Study On Invasion And Metastasis-associated Factors Of Esophageal Squamous Cell Carcinoma And Its Significance

Objectives: Esophageal carcinoma is among the most common malignant tumors, with rapid progress﹑early lymphatic spread and poor prognosis, this is due to its high malignant biologic behavior . Invasion and metastasis are the most important characters of cancer cells and closely associated with tumor prognosis. We intend to study invasion and metastasis-associated factors of esophageal carcinoma from the following four aspects.: 1 Abnormality of Cell Cycle Regulation and Over-proliferation: Over-proliferation of tumor cells is the base of invasion, can cause high pressure of tumor inside, thus promotes invasion and metastasis. Proliferation of tumor cells is achieved by cell cycle. Unbalance of cell cycle regulation results in over-proliferation of tumour cells. We divide this aspect into two parts:⑴Detecting expressions of cell cycle regulatory proteins, including Cyclin E,CyclinD1,CDK4,P27 proteins in esophageal squamous cell caicinoma(ESCC) by flow cytometry and studing their relationship to tumor differentiation, lymph nodes metastasis . ⑵Examining expression and prognostic significance of a new proliferation marker MCM2 in ESCC by immunohistochemistry. 2 Signal Transduction: Signal transduction plays a critical role in invasion and metastasis of tumour. Many studies have demonstrated that every stage of cancer metastasis, including interaction with matrix, cell motility, adhesion and angiogenesis is associated with signal transduction. We divide this aspect into two parts: ⑴c-Src: c-Src is one of non-receptor protein tyrosine kinases, mediating a variety of signal transduction pathways. Using immunohistochemistry and in situ hybridization(ISH) , we examine the expressions of c-Src and its related gene proteins, including MCM2,NOS2,E-Cadherin ,VEGF to study their relationships and influences on the differentiation,stage and survival of ESCC. ⑵NO: NO is an important signaling molecule that can transmit information among cells and cell inside. NOS2 is NO synthetase and has been found at higher expression levels in many cancers, it is suggested to contribute tumour angiogenesis and metastasis. We examine the expression of NOS2 in ESCC by flow cytometry and study its correlation with COX-2,tumour differentiation and lymph nodes metastasis. 3 Prostaglandin(PG) and Apoptosis: The level of PG is higher in tumors than in normal tissues . COX2 is synthetase of PG, its overexpression increases synthesis of PGE2, PGE2 probably promote invasion and metastasis of cancers by inducing cell proliferation,increasing the expression of Bcl-2 to make unbalance of proliferation and apoptosis . PGE2 also stimulates angiogenesis and activates MMP-2 to degrade the extracellular matrix. We study expressions and significance of COX2 and Bcl-2 by flow cytometry and analyze their relationship to tumor differentiation, lymph nodes metastasis . 4 Neuroendocrine(NE) Differentiation: Today the most important prognostic parameters in tumours are still clinical tumour stage and histologic grade. However, estimation of prognosis especially in moderately differentiated tumours is still a problem. Recent years, the significance of NE differentiation has become popular. NE differentiation is that scattered neuroendocrine cells appear in typical squamous cell carcinoma or adenocarcinoma. Neuroendocrine cells produce a variety of hormones like insulin, glucagons, calcitonin and peptides such as NSE and CgA. According to some investigators these hormones play a regulatory role in the growth and differentiation of tumours, but there is still debate on the prognostic value of NE differentiation. Our study investigate expressions and significance of CgA and Syn in early ESCC . Methods 1 Materials ⑴Tissue specimens were obtained from 65 patients aged 36～73 years that underwent surgery for ESCC between 1999 and 2000 in the fourthhospital of Hebei medical university. Each specimen was divided into two pieces and were fixed by 10% formaldehyde and 70% alcohol respectively. The specimens fixed by formaldehyde were paraffin-embedded ,HE stained, graded as differentiated(GradeⅠandⅡ,n=46) and poor-differentated(GradeⅢ,n=19). Specimens fixed by 70% alcohol were used in flow cytometry to detect expressions of Cyclin D1,Cyclin E,CDK4 ,P27,NOS2,COX2 and Bcl-2. ⑵134 patients aged 39～75 years that underwent surgery for ESCC between 1997 and 1998 in the fourth hospital of Hebei medical university were taken into study. They have completed follow-up materials. Histologic grade was divided into GradeⅠ(n=26), GradeⅡ(n=64), GradeⅢ(n=44) according to WHO standard. Clinical tumour stage was divided into stageⅠ(n=13), stageⅡa (n=72), stageⅡb (n=15) and stageⅢ(n=34) according to TNM standard. These specimens were used in immunohistochemistry to examine expressions of c-Src,MCM2,PCNA,NOS2,E-Cadherin and VEGF. ⑶Tissue specimens were obtained from 33 patients that underwent surgery for ESCC in 2004 in the fourth hospital of Hebei medical university. The specimens were fixed by 4% paraformaldehyde (containing 1/1000 DEPC) within 10 minutes after operation to be used in examining mRNA of c-src,nos2,e-cadherin and vegf by ISH. ⑷Tissue specimens were obtained from 48 patients aged 32～74 years that underwent surgery for early esophageal squamous cell carcinonmas between 1993 and 1997 in the fourth hospital of Hebei medical university. The specimens were fixed by formaldehyde,paraffin-embedded, including carcinoma in situ 13 cases, invasion to the muscularis mucosae 10 cases and invasion to the submucosa 25 cases. According to differentiation, they were divided into well-differentiated(GradeⅠ)11cases, moderately-differentiated (grade Ⅱ) 21cases and poor-differentiated (Grade Ⅲ)16 cases. These specimens were used in checking expressions of CgA and Syn by immunohistochemistry. 2 Flow cytometry(FCM): The sample fluorescence staining wasperformed using indirect immunofluorescence labeling method. The stained samples were analyzed in a FACS 420 flow cytometer(FACS 420 Fluorescence Activated Cell Sorting, Becton. Dickinson, Sunnyvale. California, U S A). The analytic data were collected in log form and processed with a HP-300 consort 300 computer. The CV of the instrument was adjusted within 5% PI staining chicken red blood cells. 3 Immunohistochemistry: We adopted three-step method of SP (streptomycin antibiotinprotein-peroxidase linked method). 4 In situ hybridization(ISH): Dewaxing paraffin-sections used in ISH to water, exposing mRNA fragments, prehybridization, hybridization, post hybridization treatment, dripping inclusion fluid, dripping biotin mouse antidigoxin, dripping SABC, DAB visualization, hematoxylin contrast stain, dehydration, making transparent, coverslip. Above-mentioned three methods even had negative control and positive control. Results 1 ⑴Expressions of Cyclin E,CyclinD1,CDK4 and P27 in ESCC by Flow Cytometry: The FI value( and positive rate )of three proteins in differentiated ESCC were 1.21±0.10(97.8%),1.36±0.10(100%),1.17±0.10(91.3%) respectively; but in poorly-differentiated ESCC, they were 1.36±0.07(100%),1.43±0.13(100%),1.23±0.11(94.7%) , significantly higher than those in differentiated ESCC(P=0.0001,0.0275,0.0174). The FI value (and positive rate) of P27 in differentiated and poorly differentiated ESCC were 0.89±0.09(2.17%)﹑0.81±0.10(0%) respectively , P27 expression in poorly-differentiated ESCC was lower than that of differentiated ESCC(P=0.0042). Expressions of the four proteins were not correlated with Lymph nodes metastasis(P>0.05). Positive correlation was found between Cyclin E and CyclinD1(r=0.3746,P=0.0021), CyclinD1 and CDK4(r=0.6681,P=0.0001) ; There was negative correlation between CyclinD1 and P27(r=-0.2961,P=0.0166). ⑵Detecting Expression of MCM2 by Immunohistochemistry:Expression of MCM2 was closely associated with differentiation of ESCC,GradeⅠ: (-)5,(+)10,(++)7,(+++)4; GradeⅡ:(-)6,(+)16,(++)18,(+++)24; GradeⅢ:(-)2,(+)2,(++)13,(+++)27,The expression level was significantly higher in poorly-differentiated ESCC than in well-differentiated ESCC(P=0.001); It was not associated with tumor clinical stage(P=0.456); The relationship between MCM2 and PCNA showed a significantly positive correlation(r=0.701,P=0.00); MCM2 and tumour stage were independent prognostic factors. 2 ⑴Expressions of C-Src and Its Related Factors in ESCC by Immunohistochemistry and ISH : Immunohistochemistry results: ①Immunhistochemical studys have demonstrated that the positive rate of c-Src in 134 ESCC patients was 98.5%; Expression of c-Src was closely associated with differentiation of ESCC,GradeⅠ: (-)0,(+)9,(++)11,(+++)6, GradeⅡ:(-)1,(+)11,(++)23,(+++)29, GradeⅢ:(-)1,(+)2,(++)5,(+++)36,the expression level was significantly higher in poorly-differentiated ESCC than in well-differentiated ESCC(P=0.001); Overexpression of c-Src was related to clinical stage, StageⅠ: (-)0,(+)6,(++)3,(+++)4, StageⅡa: (-)0,(+)13,(++)19,(+++)40, StageⅡb: (-)2,(+)2,(++)5,(+++)6, StageⅢ: (-)0,(+)1,(++)12,(+++)21, the expression level was higher in StageⅢthan in StageⅠ(P=0.041); Positive correlation was found between c-Src and MCM2(r=0.43184,P=0.0001),c-Src and VEGF(r=0.21795,P=0.0114),c-Src and NOS2(rs=0.17149,P=0.0476). C-Src was not correlated with E-cadherin (r=-0.156,P=0.072). ②90.3% patients stained positively for MCM2; Expression of MCM2 was related to histological grade (P=0.001), not related to clinical stage(P=0.456). ③99% patients stained positively for NOS2; Expression of NOS2 was not related to histological grade and clinical stage(P=0.093,0.100). ④75.3% patients stained positively for VEGF; Expression of VEGF was not related to histological grade and clinical stage(P=0.322, 0.350).⑤E-Cadherin abnormally expressed in 43.8% patients; Expression of E-cadherin was related to histological grade (P=0.001), but notrelated to clinical stage(P=0.305). ⑥Cox regression revealed that clinical stage,c-Src and MCM2 were independent prognostic factors. The Results of ISH: ①Oligonucleotide probe has detected that c-src mRNA positively stained in cytoplasm of 75.3% patients, compared with c-Src protein expression , the coincidence rate was 75.4%.②Oligonucleotide probe has detected that nos2 mRNA positively stained in cytoplasm of 81.2% patients, compared with NOS2 protein expression , the coincidence rate was 81.8%. ③Oligonucleotide probe has detected that vegf mRNA positively stained in cytoplasm in 70.1% patients, compared with VEGF protein expression , the coincidence rate was 93.3%. ④Oligonucleotide probe has detected that e-cadherin mRNA abnormally expressed in cytoplasm of 53.2% patients, compared with E-cadherin protein expression , the coincidence rate was 83%. ⑵Expression of NOS2 in ESCC by Flow Cytometry: Expression of NOS2 in poorly-differentiated ESCC(FI=1.35±0.12) was significantly higher than that in differentiated ESCC(FI=1.28±0.12), P=0.0385. It was not correlated with Lymph nodes metastasis(P=0.82). There was positive correlation between NOS2 and COX2(r=0.4207,P=0.0005). 3 Expressions of COX2 ,Bcl-2 in ESCC by Flow Cytometry: Expressions of COX2 ,Bcl-2 in poorly-differentiated ESCC(FI=1.32 ±0.11,1.32 ±0.13) were significantly higher than those in differentiated ESCC(FI=1.13 ±0.09,1.20 ±0.15), P=0.0001 , 0.0036. They were not correlated with Lymph nodes metastasis(P=0.7009,0.3992). There was positive correlation between COX2 and Bcl-2(r=0.3054,P=0.0134). 4 Expressions of CgA and Syn in ESCC by Immunohistochemistry: ⑴In 48 patients of early ESCC, 32 cases showed nagative expression of CgA ,12 cases were <1% positive expression and 4 cases were >1% positive expression . Expression of CgA was not correlated with the depth of invasion and differentiation(P=0.601, 0.091). ⑵In 48 patients of early ESCC, 37 cases showed nagative expression of Syn ,12 cases were <1% positive expression and 4 cases were >1% positive expression. Expression of Syn was not correlated with the depth of invasion (P=0.663), but correlated withdifferentiation(P=0.033).⑶CgA and Syn were correlated with PCNA(r=0.299, P=0.039; r=0.288, P=0.047). ⑷Cox regression only revealed that tumor differentiation and stage were independent prognostic factors. Conclusions 1 ⑴We put the four main cell cycle regulatory proteins together to study their expressions and interaction in ESCC by flow cytometry. Our study showed that expressions of Cyclin D1,CyclinE,CDK4 and P27 were closely related to the differentiation of ESCC, and could be used to estimate the malignant degree of ESCC; Low expression of P27 and overexpression of Cyclin D1 revealed self-regulatory mechanism of malignant tumours. ⑵MCM2 is a key factor that initiates DNA replication. It was considered a new proliferation marker that is superior to Ki67. We have not found any reports about MCM2 in our country. Our study showed that expression of MCM2 in ESCC was closely associated with differentiation; MCM2 could be considerde as an independent prognostic factor of ESCC. 2 ⑴As a signal transduction protein, c-Src becomes popular recent years. We detected c-Src mRNA and protein levels in ESCC by ISH and immunohistochemistry, analyzed its correlation with MCM2,E-Cadherin,NOS2 and VEGF ,studied its role in tumour invasion and metastasis. No report like this has been found in our country. The result showed that c-Src was closely related to the differentiation and stage of ESCC. c-Src,MCM2,NOS2 and tumour stage were independent prognostic factors. By studying the correlationship between c-Src and its related factors(MCM2,NOS2,VEGF,E-cadherin) , we demonstrated that c-Src could promote invasion and metastasis of tumors by a varerty of pathways, including stimulating proliferation,motility and angiogenesis, ruducing adhesion of tumour cells . ⑵Our study revealed that NOS2 was closely related to differentiation of ESCC. NOS2 was positively correlated with Cox2, NOS2 and Cox2 were suggested to coorperate to facilitate tumour angioigenesis. 3 Our study revealed that Cox2 and Bcl2 were closely related to differentiation of ESCC. There was positive correlation between Cox2 and...