The Expression Of FLNA,YAP1 And β-Catenin In Breast Phyllodes Tumors

Background and Purpose:Phyllodes tumor(PT)is a rare fibroepithelial tumor of the breast,accounting for about 0.3-1.0%of breast tumors.The disease mainly occurs in middle-aged women,with an average age of 37.2 years.The incidence of Asian women is higher than that of most European and American countries,which may be related to geography and race.According to the distribution and density of interstitial fibroblasts,nuclear mitosis count and cell heterogeneity,they were divided into benign,borderline and malignant phyllodes tumors.In recent years,the incidence of breast phyllodes tumors has increased year by year,and the age of onset has also tended to be younger.The size of tumors ranging from 1cm to 40cm has been reported.Benign phyllodes tumors grow slowly and the onset is not easy to be found.The history can be several years or even decades,but it can also increase rapidly.Borderline and malignant phyllodes tumors grow rapidly to a larger volume.The main treatment is surgical treatment,which is more likely to relapse than Fibroadenoma(FA).The biological behavior of the tumor after recurrence is worse and the histological grade is higher.Malignant phyllodes tumors are prone to distant metastasis,and the risk of distant metastasis is as high as 22%.Lung is the most common metastatic organ.At present,the diagnosis of phyllodes tumors is mainly based on histological grading,and there are no reliable molecular markers for differential diagnosis.We first collected paraffin specimens from patients with malignant,borderline,benign phyllodes tumors and fibroadenoma of the breast,and performed whole exome sequencing(WES)and comparison of mutant differential genes.We found that changes in FLNA,MED 12,KMT2D,KMT2C,RARA,SETD2 and other genes were associated with the occurrence and development of phyllodes tumors.Abnormal expression of FLNA(filamin A)protein plays an important role in the progression of breast cancer,renal cell carcinoma,lung cancer,ovarian cancer and other tumors.Recent studies have reported that FLNA can promote the vascularization of mesenchymal cells by binding F-actin and activating the F-actin/YAP1/β-catenin signaling pathway axis.Therefore,we speculate that FLNA gene mutation and FLNA/YAP1/β-catenin activation may play an important role in the occurrence and development of phyllodes tumors.In order to verify the above theoretical speculation,we detected the expression of FLNA in breast phyllodes tumors and fibroadenomas by immunohistochemistry(IHC),and explored the application value of FLNA as a diagnostic marker for breast phyllodes tumors.At the same time,the relationship between FLNA and YAP1 and βcatenin protein expression was detected,and the role of FLNA/YAP1/β-catenin signaling pathway in the occurrence and development of phyllodes tumors was further analyzed.Research Methods:1.Collected 18 cases of benign,19 cases of borderline,8 cases of malignant phyllodes tumors,and 16 cases of fibroadenoma as controls,performed WES detection and differential gene comparison,and analyzed FLNA gene mutation frequency and mutation sites in different pathological morphological groups.2.FLNA/YAP1/β-catenin signaling pathway protein detection.54 cases of breast phyllodes tumors(including 28 cases of benign,16 cases of borderline,10 cases of malignant)and 19 cases of fibroadenoma were collected.The clinicopathological data were improved,including age,tumor size and recurrence.The relationship between clinicopathological data and histological grade of phyllodes tumor was analyzed.The expression levels of FLNA,YAP1 and β-catenin in 73 cases of fibroepithelial tumors were detected by IHC.The expression of the above three proteins in different groups of tumors was analyzed,and the correlation between the three was studied.And the relationship between phyllodes tumor occurrence and malignant progression.Results:1.The mutation of FLNA gene in breast phyllodes tumor and fibroadenoma.Whole exome sequencing of breast phyllodes tumors and fibroadenomas revealed multiple high-frequency mutant genes including FLNA,MED 12,KMT2D,KMT2C,RARA,and SETD2.The mutation rate of FLNA exon mutation in the four tumors was 18.7%in fibroadenomas,14.3%in benign phyllodes tumors,22.7%in borderline phyllodes tumors,and 25.0%in malignant phyllodes tumors.Nine mutation sites of FLNA gene were found in the four groups of samples,among which c.1582 G>A was the common mutation site in the four groups.2.The expression of FLNA and downstream pathway proteins YAP1 and βcatenin in phyllodes tumor and fibroadenoma.2.1 The positive expression rates of FLNA in breast fibroadenoma,benign phyllodes tumor,borderline phyllodes tumor and malignant phyllodes tumor were 15.8%,64.3%,81.3%and 90%,respectively.The higher the histological grade,the higher the positive rate of FLNA.2.2 FLNA expression differences in different groups of tumors.The positive expression rate of phyllodes tumor was significantly higher than that of fibroadenoma,and the difference was statistically significant.The positive expression rate of borderline and malignant phyllodes tumors was significantly higher than that of benign phyllodes tumors,and the difference was statistically significant.The positive rate of benign phyllodes tumor was higher than that of breast fibroadenoma,and the difference was statistically significant.The positive rate of borderline phyllodes tumors was higher than that of benign phyllodes tumors,and the difference was statistically significant.There was no significant difference in the positive expression rate of FLNA between malignant and borderline phyllodes tumors.2.3 Correlation between FLNA expression level and clinicopathological parameters of four tumor patients.The expression level of FLNA was positively correlated with age,tumor size,tumor recurrence and histological grade of phyllodes tumor.2.4 The positive expression rates of YAP1 in breast fibroadenoma,benign phyllodes tumor,borderline phyllodes tumor and malignant phyllodes tumor were 52.6%,78.6%,93.7%and 90%,respectively.2.5 Differences of YAP 1 expression in different groups of tumors.The positive expression rate of phyllodes tumor was higher than that of fibroadenoma,and the difference was statistically significant.The positive expression rate of borderline and malignant phyllodes tumors was significantly higher than that of benign phyllodes tumors,and the difference was statistically significant.Benign positive expression rate was higher than that of fibroadenoma,the difference was statistically significant.2.6 Correlation between YAP1 expression level and clinicopathological parameters of patients with four tumors.The expression level of YAP 1 was positively correlated with the histological grade of phyllodes tumor,and the difference was statistically significant.There was no significant difference with other clinicopathological parameters,including patient age,tumor size,and recurrence.2.7 The positive expression rates of β-catenin in breast fibroadenoma,benign phyllodes tumor,borderline phyllodes tumor and malignant phyllodes tumor were 31.6%,50.0%,87.5%and 90%,respectively.The higher the histological grade,the higher the positive rate of FLNA.2.8 The difference of β-catenin expression in different groups of tumors.The positive expression rate of phyllodes tumor was higher than that of fibroadenoma,and the difference was statistically significant.The positive expression rate of borderline and malignant phyllodes tumors was significantly higher than that of benign phyllodes tumors,and the difference was statistically significant.The positive expression rate of borderline was higher than that of benign,with statistical significance;there was no significant difference in the positive expression rate of β-catenin between benign and fibroadenoma,malignant and borderline.2.9 The correlation between the expression level of β-catenin and the clinicopathological parameters of four tumor patients.The expression level of βcatenin was positively correlated with the histological grade of phyllodes tumor,and the difference was statistically significant.There was no significant difference with other clinicopathological parameters,including patient age,tumor size,and recurrence.3.The correlation between the expression levels of FLNA,YAP1 and β-catenin in phyllodes tumor and fibroadenoma.In all 73 cases of breast fibroepithelial tumors,the expression levels of FLNA protein and YAP1 protein,FLNA protein and β-catenin protein,YAP 1 protein and β-catenin protein were positively correlated,and the difference was statistically significant.Conclusion:1.FLNA gene has a high mutation frequency in breast fibroepithelial tumors,suggesting that FLNA may be involved in the occurrence of the tumor.2.The expression rates of FLNA,YAP1 and β-catenin protein in breast phyllodes tumors were significantly higher than those in fibroadenoma,and were positively correlated with the histological grade of phyllodes tumors,suggesting that the three play an important role in the progression of phyllodes tumors.3.The expression of FLNA,YAP1 and β-catenin was significantly correlated to each other.Combined with literature analysis,we speculate that FLNA/YAP1/βcatenin signaling pathway plays an important role in the development of phyllodes tumors.