Expression Of CD105 In Breast Foliar Tumors And Application Of Exon Sequencing

Background and objective:Phyllodes tumor(PT)is a fibroepithelial tumor of the breast 1,which is more likely to occur in middle-aged women(35～50 years old).According to its histological and biological characteristics,it is divided into three types:benign PT,borderline PT and malignant PT 2.Benign PTs are hard to be detected 3,while borderline and malignant PTs grow rapidly.Benign,borderline and malignant PTs are prone to relapse.After relapse,the biological behavior of the tumor is worse and the histological grade is higher 4.Malignant PT is highly aggressive and likely to distant metastases,mostly metastasizing to the lung,bone and brain.The overall survival time of the patients with distant metastasis will be significantly shortened 5.The subjectivity in the diagnosis of PT is high,and the repeatability of the diagnostic results is not very high.At present,biological markers about the invasion and metastasis of PT have not been reported.Therefore,an effective immuno-histochemical marker is urgently needed for auxiliary diagnosis of the disease,which is also the content in our study.The formation of neovascularization in the tumor is closely related to its invasion,infiltration and metastasis.The proliferation of tumor microvessels can be expressed by microvascular density.Microvascular density is determined by selectively marking related components of endothelial cells using immunohistochemical staining,which is the gold standard for quantitative analysis of neovascularization in tumors.CD 105 can be used to mark the neovascular endothelium,showing higher specificity and sensitivity than CD31 and CD34.CD 105 can be expressed not only in neovascular endothelial cells,but also in parenchymal cells of tumors.It can also be expressed in interstitial cells of PT.Many tumors present intratumoral heterogeneity.Poorly differentiated tumors are characterized by poor biological behavior,strong invasiveness,and susceptible local recurrence and distant metastasis and poor prognosis.The instability of gene phenotypes is the main factor leading to intratumoral heterogeneity 6.This study aims to explore the relationship of microvascular density and CD 105 expression with the histological grade of PT,and intratumoral heterogeneity of PT.Research methods:1.Data of 54 patients with PT were collected,including clinicopathological data,sections for pathological diagnosis and paraffin-embedded tissue specimens.The relationship between clinicopathological parameters and the histological grade of PT was analyzed by the statistics of clinical data.CD 105 in 54 cases of PT specimens was stained using immunohistochemical staining.The relationships of microvascular density and CD 105 expression in interstitial cells with the histological grade of PT were observed.The intratumoral heterogeneity of PT was analyzed by microscopic observation of histology and CD 105 expression as well as case reports.2.Samples of 10 cases of PT collected were selected to carry out exon sequencing.Mainly consists of paraffin tissue DNA extraction,using high-throughput exon sequencing method to highly sequence the extracted DNA samples;analyzing the gDNA extraction,matching validation,establishing the DNA library,enrichment of exon,superior sequencing and bioinformatics analysis,and according to the biological function and mutation frequency factors,furtherly summarize and elect the interesting candidate genes.Results:1.The microvessel density of benign phyllodes tumors was 18.1,the microvascular density of borderline phyllodes tumors was 48.1,and the microvessel density of malignant phyllodes tumors was 51.4.The microvessel density of borderline and malignant phyllodes tumors was significantly higher than that of benign phyllodes tumors.2.In 11 cases of borderline phyllodes tumors and 10 cases of malignant phyllodes tumors,all of the stromal cells had different levels of CD 105 expression.However,in 33 cases of benign phyllodes tumors,only 8 cases showed CD 105 expression,and The intensity is weaker.3.Among the 54 phyllodes tumors,there were 3 cases of phylldes tumors with obvious histological differentiation.The majority of tumors showed benign differentiation,and a small part of the tumor showed borderline or malignant differentiation.4.Using exome sequencing technology,according to the experimental group VS.Control group,found in 5 groups of comparison,respectively 10,17,12,15,8 genes meet the requirements,each group has a number of genes mutations occur.5.From the point of view of tumor heterogeneity,the following genes were screened for:PSMG1,FOSL2,PHGR1,CGB2,and FRG1 by comparison of 1VS.1 and 5VS.5,and selection of high-frequency mutation gene sets and tumor-driven gene sets.Conclusion:1.The higher the histological grade of phyllodes tumors,the higher the microvessel density.2.The higher the histological grade of phyllodes tumors,the more pronounced the expression of CD 105.3.There is intratumoral heterogeneity in phyllodes tumors,and CD 105 is focally expressed in stromal cells.Within the same tumor,expression is evident in high histological grades.4.Through exome sequencing technology,it was found that in the same tumor,poorly differentiated areas(experimental group)had gene mutations compared to well-differentiated areas(control group),indicating that gene changes are closely related to tumor heterogeneity.5.the genes selected have contributed to a certain degree of heterogeneity in phyllodes tumor,which is further confirmed by the expanded sample size at the later stage.