Expressions And Clinicopathological Significance Of CD47,E-Cadherin And MMP-2 Protein In Endometrioid Carcinoma With MELF Pattern Of Invasion

Background:Endometrial cancer is one of the most common malignant tumors of the reproductive system in women in my country,and its incidence is on an increasing trend.At present,the vast majority of endometrial cancers can be divided into two types: type I and type II based on clinicopathological characteristics and molecular mechanisms.Type I endometrial cancer is the main type of endometrioid cancer.Compared with type II,the overall prognosis is better,but 5%-18% of type I still have a poor prognosis.Recent studies have suggested that the invasion pattern of endometrial cancer may affect its prognosis.Murray et al.first described a special pattern of endometrial cancer infiltrating myometrium in 2003.The infiltrating glands are microcystic,elongated and fragmented,referred to as MELF pattern.MELF pattern often occurs in low-grade endometrial cancer and is related to vascular invasion and lymph node metastasis.At present,there are not many reports on the clinicopathological characteristics of MELF endometrial cancer at home and abroad,and the molecular mechanism related to its invasion potential is not very clear.Whether the histological characteristics of MELF related to its aggressiveness has not been reported yet.CD47 protein,also known as integrin-associated protein(IAP),is an immunoglobulin-like protein widely distributed on the surface of different cells in human tissues and belongs to the immunoglobulin superfamily.The ligand of CD47 protein is signal regulatory protein α(SIRPα),which is expressed on the cell membrane surface of nerve cells and myeloid cells,so it can be highly expressed in macrophages.When tumor cells overexpress the CD47 protein to release the "don’t eat me signal",it binds to the macrophage surface ligand SIRPα protein to increase the signal to inhibit cell phagocytosis,making the body mistakenly think it is the "self",thereby preventing macrophages from phagocytosis.To achieve the purpose of escaping the body’s immunity.Studies have shown that CD47 protein is overexpressed in some hematological tumors,lymphomas,gastric cancer,breast cancer,ovarian cancer,liver cancer and bladder cancer,and is closely related to tumor invasion and metastasis.At present,there are few reports on the expression of CD47 protein in endometrial cancer at home and abroad.It is not known whether the molecular mechanism of endometrial cancer with MELF is related to CD47 protein.As a cell adhesion protein,E-Cadherin is normally expressed on the cell surface and participates in cell stability and polarity.Studies have shown that the lack of E-Cadherin protein expression in tumor cells is one of the important evidences for the epithelial-mesenchymal transition(EMT)of tumors.It has been reported that the expression of E-Cadherin protein in MELF glands is missing,suggesting that MELF pattern may be related to EMT.Matrix metalloproteinases(MMPs)can almost degrade various protein components in extracellular matrix(ECM)and play a key role in tumor invasion and metastasis.MMPs can be directly secreted by tumor solid cells,but are most often synthesized and secreted by reactive mesenchymal cells including fibroblasts and inflammatory cells.Matrix metalloproteinase-2(MMP-2)in MMPs,overexpression can be observed in a variety of malignant tumors,is associated with unfavorable clinicopathological factors,and increases the potential for tumor metastasis.Objectives:1.To explore the clinicopathological characteristics and prognostic differences between MELF and non-MELF endometrial cancer.2.To explore the expressions of CD47,E-Cadherin and MMP-2 protein in endometrial carcinoma with MELF pattern and their clinicopathological significance.Methods:1.Retrospective collection of 180 cases of type I endometrial cancer admitted to Dalian Maternity Hospital Affiliated to Dalian Medical University between 2009-2012 and 2017-2019,with complete clinicopathological data.According to the diagnostic criteria of MELF pattern,52 cases of endometrial cancer with MELF pattern were screened as the experimental group.25 cases in each of the two time periods,a total of 50 cases of non-MELF endometrial cancer,were screened randomly as the control group.The clinicopathological characteristics and prognosis of cancer were compared and analyzed.2.The expressions of CD47,E-Cadherin and MMP-2 protein were detected by SP immunohistochemical staining.According to the percentage of stained cells and staining intensity,the results were judged by semi-quantitative grading method.The expression differences of these markers in MELF pattern and their clinicopathological significance were analyzed.Results:1.Comparison of general clinicopathological characteristics of endometrial cancer in the two groups: The incidence of MELF pattern was 14.4%(52/360).The average age of patients with MELF pattern was 58.88±7.6 years old,which was significantly different from 55.72±7.2 years old with non-MELF pattern(P=0.034).There were 26 patients with preoperative CA125 concentration ≥35 KU/L in MELF pattern,and the difference was significant compared with 10 patients without MELF pattern(P=0.002).The number of patients with cervical stromal invasion(P=0.021),≥1/2 myometrial invasion(P<0.001),vascular invasion(P<0.001),lymph node metastasis(P<0.001)and FIGO stage III(P<0.001)increased significantly in MELF pattern.There was no significant difference in menopause(P=0.058)and tumor size(P=0.81)between the two groups.2.Histopathological characteristics of endometrial carcinoma with MELF pattern:Compared with non MELF pattern,there was no statistical difference in the general morphology and tumor proportion of ≥2 cm(P=0.169),and tumor size(P=0.81).Under the light microscope,MELF glands were mainly located in the front edge of the tumor infiltrating foci,showing characteristic microcapsules,elongated or fragmented.There were obvious interstitial reactions around the infiltrating glands,often accompanied by vascular invasion.The fragmented epithelial cell clusters in the invasive muscle layer are similar to the tumor thrombus and lymph node metastasis.In 52 cases of MELF pattern,the number of lesions of the invasive muscle layer of fragmented epithelial cell clusters ranged from 1 to 11.Univariate analysis showed that the number of invasive muscle layer of fragmented epithelial cell clusters ≥7 was significantly associated with lymph node metastasis(P=0.006).3.In 52 cases of MELF pattern endometrial carcinoma,31 cases were positive for CD47 protein expression,7 cases were(+++),7 cases were(++),17 cases were(+),the positive rate was 59.62%.In non MELF region,9 cases were positive for CD47 protein expression,the positive rate was 17.31%.There was significant difference between MELF region and non MELF region(P<0.001).In the univariate analysis of lymph node metastasis or not,the expression of CD47 protein in primary endometrial carcinoma of MELF pattern was(++~+++)and(-),the difference was statistically significant(P=0.021).4.The expression of E-Cadherin was absent in 29 of 52 MELF cases(55.77%).The staining of cell membrane in non MELF area was brown,continuous and complete,and no clear loss of expression region was found.The expression of E-Cadherin in MELF region was significantly lower than that in non MELF region(P<0.001).The expression of CD47 was consistent with the deletion of E-Cadherin,and the positive expression of CD47 in MELF was correlated with the deletion of E-Cadherin(P<0.001).5.The expression of MMP-2 protein was observed in the interstitial reaction area around MELF gland,but not in the non MELF area.The number of MMP-2 positive foci ranged from 2 to 8 in 52 cases of MELF.Spearman analysis showed that there was a negative correlation between the number of MMP-2 positive foci and the distance between the deepest tumor infiltration and uterine serosa(Rs=-0.859,P<0.001).Univariate analysis showed that the number of MMP-2 positive lesions(≥4 lesions)and the depth of invasive muscle layer(≥1/2)were statistically significant(P<0.001).6.Multivariate analysis showed that cervical stromal invasion,vascular invasion and MELF pattern were independent factors affecting lymph node metastasis of endometrial carcinoma(P=0.024,0.02 and 0.031 respectively).7.CD47 protein(++~+++)and fragmented epithelial cell clusters(≥7 foci)were the risk factors of lymph node metastasis in MELF endometrial carcinoma(P=0.038,P=0.049respectively).8.There was no significant difference in 5-year OS and DFS between MELF group and non MELF group(P=0.977,P=0.906 respectively).Conclusions:1.Endometrial cancer with MELF is closely related to the average age of patients,high preoperative serum CA125 level(≥35 KU/L),cervical stromal invasion,deep myometrial invasion,vascular invasion,lymph node metastasis and high FIGO stage.2.The high expression of CD47 protein was positively correlated with the invasion of MELF.The intensity of CD47 protein expression in primary lesions of MELF was positively correlated with lymph node metastasis.3.The loss of E-Cadherin expression was positively correlated with MELF infiltration.The positive expression of CD47 protein in MELF was correlated with the deletion of E-Cadherin protein.4.The multifocal expression of MMP-2 protein was positively correlated with the depth of tumor invasion.5.Cervical stromal invasion,vascular invasion and MELF pattern are risk factors for lymph node metastasis of FIGO Ⅰ-Ⅱ endometrial carcinoma.The strong positive expression of CD47 protein and the presence of multifocal fragmented epithelial cell clusters are the risk factors of lymph node metastasis in endometrioid carcinoma with MELF pattern of invasion.6.The prognosis of endometrial carcinoma with MELF was not significantly different from that of non MELF.Because of the high invasiveness of MELF and the uncertainty of its clinical significance,pathologists should strengthen their understanding and provide more accurate diagnosis results for clinic.