摘要
目的:1.本课题是通过蛋白芯片(protein chip)技术检测乳腺癌(breast cancer)患者、乳腺良性病变患者和正常女性血清中肿瘤标志物(tumor markers)表达的差异,筛选出对乳腺癌有临床价值的肿瘤标志物,分析其诊断价值,探讨其升高的可能原因;2.通过蛋白芯片技术检测乳腺癌患者血清肿瘤标志物水平与肿瘤大小、淋巴结转移状况的相关性,探讨肿瘤标志物对病情判断的临床意义;3.检测乳腺癌患者手术前后、术后复发转移血清肿瘤标志物水平的变化并进行分析,探讨肿瘤标志物在乳腺癌病情监测,预后判断的临床意义;4.通过蛋白芯片技术的应用,分析蛋白芯片技术的临床价值。
方法:采用多肿瘤标志物蛋白芯片诊断系统,检测分析90例乳腺癌患者,60例乳腺良性病变和100例正常对照者血清12种常见肿瘤标志物,包括糖类抗原199(CA199)、甲胎蛋白(AFP)、神经原特异性烯醇化酶(NSE)、游离前列腺特异抗原(f-PSA)、前列腺特异杭原(PSA)、癌胚抗原(CEA)、糖类抗原242(CA242)、糖类抗原125(CA125)、糖类抗原153(CA153)、铁蛋白(FER)、人生长激素(HGH)和人绒毛膜促性腺激素(HCG);首先静脉采血,静置离心;试验前将标准品稀释;将各待测血清或不同浓度的标准品滴加到不同的芯片分格内;将蛋白芯片温育振荡,洗涤;芯片小方格内各加入反应液,温育振荡,取出弃去孔内液体;剥离蛋白芯片集成块的上部,放入洗盒中,洗涤4次;加入检测液,用HD-2001A蛋白芯片仪对蛋白芯片读取数据,获得结果。采用SPSS 13.0统计软件进行数据分析,计数资料采用χ~2检验;3组样本率的比较采用χ~2分割法;计量资料采用t检验;3组样本均数间的多重比较采用SNK-q检验;应用logisitic逐步回归筛选出有临床价值的肿瘤标志物,获得相应的数学模型即回归方程,通过ROC曲线分析CEA,FER,CA125,CA153及logistic回归结果的曲线下面积(AUC)。
结果:乳腺癌组CEA、CA125、FER、CA153水平均显著高于乳腺良性病变组和正常对照组,有显著统计学差异(P＜0.01);乳腺良性病变组CEA、CA125、FER、CA153与正常对照组差异无显著性意义(P＞0.05);而CA199、NSE、CA242、HCG、AFP、f-PSA、PSA、HGH在乳腺癌组、乳腺良性病变组和正常对照组间差异未见统计学意义;乳腺癌组与正常对照组血清CEA、CA125、FER、CA153的logistic回归方程P_1=1/1+e~(-(-1.29+2.25×CEA+3.12×CA125+10.59×CA153));乳腺癌组、正常对照组CEA、CA125、FER、CA153和联合CEA、CA125、CA153三种肿瘤标志物的logistic回归曲线转化参数P_1的ROC曲线中,单独检测时CA153的AUC最大,有统计学意义(P=0.000),CA125、CA153、CEA、FER联合检测的AUC大于各项肿瘤标志物单项检测的AUC,有统计学意义(P=0.000)。
乳腺癌患者腋淋巴结转移组CEA、CA125、FER、CA153水平及阳性率高于无转移组,有显著统计学差异(P＜0.01);淋巴结转移组与无淋巴结转移组血清CEA、CA125、FER与CA153的logistic回归方程P_2=1/1+e~(-(-2.27+2.05×CEA+1.94×FER+1.06×CA125+1.84×CA153));淋巴结转移组与无淋巴结转移组血清CEA、CA125、FER、CA153和四种肿瘤标志物的logistic回归曲线转化参数P_2的ROC曲线中,单独检测时FER的AUC最大,有统计学意义(P=0.000),四种肿瘤标志物联合检测的AUC大于各项肿瘤标志物单独检测的AUC,有统计学意义(P=0.000);乳腺癌患者肿瘤直径d＞3cm组CEA、CA125、FER、CA153水平均显著高于肿瘤直径d＜3cm组,有显著统计学差异(P＜0.01)。单因素logistic回归分析CEA,CA125,FER,CA153是肿瘤大小的危险因素。
乳腺癌患者术后组血清CEA、FER、CA125和CA153水平明显降低,与术前组比较,差异均有显著性意义(P＜0.01);手术后3个月患者血清CEA、FER、CA125和CA153水平与正常对照组比较,差异无显著性意义(P＞0.05);乳腺癌复发转移组患者血清CEA、FER、CA125、CA153水平与无复发转移组比较、差异均有显著性意义(P＜0.01);复发转移组与无复发转移组血清CEA、CA125与CA153的logistic回归方程P_3=1/1+e~(-(-21.00+11.50×CEA+20.31×CA125+21.70×CA153));复发转移组与无复发转移组CEA、CA125、FER、CA153和联合四种肿瘤标志物的logistic回归曲线转化参数P_3的ROC曲线中,单独检测CA153的AUC最大,有统计学意义(P=0.000);四种肿瘤标志物联合的AUC大于肿瘤标志物单项检测的AUC,有统计学意义(P=0.000)。
结论:应用蛋白芯片技术联合检测肿瘤标志物对乳腺癌的诊断、监测其复发和转移均有较高的临床价值;血清CA125、CA153、CEA、FER是乳腺癌辅助诊断的理想指标,其联合检测有利于提高乳腺癌的诊断率;血清CA125、CA153、CEA、FER为乳腺癌患者腋淋巴结转移的高危因素,与肿瘤大小正相关,其血清水平反映了机体的肿瘤负荷;动态监测CA125、CA153、CEA、FER对乳腺癌预后具有重要临床价值;联合检测CA125、CA153、CEA、FER对于乳腺癌淋巴结转移的判断,手术前后、复发转移的病情监测是比较理想的检测指标,是监测乳腺癌复发转移较好的肿瘤标志物组合;CA199,NSE,CA242,HCG,AFP,f-PSA,PSA,HGH这些指标对乳腺癌诊断价值较低;作为一种统计手段,logistic回归可改善诊断的灵敏度和特异性。
Objective:1.The research is to detect tumor markers(TM)in serum of the patients with breast cancer,the patients with benign breast disease and control groups by the multi-tumor marker protein biochip detective system,then choose tumor markers which have clinical value for breast cancer,analyze the diagnosis value and the cause of abnormity; 2.Tumor markers in serum of the patients with breast cancer are detected by the multi-tumor marker protein biochip detective system,then analyze the relations between diameter of primary tumor,axillary lymph node metastases(ALNM)and tumor markers,and investigate the clinical value of tumor markers about the severity of breast cancer;3.Tumor markers of the patients with breast cancer both before and 3 months after operation and the patients with recurrent breast cancer are detected by the multi-tumor marker protein biochip detective system,then investigate the prognostic value of tumor markers in breast cancer;4.We assess the clinical value of protein chip technology by applying to clinic.
Method:By the multi-tumor marker protein biochip diagnostic system,we detected 12 kinds of tumor markers,which included carbohydrate antigen 199(CA199),alphafetoprotein(AFP), neuron-specific enolase(NSE),free prostate specific antigen(f-PSA), carcinoembryonic antigen(CEA),prostate specific anitgen(PSA), carbohydrate antigen 242(CA242),carbohydrate antigen 125(CA125), carbohydrate antigen 153(CA153),FER(Ferritin),human growth hormone(HGH),and human chorionic gonadotropin(HCG)in 90 cases of breast cancer,60 cases of benign breast disease and 100 cases of healthy individuals as control groups;First,we get blood samples from the patients,then centrifuge the samples;We dilute the standard samples before testing;Put the samples into the different compartments of protein chip;Incubate and surge the protein chip,then wash it;Put the reagent into the compartments of protein chip;Peel off the top of protein chip and wash it;Put testing reagent into the compartments,then gain the results by the multi-tumor marker protein biochip detective system.SPSS version 13.0 was used for statistical analysis;Use Chi-square test for enumeration count data;Use partition ofχ2 method for comparing rates of three groups;Use t-test for measurement data;use SNK-q test for comparing means of three groups;Assess the clinical value of tumor markers for breast cancer by logistic regression and establishing logistic regression equation;The area under the ROC curve(AUC)of CEA,FER, CA125,CA153 from logistic regression results were compared.
Results:①The serum levels of CEA,FER,CA125,CA153 in the patients with breast cancer were significantly higher than that in control groups and the patients with benign breast disease(P＜0.01);There was no significant difference between the patients with benign breast disease and control groups(P＞0.05);The serum levels of CA199,NSE,CA242, HCG,AFP,f-PSA,PSA,HGH had little significant difference between the patients with breast cancer,the patients with benign breast disease and control groups(P＞0.05);The logistic regression equation of groups of breast cancer and control groups is P_1=1/1+e~(-(-1.29+2.25×CEA+3.12×CA125+10.59×CA153));In cancer-control group,the AUC of combination of CEA,FER, CA125 and CA153 was larger than any AUC of CEA,FER,CA125 or CA153 alone(P=0.000),the AUC of CA153 was larger than AUC of CEA,FER,CA125(P=0.000);②The serum levels and positive rates of the CEA,CA125,FER,CA153 in the patients with axillary lymph node metastases(ALNM)were significantly higher than that in the patients without ALNM(P＜0.01);The logistic regression equation of groups with ALNM and groups without ALNM is P_2=1/1+e~(-(-2.27+2.05×CEA+1.94×FER+1.06×CA125+1.84×CA153));In groups with ALNM and groups without ALNM,the AUC of combination of CEA,FER,CA125 and CA153 was larger than any AUC of CEA,FER,CA125 or CA153 alone(P=0.000),the AUC of FER was larger than AUC of CEA,FER,CA125(P=0.000).The serum levels of the CEA,CA125,FER,CA153 in the patients with the diameter of primary tumor over 3 centimeters were significantly higher than that in patients with the diameter of primary tumor below 3 centimeters(P＜0.01).③The serum levels of CEA,FER,CA125 and CA153 was significantly lower after operation than that before operation in the patients with breast cancer(P＜0.01);There was no significant difference between the patients 3 months after operation and control groups(P＞0.05);The serum levels of CEA,FER,CA125,CA153 in the patients with recurrent breast cancer were significantly higher than that in the patients without recurrent breast cancer(P＜0.01);The logistic regression equation of groups with recurrent breast cancer and groups without recurrent breast cancer is P_3=1/1+e~(-(-21.00+11.50×CEA+20.31×CA125+21.70×CA153)),In groups with recurrent breast cancer and groups without recurrent breast cancer,the AUC of combination of CEA,FER,CA125 and CA153 was larger than any AUC of CEA,FER,CA125 or CA153 alone(P=0.000),the AUC of CA153 was larger than AUC of CEA,FER,CA125(P=0.000).
Conclusion:①The application of C-12 multi-tumor marker protein biochip diagnostic system in diagnosis,observing recruuent and metasetases of breast cancer is valuable;②CEA,FER,CA125 and CA153 can be regarded as valuable tumor markers for breast cancer; Combining detection of CA125,CA153,CEA,FER could increase the diagnostic sensitivity of breast cancer;③The serum levels of CEA,FER, CA125 and CA153 are risk factors of ALNM,and related with the diameter of primary tumor;④Detection of serum CA153,CA125,FER and CEA levels is valuable for prognosis in the patients with breast cancer.⑤Combined detection of CEA,FER,CA125 and CA153 is valueable for judging ALNM,evaluation the effect of operation,observing recurrence and metastasis and evaluation of the progress and prognosis of breast cancer;CEA,FER,CA125 and CA153 is a better combination of tumor markers for breast cancer;⑥CA199,NSE,CA242,HCG,AFP,f-PSA, PSA,HGH have little value for breast cancer;⑦As an advanced statistical method,logistic regression can improve the diagnostic sensitivity and specificity.
引文
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