 100 differentially expressed microRNAs between malignant glioma serum exosomes and normal controls serum exosomes,186 differentially expressed microRNAs between intracranial lymphoma serum exosomes and normal controls serum exosomes,140 differentially expressed microRNAs between intracranial lymphoma serum exosomes and malignant glioma serum exosomes.The difference of microRNAs between malignant glioma serum exosomes and normal control serum exosomes and the difference of microRNAs between malignant glioma serum exosomes and normal control serum exosomes were statistically analyzed.The number of microRNAs of malignant glioma and lymphoma distinguishing from normal control in serum exosomes was 44.Mi-20b-5p,Mi-96-5p,Mi-550a-3-5p and Mi-550a-5p were found up-regulated,and Mi-203a-3p,Mi-203b-5p,Mi-1255a,Mi-1296-5p,Mi-6516-5p and Mi-376b-5p were down-regulated.The number of microRNAs specific to gliomas found in serum exosomes was 56,including microRNAs-375,microRNAs-532-3p,microRNAs-30c-1-3p,microRNAs-23b-5p,microRNAs-330-5p and microRNAs-3074-3p.Cluster analysis showed that there were 56 specific microRNAs in serum exosomes of patients with malignant glioma compared with those of patients with intracranial lymphoma and normal controls.KEGG enrichment analysis revealed 20 significant pathways involved in glioma regulation.It involves Focal adhesion,Proteoglycans in cancer,Pathways in cancer and other pathways related to physiological and pathological angiogenesis.MiR-376b-5p validates its potential as a common anti-angiogenesis target for intracranial lymphoma and malignant glioma.In 12 patients with intracranial lymphoma,32 patients with malignant glioma and 20 healthy controls,the levels of miR-532-3p in serum exosomes were down-regulated in intracranial lymphoma(P = 0.02),up-regulated in malignant glioma(P<0.001),and Mi-376b-5p was down-regulated in lymphoma and glioma(P = 0.02,P = 0.04).The AUC value of ROC curve of miR-532-3p for glioma was 0.8203(P<0.001).Conclusion:MiR-532-3p can be used as a marker for the distinguishing diagnosis of malignant glioma and intracranial lymphoma.Mi-376b-5p may be a new target for anti-angiogenesis therapy of intracranial lymphoma and malignant glioma.