Study On Gene Sequencing,Clinical Features And Immune Microenvironment Of Craniopharyngioma

Part 1 Exon Sequencing and Bioinformatics Analysis of CraniopharyngiomaObjective: Craniopharyngioma is a common tumor in the saddle area of children.Although it is a benign tumor,it has complicated clinical manifestations and is highly easy to relapse.At present,due to the lack of mature tumor cell lines and the difficulty in the culture of primary tumor cells,the study on the mechanism of cell and molecular functions in craniopharyngioma in vitro is impossible.The second-generation full exon sequencing technology and bioinformatics analysis were used to search for the differentially expressed genes and molecules in craniopharyngioma with high frequency,followed by the exploration of its molecular biological characteristics and pathogenesis.Methods: Blood and tumor tissue samples from 32 patients with craniopharyngioma were collected.After extracting tissue DNA,a database was established and full exon sequencing was performed.Meanwhile,bioinformatics analysis was performed to screen differentially expressed genes and molecules in craniopharyngioma.Results: Among the 26 patients with adamantinomatous craniopharyngioma,18 patients carried CTNNB1 mutation,all of which appeared on CHr3,and there were 8 types of mutation base changes.Among them,CC,CC and C were replaced by GG,GA and T in the most cases,including 4 males and 1 female,with a total of 5 patients.BRAF mutation was carried in 5 cases of squamous papillary craniopharyngioma,and 1 type of mutated base change was AA,AA and A replaced by TT,TT and T.The driving genes were predicted to be CTNNB1 and MYO15 A.Gene co-expression network analysis revealed that CTNNB1 and NOTCH2 genes covered 84.38% of the samples.CTNNB1,OR2T3 and SPDYE1 are high frequency pathogenic genes of craniopharyngioma.Conclusions: CTNNB1 and BRAF are high frequency differentially expressed genes of adamantinomatous and squamous papillary craniopharyngioma.MYO15 A,OR2T3,SPDYE1 and MY015 A should be further verified in tumor specimens of craniopharyngioma.Part 2 Study of Correlation between molecular biomarkers and clinical features of craniopharyngiomaObjective: Through the second-generation full exon sequencing technology and bioinformatics analysis of 32 craniopharyngioma samples,we found that adamantinomatous and papillary craniopharyngioma mainly carried CTNNB1 and BRAF gene mutations,respectively.CTNNB1 and BRAF mutations have become new molecular diagnostic markers for adamantinomatous and papillary craniopharyngiomas.This study investigated the correlation between molecular biomarkers and clinical features of craniopharyngioma.Methods: The whole exon second generation sequencing and analysis were performed in 32 patients with craniopharyngioma.According to the sequencing results,the patients were divided into CTNNB1 mutant group,CTNNB1 wild type group,BRAF mutant group and BRAF wild type group,and the relationship between the characteristics and clinical characteristics of each group was evaluated.Results: BRAF mutations do not occur in cystic component-based craniopharyngioma.CTNNB1 wild-type tumors have no calcification,and calcified tumors do not cause BRAF mutations.Patients with CTNNB1 mutations were mostly without overweight.CTNNB1 mutant patients and CTNNB1 wild-type patients have statistically significant differences in cholesterol and low-density lipoprotein.CTNNB1 wild-type patients have higher cholesterol and low-density lipoprotein levels,and are more prone to hyperlipidemia.CTNNB1 wild-type patients had higher cholesterol and low-density lipoprotein levels,and were more prone to hyperlipidemia.CTNNB1 encodes ?-catenin and BRAF encoded BRAF V600 E proteins are all expressed in adamantinomatous and papillary craniopharyngiomas.Conclusions: Each of the craniopharyngioma carrying different molecular markers has some specific clinical features.Understanding the clinical features of these types of tumors provides some clues for non-invasive diagnosis and treatment.Detailed follow-up data should be included in the subsequent correlation study.There is a difference in the level of gene sequencing and the level of its encoded protein.Epigenetics may also be a causative factor in craniopharyngioma.Part 3 Immune microenvironment of primary and recurrent craniopharyngiomas: A study of the differences and clinical significanceObjective: This study explored the differences between the immune microenvironments of primary and recurrent craniopharyngiomas(CPs).In addition,we investigated the relationship between the immune microenvironment and clinical characteristics of CP.Methods: We collected 52 specimens from 26 patients with CPs.For each patient,specimens for both primary and recurrent CP were obtained.We performed an immunohistochemical analysis of these specimens to determine the distributions of M2 macrophages,CD8+ T cells,programmed cell death 1 ligand 1(PD-L1),and Ki67.Results: In recurrent CP specimens,the distributions of M2 macrophages,Ki67,and PD-L1 increased compared with primary CP specimens(P=0.019,P=0.0084,and P=0.0319,respectively).Moreover,the distributions of M2 macrophages,CD8+T cells,and PD-L1 in papillary craniopharyngiomas(PCPs)were higher than those observed in adamantinomatous craniopharyngiomas(ACPs)(P=0.0317,P=0.0359,and P< 0.0001,respectively).In the adult ACP group,M2 macrophages,CD8+T cells,and PD-L1 were more abundant/expressed than in the child ACP group(P=0.0159,P=0.0215,and P< 0.0088,respectively).A positive correlation was found between M2 macrophages and CD8+T cells(r=0.4079 P=0.0027).Correspondingly,M2 macrophages and CD8+T cells were both positively correlated with PD-L1(r=0.4564 P=0.0007 and r=0.3987 P=0.0034,respectively).The observed high-expression of M2 macrophages in primary CPs suggests a shortened time for tumor recurrence(P=0.0131).Conclusions: The microenvironment of recurrent CP varies from that of primary CP.The abundance of M2 macrophages in primary CP may indicate a risk of early recurrence.Therefore,it is recommended to increase the frequency of follow-up examinations in these patients.