Detection And Clinical Significance Of Hsp90α In Nipple Discharge,Blood And Tissues Of Breast Cancer Patients

According to the latest global cancer statistics in 2020,breast cancer has surpassed lung cancer as the most common cancer in the world,and at the same time it has become the world’s fifth leading cause of cancer deaths,causing a heavy burden on the economic and social development of countries around the world.Breast cancer is a malignant tumor that originates from the endothelium of the breast ducts.Early lesions are often confined to the ducts of the breast and grow along the ducts.At this time,surgical operations can be used to achieve a cure.However,once the tumor breaks through the basement membrane of the catheter and infiltrates,some tumor cells may enter the systemic blood circulation and progress to a systemic disease.At this time,even after standard surgical operations and systemic adjuvant treatments,a considerable number of patients still die from distant recurrence and metastasis.Therefore,early detection,early diagnosis,and early treatment are the keys to improving the prognosis of breast cancer patients.Nipple discharge is the most common main complaint of breast cancer patients except breast masses.The final pathological results of breast cancer patients with nipple discharge are intraductal cancer.Therefore,improving the diagnosis and treatment of breast cancer patients with nipple discharge is beneficial to improve the overall survival rate of breast cancer.At present,the specific auxiliary examination methods for nipple discharge mainly include:cytology,ductography,ductoscopy,etc..These examination methods have their own advantages and application limitations.Nipple discharge cytology smear is a qualitative examination.The detection of cancer cells means a clear indication for surgery,but the overall positive detection rate is only about 10%;the characteristic images in ductography can be used for diagnosis and Surgery indications can be grasped,but it is not possible to judge benign or malignant based on images alone;as an invasive method,ductoscopy can only observe the conditions in the main duct and branch ducts,and for ducts that originate in the terminal duct lobular unit.The internal lesions cannot be observed and cannot be characterized.Therefore,it is imperative to develop a highly sensitive and rapid diagnosis method for early breast cancer in nipple discharge.Tumor markers are of great practical value in tumor screening,diagnosis,prognosis and outcome,evaluation of curative effect,and follow-up observation of high-risk groups due to their non-invasive and convenient acquisition and strong patient compliance.At present,the clinical application of tumor markers is becoming more and more common.For example,alpha-fetoprotein is a tumor marker for liver cancer;CYFRA21-1 is a marker for non-small cell lung cancer;neuron-specific enolase(NSE)and gastrin release Peptide precursor(ProGRP)is a marker of small cell lung cancer;squamous cell carcinoma antigen(SCC)is a marker of squamous cell carcinoma.At present,the commonly used tumor markers for breast tumors are CEA,CA153,etc.,but because of their low sensitivity and specificity,they are of limited value in the differential diagnosis of early breast cancer.They are often used to prompt prognosis,monitor recurrence,and evaluate treatment effects.Clinically,there is a lack of tumor markers for the differential diagnosis of benign and malignant nipple discharge,so there is an urgent need to find tumor markers with application potential for breast cancer screening in nipple discharge.Human heat shock protein 90 alpha(Hsp90α)is a protein molecule composed of 732 amino acids and is one of the most abundant proteins in cells.In normal cells,Hsp90α,as a molecular chaperone protein,can assist protein folding and maintain the stability of a variety of signaling proteins in the cell,thereby promoting cell survival and growth.However,in tumor cells,Hsp90α can help over-activated or mutated signaling proteins to maintain their activity,thereby accelerating the malignant transformation of tumor cells.Hsp90α can be secreted by tumor cells outside the cell,enter body fluids,and play an important role in promoting tumor invasion and metastasis.A number of studies have confirmed that Hsp90α is up-regulated in a variety of tumor tissues and plasma,and its content is closely related to tumor occurrence,progression,malignancy,especially metastasis,and can be used as a potential tumor marker.The research of Hsp90α in breast cancer is relatively concentrated in the tissues,and there are few studies in the plasma,and the conclusions are not uniform,which is still controversial.Studies have reported that the level of Hsp90α in breast cancer plasma is significantly higher,however,the results of some foreign scholars suggest that there is no significant difference in plasma Hsp90a levels in patients with breast benign and malignant lesions.Further searching the literature,there is no relevant research report about Hsp90α in nipple discharge.Whether Hsp90α exists in nipple discharge,its concentration level,and its diagnostic value are still unclear.This will be the main innovation and focus of our research on this subject.At the same time,there is no report on the correlation between the level of Hsp90α in nipple discharge and the expression of Hsp90α in matched tumor tissues.This study intends to detect the level of Hsp90α in nipple discharge and plasma,as well as its expression in nipple discharge-related tumor tissues,and to explore the relationship between it and clinicopathological parameters.At the same time,we used proteomics technology to analyze the differential proteins in 15 cases of malignant nipple discharge and 20 cases of benign nipple discharge to further verify the difference in Hsp90a levels in benign and malignant nipple discharge.Part I:Detection of Hsp90α in nipple discharge,proteomics verification and clinical value analysis(1)Detection and clinical value analysis of Hsp90α in nipple dischargeMETHODS:This study included 110 patients with nipple discharge who were hospitalized in the breast surgery department of Qilu Hospital of Shandong University from January 2019 to March 2021.Among them,18 patients had bilateral discharge,and a total of 128 cases of nipple discharge were obtained.All patients signed an ethical informed consent form and performed surgical treatment.Collect nipple discharge specimens and clinicopathological data.The 128 samples included 32 cases of breast cancer(15 cases of invasive ductal carcinoma,15 cases of intraductal carcinoma,and 2 cases of lobular carcinoma in situ)and 96 cases of benign breast disease(72 cases of intraductal papilloma,11 cases of ductal dilatation,7 cases of fibroadenoma and 6 cases of breast hyperplasia).All specimens were tested for Hsp90α using enzyme-linked immunosorbent assay(ELISA),and CEA and CA153 were tested using sandwich electrochemical immunoluminescence assay(ECLIA).At the same time,retrospectively obtain all plasma Hsp90α,serum CEA,CA153 data in the case data.Perform data statistics on the benign breast disease group and breast cancer group,and analyze the diagnostic efficacy of Hsp90α,CEA,and CA153 in nipple discharge.RESULTS:1.Compared with benign discharge,the level of Hsp90α in malignant discharge was significantly increased(P<0.0001).The level of CEA in malignant discharge was significantly increased(P=0.0005),while the level of CA153 in benign and malignant discharge was not significantly different(P=0.1928).2.ROC curve analysis showed that the area under the curve of Hsp90α and CEA to distinguish benign and malignant nipple discharge were 0.8268 and 0.7086,respectively.The best diagnostic cut-off value of Hsp90α is 281.7 ng/ml,and its sensitivity is 84.38%and specificity is 84.38%.The best diagnostic cut-off value of CEA is 494.5 ng/ml,its sensitivity is 51.61%,and its specificity is 90.22%.It can be seen that the diagnostic value of Hsp90α in nipple discharge is better than CEA.The area under the ROC curve for the combined diagnosis of breast cancer is 0.857,and the diagnostic value is better than Hsp90α and CEA.3.From benign lesions to breast carcinoma in situ,and then to invasive ductal carcinoma,the level of Hsp90α in nipple discharge showed an increasing trend,with statistical differences(P<0.0001).4.Hsp90a level in nipple discharge is positively correlated with Ki67 expression(r=0.3639,P<0.0480).5.Hsp90α tends to be highly expressed in patients with negative ER or PR,but neither is statistically significant(P=0.0556,P=0.0584,respectively).There was no significant correlation between Hsp90α and patient age,body mass index,tumor diameter,lymph node metastasis,HER-2 expression,molecular typing,T stage,N stage,and clinical stage(all P>0.05).6.The level of Hsp90α in nipple discharge was significantly higher than that in blood(P=0.0003).(2)Proteomics technology verifies the expression difference of Hsp90α in benign and malignant nipple dischargeMETHODS:A total of 35 nipple discharge samples were collected from hospitalized patients with breast surgery in Qilu Hospital of Shandong University from January 2019 to March 2021,including 15 cases of breast cancer and 20 cases of intraductal papilloma.The specimens were put into operation immediately after collection Store in liquid nitrogen tank.All patients signed an ethical informed consent form,and the specimens were entrusted to Shanghai Baiqu Company for proteomics analysis to find differential proteins.RESULTS:A total of 859 differential proteins were screened,including Hsp90α.The nipple discharge in the breast cancer group was up-regulated by 13.74 times compared with the intraductal papilloma group,with a P value of<0.01.CONCLUSIONS:The level of Hsp90α in nipple discharge of breast cancer patients is elevated,and the level of Hsp90α is increasing from benign lesions to breast carcinoma in situ,and then to invasive ductal carcinoma.The detection of Hsp90α combined with CEA in nipple discharge can be used as a potential diagnostic marker for breast cancer.Part II:Analysis of plasma Hsp90a level and clinical value in breast cancer patientsMETHODS:The case data of 273 patients who underwent breast surgery in Qilu Hospital of Shandong University from January 2019 to March 2021 were retrospectively collected.All patients in the group had clear pathological diagnosis,and the plasma Hsp90α level was routinely checked before operation.91cases of benign(46 cases of intraductal papilloma,31 cases of fibroadenoma,8 cases of duct dilatation,4 cases of adenopathy,1 case of breast hyperplasia,1 case of adenomyosis),182 cases of malignancy(29 cases of intraductal carcinoma,136 cases of invasive ductal carcinoma,6 cases of invasive lobular carcinoma,6 cases of mucinous adenocarcinoma,2 cases of invasive apocrine carcinoma,1 case of lobular carcinoma in situ,1 case of occult carcinoma,1 case of adenoid cystic carcinoma).Collect the data of Hsp90α,CEA,CA153 in the blood,as well as the clinical pathological parameters of the patient,such as age,body mass index,tumor diameter,lymph node metastasis,etc.,make statistics on the specimens between the benign breast disease group and the breast cancer group,and analyze Hsp90α in the diagnostic efficacy in the blood was compared with CEA and CA153,and the relationship between Hsp90αand the patient’s clinicopathological parameters was analyzed at the same time.RESULTS:1.Compared with the benign breast disease group,the plasma Hsp90a level in the breast cancer group did not have a significant difference(P=0.1057).The serum levels of CEA and CA153 in breast cancer were significantly increased,with statistical differences(all P values<0.05).2.The level of Hsp90α in the blood of patients with stage 0 to Ⅲ breast cancer has an increasing trend,but there is no statistical difference(P>0.05),but the expression in the blood of stage Ⅳ patients is significantly increased(especially compared with patients of stage 0,Ⅰ,Ⅱ,the P values are all<0.05).3.The level of Hsp90α in the blood of PR-negative breast cancer patients was significantly higher than that of PR-positive patients(P<0.05).4.There was no significant correlation between the level of Hsp90α in the blood and the patient’s age,body mass index,tumor diameter,lymph node metastasis,ER expression,ki67 expression,HER-2 expression,molecular typing,and Pathological Stage(all P values>0.05).CONCLUSIONS:There is no statistical difference in plasma Hsp90a levels between breast cancer group and benign breast disease group,but it can be significantly increased when breast cancer occurs distant metastasis.Hsp90α in the blood is not suitable for the differential diagnosis of breast cancer as a tumor marker,but it has the potential value of suggesting distant metastasis.Part Ⅲ:Hsp90a expression in breast cancer tissue and clinical significance analysisMETHODS:The surgical tissue samples of breast cancer patients and nipple discharge patients in the first part of the study cases were cut from the wax blocks stored in the pathology department.Due to the small lesions of some specimens,after routine pathological examinations,the lesion tissue could not be removed.A total of 29 breast cancer samples were collected(14 cases of invasive ductal carcinoma,13 cases of intraductal carcinoma,and 2 cases of lobular carcinoma in situ).There were 33 samples of intraductal papilloma.The immunohistochemical staining method was used to detect the expression level of Hsp90α in breast benign and malignant disease tissues with nipple discharge,and according to the results,the differences in expression of Hsp90α in breast benign and malignant tissues were analyzed,and the relationship of the level of Hsp90α and clinicopathological parameters in nipple discharge were discussed.RESULTS:1.There is no significant difference in the expression of Hsp90α in breast cancer and intraductal papilloma tissues(P=0.0649).2.The expression of Hsp90α in breast cancer tissues has no significant correlation with its level in nipple discharge(r=0.1605,P=0.2167).3.The expression of Hsp90α in breast cancer tissue related to nipple discharge has no significant correlation with tumor diameter,lymph node metastasis,ER expression,PR expression,ki67,HER-2 expression,molecular typing,and pathological grade(all P>0.05).CONCLUSIONS:There is no significant difference in the expression of Hsp90α in breast cancer and intraductal papilloma tissues,and there is no significant correlation with its concentration level in nipple discharge.The level of Hsp90α in nipple discharge may not directly reflect its expression in tissues.