TMPRSS2和KLK11基因表达水平在良性前列腺增生症与前列腺癌中的应用
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摘要
研究背景和目的：
前列腺癌(PCa)和前列腺增生(BPH)是泌尿外科常见的老年男性疾病,我国13.6%的老年男性患前列腺增生症；在西方国家,前列腺癌死亡人数仅次于第一位的肺癌。当前临床诊断中主要使用前列腺特异抗原(PSA)作为前列腺癌和前列腺增生的主要鉴别诊断手段,用Gleason评分来进行前列腺癌病理分级,用肿瘤恶性程度进行前列腺癌细胞恶性分级,用TNM分期进行前列腺癌临床分期。然而由于PSA并非前列腺癌细胞所特有,前列腺增生、前列腺炎,甚至乳腺癌等多种疾病亦会引起血浆中PSA升高。当PSA在4-10ng/ml时,难以区分PCa和BPH。而进行Gleason评分时以及肿瘤细胞的恶性程度受到病理医生的主观看法和经验的影响,也不可能对前列腺癌的恶性程度进行客观,准确的判定。因此临床有必要寻找比PSA更可靠的诊断指标,比Gleason评分及肿瘤恶性程度分级更客观的病理分级指标。
近年来,人组织激肽释放酶(HK)家族日益被人们所重视。其中的KLK11基因在前列腺组织中特异性表达。雄激素通过激活雄激素受体(AR),一方面引发级联反应,导致前列腺癌的形成。另一方面,KLK11基因其5’端增强子的雄激素反应元件,与雄激素特异性地结合,激活基因表达KLK11分子,使KLK11表达量也在前列腺癌组织中上调表达。提示检测前列腺组织中KLK1 1基因的表达值可以有助于前列腺癌的早期诊断和恶性程度的评估。
TMPRSS2是一种跨膜丝氨酸蛋白酶,它在正常前列腺细胞和前列腺癌细胞中均有表达,并且也受到雄激素的调控。它通过调节细胞的增生、分化、凋亡以及与细胞间的相互作用在许多生理和病理过程中发挥重要的调控作用。
实时荧光定量PCR技术(fluorescence quantitative realtime polymerase chain reaction, FQ-RT-PCR),是指在PCR反应体系中加入荧光基团或染料,利用荧光信号的变化实时监测PCR反应过程,最后通过标准曲线对未知模板进行定量分析的方法。定量原理是PCR反应时发射的荧光信号达到检测域值时的循环数(Cycle threshold, Ct),即Ct值与模板的起始拷贝数的对数存在线性关系。起始拷贝数越多,Ct值越小。实时荧光定量RT-PCR在反转录反应后,进行实时荧光定量PCR反应,反应结果与标准曲线对照,可获得样品的起始模板量。
本论文利用实时荧光定量PCR对良性前列腺增生和前列腺癌的组织中TMPRSS2和KLK11基因表达量进行定量检测。结合临床中良性前列腺增生的病理诊断和前列腺癌的Gleason评分,肿瘤恶性程度分级以及前列腺癌的临床分期,探讨TMPRSS2和KLK11基因在前列腺癌的诊断和病理分级中的意义,对肿瘤的无进展生存期的指导作用,进一步认识TMPRSS2和KLK11基因的生物行为学特征。并探讨其数值对前列腺癌各项指标的临床指导意义。
材料和方法：
收集广州市第一人民医院泌尿外科2004年12月至20009年12月收治的74例前列腺癌病人和80前列腺良性增生病人的临床开放手术,TURP(经尿道前列腺切除术)切下的组织、穿刺活检组织。正常前列腺组织由行膀胱癌根治术后标本获得。取得样本后,马上放入液氮瓶中速冻收集保存。其中PCa患者平均年龄75岁；BPH患者平均年龄67岁；正常前列腺3例,平均年龄53岁。并记录石蜡切片观测样本的病理恶性程度分级,Gleason评分,记录病例的临床分期和肿瘤的无进展生存期。患者的随访终点为：PSA连续两次随访再次升高,以第一次升高的时间为随访终点；或者核素骨扫描提示骨转移；或者经直肠超声、CT、MR等影像学检查提示前列腺癌局部进展或死亡。随访最后截止日期：2009年12月31日。
运用实时荧光定量PCR测定标本中TMPRSS2和KLK11基因的表达量比较良性前列腺增生和前列腺癌TMPRSS2和KLK11基因表达值：(1)以临床样本提取组织样本。(2)应用Takara Taq HS试剂。(3)使用仪器为荧光定量PCR仪RotorGene 2000 (Corbett公司),荧光定量PCR分析软件用Rotor-gene v5。(4)提取mRNA。(5)从NCBI的gene bank中检索TMPRSS2和KLK11及β-actin基因的uniGene资料,从中获得各基因的Normal mRNA序列及相关uniSTS。用Vector NT6.0软件(Informax)定位相关uniSTS扩增片段在Normal mRNA中的位置,选择长度在100-300 bp,且位置近mRNA 3'端的uniSTS序列。最后再经NCBI网站的Blast软件分析和e-PCR软件确认该片段的特异性,剔除特异性较差和e-PCR结果中有重复的uniSTS引物。根据TMPRSS2和KLK11基因的mRNA序列及相关UniSTS序列,并考虑杂交和反转录标记的要求,以及ePCR结果,分析筛选到具有较好的特异性的序列。制作相应的TMPRSS2和KLK11探针序列和其相应的UniSTS引物序列。(6)对RNA样本进行实时荧光定量PCR。(7)制作标准曲线。(8)运用相对定量方法对数据进行处理。
统计学处理：
良性前列腺增生症和前列腺癌的TMPRSS2和KLK11 mRNA表达量(Ct值)进行独立样本t检验。将肿瘤恶性程度分为G1-G2以及G3两组,Gleason评分分为大于6以及小于等于6组,临床病理分期分为Ⅰ-Ⅱ组以及Ⅲ-Ⅳ组。对其肿瘤组织中的TMPRSS2和KLK11 mRNA表达量(Ct值)进行独立样本的t检验。对肿瘤恶性程度(G1-G2和G3), Gleason评分(小于等于6和大于等于6),肿瘤的临床分期(Ⅰ-Ⅱ和Ⅲ-Ⅳ)分组不同的无进展生存期差异,进行log-rank检验,并分别采用Kaplan-Meier方法绘制生存曲线；多因素分析采用Cox比例风险回归模型进行分析。将单因素分析中有统计学意义的临床病理因素：Gleason评分、临床分期、TMPRSS2/KLK11 Ct值纳入Cox比例风险回归模型进行综合分析,变量筛选采用逐步向前回归法,以P≤0.05为纳入标准,P≥0.10为剔除标准。
结果：
1、对良性前列腺增生症和前列腺癌的TMPRSS2和KLK11 mRNA表达量(Ct值)进行独立样本t检验,发现TMPRSS2和KLK11 mRNA表达量在良性前列腺增生症和前列腺癌中的表达量有显著性差异(P<0.05), TMPRSS2和KLK11 mRNA表达量在前列腺癌组织中比良性前列腺增生症组织中要高。
2、对TMPRSS2和KLK11 mRNA表达量(Ct值)在前列腺癌中不同的病理因素进行独立样本的t检验。发现在临床分期,Gleason评分,肿瘤恶性程度的不同分组中TMPRSS2和KLK11 mRNA表达量(Ct值)均有显著性差异(P<0.05)。TMPRSS2 mRNA表达量(Ct值)随临床分期,Gleason评分,肿瘤恶性程度升高而升高。KLK11 mRNA表达量(Ct值)随随临床分期,Gleason评分,肿瘤恶性程度升高而降低。
3、对各临床病理因素分组不同的无进展生存期差异,进行log-rank检验,发现肿瘤恶性程度、Gleason评分、临床分期、TMPRSS2/KLK11 Ct值在单因素分析中是前列腺癌无进展生存期的影响因素。然后分别对Gleason评分、临床分期、肿瘤恶性程度和TMPRSS2/KLK11 Ct值的不同分组绘制无进展生存曲线。在所有病例中,Gleason评分≤6分的病例无进展生存期长于Gleason评分>6分的病例(P<0.01, log-rank检验)。同样地,临床分期为Ⅰ-Ⅱ的比Ⅲ-Ⅳ的病例无进展生存期长(P<0.01, log-rank检验),肿瘤恶性程度分级为G1-G2的比G3的病例无进展生存期长(P<0.01, log-rank检验)。TMPRSS2/KLK11 Ct小于2的比TMPRSS2/KLK11 Ct值大于等于2分组的无进展生存期差异也有统计学意义(P<0.01, log-rank)。
4、将单因素分析中有统计学意义的三项临床病理因素：Gleason评分、临床分期、TMPRSS2/KLK11 Ct值纳入Cox比例风险回归模型进行综合分析,变量筛选采用逐步向前回归法,以P≤0.05为纳入标准,P≥0.10为剔除标准。最终确定Gleason评分(P<0.01)和TMPRSS2/KLK11 Ct值(P<0.01)是影响前列腺癌无进展生存期的主要因素。
结论：
1、良性前列腺增生症中前列腺组织的TMPRSS2和KLK11mRNA的表达值与前列腺癌中TMPRSS2和KLK11mRNA的表达值有显著差异,通过检测组织中TMPRSS2和KLK11mRNA的表达值可作为鉴别良性前列腺增生症和前列腺癌的指标。
2、前列腺癌组织中TMPRSS2和KLK11mRNA的表达值随着Gleason评分,临床分期,肿瘤恶性程度的不同而显示出差异。TMPRSS2mRNA表达值随着Gleason评分,临床分期和肿瘤恶性程度升高而表达增强。KLK11mRNA的表达值随着Gleason评分,临床分期和肿瘤恶性程度升高而表达下降。TMPRSS2和KLK11基因表达值的比值可作为前列腺癌病人无进展生存期的临床指标。
3、TMPRSS2/KLK11 Ct值有助于判断前列腺癌的无进展生存期,其数值小于2提示无进展生存期较长,其数值大于等于2提示无进展生存期较短。
Background and objective:
Prostate cancer (PCa) and benign prostate hyperplasia (BPH) are two commom diseases for the elder men. In China 13.6% of the elder men suffered from BPH, and PCa accounts for approximately 13% of all cancer deaths in America. In clinical, PSA is used for diagnosis of PCa, and Gleason score or the tumor malignancy degree to evaluate the prostate cancer's pathologic classes. The TNM clinic classify system is used to define the cancer class. However, as a result of the insufficent specifity of PCa, elevated serum PSA levels may be the outcomes of prostatitis and BPH, even breast cancer. In particularly, it is difficult to distinguish PCa from BPH when serum PSA level is at 4-10 ng/ml. Affected by the subjective and empirical opinion of the pathologist, the Gleason score and the tumor malignancy may be inaccurate. So searching more specific biomarkers for PCa and the pathology staging is pressing.
In these years, the human tissue kallikrein family is to be highly thought of by the researches. The gene of KLK11 specify expresses in the tissue of prostate. The androgen initiates cascade reaction via the activation of androgen receptor to lead to form the prostate cancer. On the other hand, the androgen response element in the KLK11 gene 5'termination enhanser combines with the androgen. They activate the gene to express KLK11 molecule to. The KLK11 gene amount expresses up-regulation. So it hints that we can evaluate the prostate cancer diagnosis early and the malignancy degree.
The gene TMPRSS2, a transmembrane serine protease, expresses in the normal prostate cell and prostate cancer cell. It is regulated by androgen. The TMPRSS2 gene plays an important role in many physiology and pathology process by regulating the cell hyperplasy, differentiation and apoptosis or interaction between cells.
Fluorescence quantitative realtime polymerase chain reaction(FQ-RT-PCR) is a technique which is adding fluorophore or dye agent in the PCR reaction system. It utilizes the fluorescence signal changes to monitor PCR reaction in real time. Eventually the unknown temple is analyzed by standard curve. The principle of quantization is the liner correlation between the fluorescence signals achieving the domain and the logarithm of the template onset copy number. The more number the template onset copy has, and less the Ct value will be. When the fluorescence quantitative realtime polymerase chain reaction begins to inverse reaction, the PCR will be carried out soon. The sample onset template numbers will be obtain as soon as the reaction result compared with the standard curve.
In this study, fluorescence quantitative realtime polymerase chain reaction is utilized to detect the quantity of the gene of TMPRSS2 and KLK11 expression in the BPH and PCa. To approach the meaning gene of TMPRSS2 and KLK11 expression in the prostate cancer diagnosis and pathology ranking, the BPH and PCa's Gleason score, tumor malignancy grade and clinical stage, progression-free survival time are combined to analyze. The genes of TMPRSS2 and KLK11 biology ethology and clinical guidance come to be known.
Material and method:
74 cases of PCa tissue and 80 BPH tissue were collected from December 2004 to December 2009. The cases were from open operation or TURP or puncture biopsy tissue. The normal prostate tissue were obtained from radical bladder cancer operation. The tissue were put into a liquid vase as soon as they were obtained. The average age of PCa patients was 75 years old, the average age of BPH patients was 67 years old, the average age of normal prostate patients was 53 years old. The pathology malignancy grade and Gleason score detected from paraffin section were to recorded as well as the clinical stage and progression-free survival time. The end of the follow up was defined as:When the PSA value continuous arise twice, the first rise time was the end of follow up; osseous metastasis were detected by nuclide bone scan; the prostate tumor region progressed were detected by transrectal ultrasound, CT,MR; the patients died. The time of deadline was 31th December 2009.
Fluorescence quantitative realtime polymerase chain reaction was used to determine the genes expression of TMPRSS2 and KLK11 in BPH and PCa. (1) The sample of tissue were obtained from clinical operation.(2) The Takara Taq HS agent was used. (3) The implements were:fluorescent quantization PCR meter RotorGene 2000(Corbett Co.), fluorescent quantization PCR analyze software was Rotor-gene v5. (4) mRNA was extracted. (5) The gene uniGene data was obtained from NCBI gene bank. The normal mRNA sequence was obtained. Vector NT6.0 software was used to locate the correlated uniSTS amplification fragment in Normal mRNA. The uniSTS sequence about 100-300 bp long and the location closes to mRNA 3'were selected. In the end, the fragments specificity were analyzed by NCBI Blast software and e-PCR software. The uniSTS primer of poor specificity and duplicate with e-PCR were rejected. According to the genes of TMPRSS2 and KLK11's mRNA sequence and UniSTS sequence, the better sequences were obtained. The probes of TMPRSS2 and KLK11 and the reciprocal UniSTS primer sequence.(6) Fluorescence quantitative realtime polymerase chain reactions were undertook for the sample of mRNA. (7) The standard curve was mede. (8) The date were dealed with relative quantitative method.
Statistical treatment:
Independence sample t test was undertook to compare the mRNA expression quantity of TMPRSS2 and KLK11. The malignancy grade of tumor was classified into G1-G2 and G3; Gleason score was classified into exceed 6 and smaller or equal to 6; clinical pathology staging was classified intoⅠ-ⅡandⅢ-Ⅳ. The mRNA expression quantity of TMPRSS2 and KLK11 in these groups was undertook independence sample t test. The progression-free survival time of malignancy grade(G1-G2 and G3), Gleason score (exceed 6 and smaller or equal to 6) and clinical pathology staging(Ⅰ-ⅡandⅢ-Ⅳ) were undertook log-rank test. The survival curves were made by Kaplan-Meier approach. The Cox proportional hazards regression was used to analyze multiplicity. The Gleason score, clinical staging, TMPRSS2/KLK11 value were included in Cox proportional hazards regression. Variance filting was adopted by step by step forward regression. P≤0.05 was internalized standard, P>0. 10 was rejected standard.
Statistical treatment:
The mean number of two sets of specimen measurement data was analyzed by t test. Log-rank test was deployed in the different factor of progression-free survival time. The Kaplan-Meier method was utilized to draw the survival curve; Cox proportional hazards regression models was utilized in the multiplicity.
Result:
1. Independence sample t test was undertook to compare the mRNA expression quantity of TMPRSS2 and KLK11. We found that the mRNA expression quantity of TMPRSS2 and KLK11 had significant difference between BPH and PCa (P<0.05). mRNA expression quantity of TMPRSS2 and KLK11 in PCa were higher than BPH.
2. The mRNA expression quantity of TMPRSS2 and KLK11 had significant difference between clinical stage, Gleason score and tumor malignancy grade (P<0.05). When the clinical stage, Gleason score and tumor malignancy grade stepped up, the mRNA expression quantity of TMPRSS2 stepped up too. When the clinical stage, Gleason score and tumor malignancy grade stepped up, the mRNA expression quantity of KLK11 stepped down.
3. The progression-free survival time of malignancy grade(G1-G2 and G3), Gleason score (exceed 6 and smaller or equal to 6),clinical pathology staging(I-II and III-IV) and ratio of TMPRSS2/KLK11 Ct value(exceed 2 and smaller or equal to 2) were undertook log-rank test. We found that the clinical stage, Gleason score, tumor malignancy grade and ratio of TMPRSS2/KLK11 Ct value were influential factors in prostate cancer progression-free survival time. The progression-free survival time survival curve were made according to malignancy grade, Gleason score and clinical pathology staging. In the cases, the progression-free survival time was longer in Gleason score smaller or equal to 6; clinical pathology staging ofⅠ-Ⅱwas longer; malignancy grade of G1-G2 was longer, TMPRSS2/KLK11 Ct value of smaller to 2 was longer(P<0.01, log-rank).
4. Gleason score, ratio of TMPRSS2/KLK11 Ct value and clinical pathology staging were included in Cox proportional hazards regression. Gleason score, ratio of TMPRSS2/KLK11 Ct value were found to be the main factor of prostate cancer progression-free survival time. Clinical pathology staging were found to be lost statistical significance in multiplicity.
Conclusion:
1. The mRNA expression quantity of TMPRSS2 and KLK11 had significant difference between BPH and PCa. BPH and PCa can be discriminated by detected The mRNA expression quantity of TMPRSS2 and KLK11 in the tissue of BPH and PCa.
2. The mRNA expression quantity of TMPRSS2 and KLK11 change with the Gleason score, clinical stage and tumor malignancy grade. The mRNA expression quantity of TMPRSS2 stepped up when Gleason score, tumor malignancy grade and clinical stage stepped up. The mRNA expression quantity of KLK11 stepped down when Gleason score, tumor malignancy grade and clinical stage stepped up. The ratio of TMPRSS2/KLK11 Ct value can act as a clinical index for the prostate cancer progression-free survival time.
3. The ratio of TMPRSS2/KLK11 Ct value helped to indicate the prostate cancer progression-free survival time. The prostate cancer progression-free survival time will be longer when the ratio less than 2. While the time will be shorter when the ratio exceed of equal to 2.
前列腺癌(PCa)和前列腺增生(BPH)是泌尿外科常见的老年男性疾病,我国13.6%的老年男性患前列腺增生症；在西方国家,前列腺癌死亡人数仅次于第一位的肺癌。当前临床诊断中主要使用前列腺特异抗原(PSA)作为前列腺癌和前列腺增生的主要鉴别诊断手段,用Gleason评分来进行前列腺癌病理分级,用肿瘤恶性程度进行前列腺癌细胞恶性分级,用TNM分期进行前列腺癌临床分期。然而由于PSA并非前列腺癌细胞所特有,前列腺增生、前列腺炎,甚至乳腺癌等多种疾病亦会引起血浆中PSA升高。当PSA在4-10ng/ml时,难以区分PCa和BPH。而进行Gleason评分时以及肿瘤细胞的恶性程度受到病理医生的主观看法和经验的影响,也不可能对前列腺癌的恶性程度进行客观,准确的判定。因此临床有必要寻找比PSA更可靠的诊断指标,比Gleason评分及肿瘤恶性程度分级更客观的病理分级指标。
近年来,人组织激肽释放酶(HK)家族日益被人们所重视。其中的KLK11基因在前列腺组织中特异性表达。雄激素通过激活雄激素受体(AR),一方面引发级联反应,导致前列腺癌的形成。另一方面,KLK11基因其5’端增强子的雄激素反应元件,与雄激素特异性地结合,激活基因表达KLK11分子,使KLK11表达量也在前列腺癌组织中上调表达。提示检测前列腺组织中KLK1 1基因的表达值可以有助于前列腺癌的早期诊断和恶性程度的评估。
TMPRSS2是一种跨膜丝氨酸蛋白酶,它在正常前列腺细胞和前列腺癌细胞中均有表达,并且也受到雄激素的调控。它通过调节细胞的增生、分化、凋亡以及与细胞间的相互作用在许多生理和病理过程中发挥重要的调控作用。
实时荧光定量PCR技术(fluorescence quantitative realtime polymerase chain reaction, FQ-RT-PCR),是指在PCR反应体系中加入荧光基团或染料,利用荧光信号的变化实时监测PCR反应过程,最后通过标准曲线对未知模板进行定量分析的方法。定量原理是PCR反应时发射的荧光信号达到检测域值时的循环数(Cycle threshold, Ct),即Ct值与模板的起始拷贝数的对数存在线性关系。起始拷贝数越多,Ct值越小。实时荧光定量RT-PCR在反转录反应后,进行实时荧光定量PCR反应,反应结果与标准曲线对照,可获得样品的起始模板量。
本论文利用实时荧光定量PCR对良性前列腺增生和前列腺癌的组织中TMPRSS2和KLK11基因表达量进行定量检测。结合临床中良性前列腺增生的病理诊断和前列腺癌的Gleason评分,肿瘤恶性程度分级以及前列腺癌的临床分期,探讨TMPRSS2和KLK11基因在前列腺癌的诊断和病理分级中的意义,对肿瘤的无进展生存期的指导作用,进一步认识TMPRSS2和KLK11基因的生物行为学特征。并探讨其数值对前列腺癌各项指标的临床指导意义。
材料和方法：
收集广州市第一人民医院泌尿外科2004年12月至20009年12月收治的74例前列腺癌病人和80前列腺良性增生病人的临床开放手术,TURP(经尿道前列腺切除术)切下的组织、穿刺活检组织。正常前列腺组织由行膀胱癌根治术后标本获得。取得样本后,马上放入液氮瓶中速冻收集保存。其中PCa患者平均年龄75岁；BPH患者平均年龄67岁；正常前列腺3例,平均年龄53岁。并记录石蜡切片观测样本的病理恶性程度分级,Gleason评分,记录病例的临床分期和肿瘤的无进展生存期。患者的随访终点为：PSA连续两次随访再次升高,以第一次升高的时间为随访终点；或者核素骨扫描提示骨转移；或者经直肠超声、CT、MR等影像学检查提示前列腺癌局部进展或死亡。随访最后截止日期：2009年12月31日。
运用实时荧光定量PCR测定标本中TMPRSS2和KLK11基因的表达量比较良性前列腺增生和前列腺癌TMPRSS2和KLK11基因表达值：(1)以临床样本提取组织样本。(2)应用Takara Taq HS试剂。(3)使用仪器为荧光定量PCR仪RotorGene 2000 (Corbett公司),荧光定量PCR分析软件用Rotor-gene v5。(4)提取mRNA。(5)从NCBI的gene bank中检索TMPRSS2和KLK11及β-actin基因的uniGene资料,从中获得各基因的Normal mRNA序列及相关uniSTS。用Vector NT6.0软件(Informax)定位相关uniSTS扩增片段在Normal mRNA中的位置,选择长度在100-300 bp,且位置近mRNA 3'端的uniSTS序列。最后再经NCBI网站的Blast软件分析和e-PCR软件确认该片段的特异性,剔除特异性较差和e-PCR结果中有重复的uniSTS引物。根据TMPRSS2和KLK11基因的mRNA序列及相关UniSTS序列,并考虑杂交和反转录标记的要求,以及ePCR结果,分析筛选到具有较好的特异性的序列。制作相应的TMPRSS2和KLK11探针序列和其相应的UniSTS引物序列。(6)对RNA样本进行实时荧光定量PCR。(7)制作标准曲线。(8)运用相对定量方法对数据进行处理。
统计学处理：
良性前列腺增生症和前列腺癌的TMPRSS2和KLK11 mRNA表达量(Ct值)进行独立样本t检验。将肿瘤恶性程度分为G1-G2以及G3两组,Gleason评分分为大于6以及小于等于6组,临床病理分期分为Ⅰ-Ⅱ组以及Ⅲ-Ⅳ组。对其肿瘤组织中的TMPRSS2和KLK11 mRNA表达量(Ct值)进行独立样本的t检验。对肿瘤恶性程度(G1-G2和G3), Gleason评分(小于等于6和大于等于6),肿瘤的临床分期(Ⅰ-Ⅱ和Ⅲ-Ⅳ)分组不同的无进展生存期差异,进行log-rank检验,并分别采用Kaplan-Meier方法绘制生存曲线；多因素分析采用Cox比例风险回归模型进行分析。将单因素分析中有统计学意义的临床病理因素：Gleason评分、临床分期、TMPRSS2/KLK11 Ct值纳入Cox比例风险回归模型进行综合分析,变量筛选采用逐步向前回归法,以P≤0.05为纳入标准,P≥0.10为剔除标准。
结果：
1、对良性前列腺增生症和前列腺癌的TMPRSS2和KLK11 mRNA表达量(Ct值)进行独立样本t检验,发现TMPRSS2和KLK11 mRNA表达量在良性前列腺增生症和前列腺癌中的表达量有显著性差异(P<0.05), TMPRSS2和KLK11 mRNA表达量在前列腺癌组织中比良性前列腺增生症组织中要高。
2、对TMPRSS2和KLK11 mRNA表达量(Ct值)在前列腺癌中不同的病理因素进行独立样本的t检验。发现在临床分期,Gleason评分,肿瘤恶性程度的不同分组中TMPRSS2和KLK11 mRNA表达量(Ct值)均有显著性差异(P<0.05)。TMPRSS2 mRNA表达量(Ct值)随临床分期,Gleason评分,肿瘤恶性程度升高而升高。KLK11 mRNA表达量(Ct值)随随临床分期,Gleason评分,肿瘤恶性程度升高而降低。
3、对各临床病理因素分组不同的无进展生存期差异,进行log-rank检验,发现肿瘤恶性程度、Gleason评分、临床分期、TMPRSS2/KLK11 Ct值在单因素分析中是前列腺癌无进展生存期的影响因素。然后分别对Gleason评分、临床分期、肿瘤恶性程度和TMPRSS2/KLK11 Ct值的不同分组绘制无进展生存曲线。在所有病例中,Gleason评分≤6分的病例无进展生存期长于Gleason评分>6分的病例(P<0.01, log-rank检验)。同样地,临床分期为Ⅰ-Ⅱ的比Ⅲ-Ⅳ的病例无进展生存期长(P<0.01, log-rank检验),肿瘤恶性程度分级为G1-G2的比G3的病例无进展生存期长(P<0.01, log-rank检验)。TMPRSS2/KLK11 Ct小于2的比TMPRSS2/KLK11 Ct值大于等于2分组的无进展生存期差异也有统计学意义(P<0.01, log-rank)。
4、将单因素分析中有统计学意义的三项临床病理因素：Gleason评分、临床分期、TMPRSS2/KLK11 Ct值纳入Cox比例风险回归模型进行综合分析,变量筛选采用逐步向前回归法,以P≤0.05为纳入标准,P≥0.10为剔除标准。最终确定Gleason评分(P<0.01)和TMPRSS2/KLK11 Ct值(P<0.01)是影响前列腺癌无进展生存期的主要因素。
结论：
1、良性前列腺增生症中前列腺组织的TMPRSS2和KLK11mRNA的表达值与前列腺癌中TMPRSS2和KLK11mRNA的表达值有显著差异,通过检测组织中TMPRSS2和KLK11mRNA的表达值可作为鉴别良性前列腺增生症和前列腺癌的指标。
2、前列腺癌组织中TMPRSS2和KLK11mRNA的表达值随着Gleason评分,临床分期,肿瘤恶性程度的不同而显示出差异。TMPRSS2mRNA表达值随着Gleason评分,临床分期和肿瘤恶性程度升高而表达增强。KLK11mRNA的表达值随着Gleason评分,临床分期和肿瘤恶性程度升高而表达下降。TMPRSS2和KLK11基因表达值的比值可作为前列腺癌病人无进展生存期的临床指标。
3、TMPRSS2/KLK11 Ct值有助于判断前列腺癌的无进展生存期,其数值小于2提示无进展生存期较长,其数值大于等于2提示无进展生存期较短。
Background and objective:
Prostate cancer (PCa) and benign prostate hyperplasia (BPH) are two commom diseases for the elder men. In China 13.6% of the elder men suffered from BPH, and PCa accounts for approximately 13% of all cancer deaths in America. In clinical, PSA is used for diagnosis of PCa, and Gleason score or the tumor malignancy degree to evaluate the prostate cancer's pathologic classes. The TNM clinic classify system is used to define the cancer class. However, as a result of the insufficent specifity of PCa, elevated serum PSA levels may be the outcomes of prostatitis and BPH, even breast cancer. In particularly, it is difficult to distinguish PCa from BPH when serum PSA level is at 4-10 ng/ml. Affected by the subjective and empirical opinion of the pathologist, the Gleason score and the tumor malignancy may be inaccurate. So searching more specific biomarkers for PCa and the pathology staging is pressing.
In these years, the human tissue kallikrein family is to be highly thought of by the researches. The gene of KLK11 specify expresses in the tissue of prostate. The androgen initiates cascade reaction via the activation of androgen receptor to lead to form the prostate cancer. On the other hand, the androgen response element in the KLK11 gene 5'termination enhanser combines with the androgen. They activate the gene to express KLK11 molecule to. The KLK11 gene amount expresses up-regulation. So it hints that we can evaluate the prostate cancer diagnosis early and the malignancy degree.
The gene TMPRSS2, a transmembrane serine protease, expresses in the normal prostate cell and prostate cancer cell. It is regulated by androgen. The TMPRSS2 gene plays an important role in many physiology and pathology process by regulating the cell hyperplasy, differentiation and apoptosis or interaction between cells.
Fluorescence quantitative realtime polymerase chain reaction(FQ-RT-PCR) is a technique which is adding fluorophore or dye agent in the PCR reaction system. It utilizes the fluorescence signal changes to monitor PCR reaction in real time. Eventually the unknown temple is analyzed by standard curve. The principle of quantization is the liner correlation between the fluorescence signals achieving the domain and the logarithm of the template onset copy number. The more number the template onset copy has, and less the Ct value will be. When the fluorescence quantitative realtime polymerase chain reaction begins to inverse reaction, the PCR will be carried out soon. The sample onset template numbers will be obtain as soon as the reaction result compared with the standard curve.
In this study, fluorescence quantitative realtime polymerase chain reaction is utilized to detect the quantity of the gene of TMPRSS2 and KLK11 expression in the BPH and PCa. To approach the meaning gene of TMPRSS2 and KLK11 expression in the prostate cancer diagnosis and pathology ranking, the BPH and PCa's Gleason score, tumor malignancy grade and clinical stage, progression-free survival time are combined to analyze. The genes of TMPRSS2 and KLK11 biology ethology and clinical guidance come to be known.
Material and method:
74 cases of PCa tissue and 80 BPH tissue were collected from December 2004 to December 2009. The cases were from open operation or TURP or puncture biopsy tissue. The normal prostate tissue were obtained from radical bladder cancer operation. The tissue were put into a liquid vase as soon as they were obtained. The average age of PCa patients was 75 years old, the average age of BPH patients was 67 years old, the average age of normal prostate patients was 53 years old. The pathology malignancy grade and Gleason score detected from paraffin section were to recorded as well as the clinical stage and progression-free survival time. The end of the follow up was defined as:When the PSA value continuous arise twice, the first rise time was the end of follow up; osseous metastasis were detected by nuclide bone scan; the prostate tumor region progressed were detected by transrectal ultrasound, CT,MR; the patients died. The time of deadline was 31th December 2009.
Fluorescence quantitative realtime polymerase chain reaction was used to determine the genes expression of TMPRSS2 and KLK11 in BPH and PCa. (1) The sample of tissue were obtained from clinical operation.(2) The Takara Taq HS agent was used. (3) The implements were:fluorescent quantization PCR meter RotorGene 2000(Corbett Co.), fluorescent quantization PCR analyze software was Rotor-gene v5. (4) mRNA was extracted. (5) The gene uniGene data was obtained from NCBI gene bank. The normal mRNA sequence was obtained. Vector NT6.0 software was used to locate the correlated uniSTS amplification fragment in Normal mRNA. The uniSTS sequence about 100-300 bp long and the location closes to mRNA 3'were selected. In the end, the fragments specificity were analyzed by NCBI Blast software and e-PCR software. The uniSTS primer of poor specificity and duplicate with e-PCR were rejected. According to the genes of TMPRSS2 and KLK11's mRNA sequence and UniSTS sequence, the better sequences were obtained. The probes of TMPRSS2 and KLK11 and the reciprocal UniSTS primer sequence.(6) Fluorescence quantitative realtime polymerase chain reactions were undertook for the sample of mRNA. (7) The standard curve was mede. (8) The date were dealed with relative quantitative method.
Statistical treatment:
Independence sample t test was undertook to compare the mRNA expression quantity of TMPRSS2 and KLK11. The malignancy grade of tumor was classified into G1-G2 and G3; Gleason score was classified into exceed 6 and smaller or equal to 6; clinical pathology staging was classified intoⅠ-ⅡandⅢ-Ⅳ. The mRNA expression quantity of TMPRSS2 and KLK11 in these groups was undertook independence sample t test. The progression-free survival time of malignancy grade(G1-G2 and G3), Gleason score (exceed 6 and smaller or equal to 6) and clinical pathology staging(Ⅰ-ⅡandⅢ-Ⅳ) were undertook log-rank test. The survival curves were made by Kaplan-Meier approach. The Cox proportional hazards regression was used to analyze multiplicity. The Gleason score, clinical staging, TMPRSS2/KLK11 value were included in Cox proportional hazards regression. Variance filting was adopted by step by step forward regression. P≤0.05 was internalized standard, P>0. 10 was rejected standard.
Statistical treatment:
The mean number of two sets of specimen measurement data was analyzed by t test. Log-rank test was deployed in the different factor of progression-free survival time. The Kaplan-Meier method was utilized to draw the survival curve; Cox proportional hazards regression models was utilized in the multiplicity.
Result:
1. Independence sample t test was undertook to compare the mRNA expression quantity of TMPRSS2 and KLK11. We found that the mRNA expression quantity of TMPRSS2 and KLK11 had significant difference between BPH and PCa (P<0.05). mRNA expression quantity of TMPRSS2 and KLK11 in PCa were higher than BPH.
2. The mRNA expression quantity of TMPRSS2 and KLK11 had significant difference between clinical stage, Gleason score and tumor malignancy grade (P<0.05). When the clinical stage, Gleason score and tumor malignancy grade stepped up, the mRNA expression quantity of TMPRSS2 stepped up too. When the clinical stage, Gleason score and tumor malignancy grade stepped up, the mRNA expression quantity of KLK11 stepped down.
3. The progression-free survival time of malignancy grade(G1-G2 and G3), Gleason score (exceed 6 and smaller or equal to 6),clinical pathology staging(I-II and III-IV) and ratio of TMPRSS2/KLK11 Ct value(exceed 2 and smaller or equal to 2) were undertook log-rank test. We found that the clinical stage, Gleason score, tumor malignancy grade and ratio of TMPRSS2/KLK11 Ct value were influential factors in prostate cancer progression-free survival time. The progression-free survival time survival curve were made according to malignancy grade, Gleason score and clinical pathology staging. In the cases, the progression-free survival time was longer in Gleason score smaller or equal to 6; clinical pathology staging ofⅠ-Ⅱwas longer; malignancy grade of G1-G2 was longer, TMPRSS2/KLK11 Ct value of smaller to 2 was longer(P<0.01, log-rank).
4. Gleason score, ratio of TMPRSS2/KLK11 Ct value and clinical pathology staging were included in Cox proportional hazards regression. Gleason score, ratio of TMPRSS2/KLK11 Ct value were found to be the main factor of prostate cancer progression-free survival time. Clinical pathology staging were found to be lost statistical significance in multiplicity.
Conclusion:
1. The mRNA expression quantity of TMPRSS2 and KLK11 had significant difference between BPH and PCa. BPH and PCa can be discriminated by detected The mRNA expression quantity of TMPRSS2 and KLK11 in the tissue of BPH and PCa.
2. The mRNA expression quantity of TMPRSS2 and KLK11 change with the Gleason score, clinical stage and tumor malignancy grade. The mRNA expression quantity of TMPRSS2 stepped up when Gleason score, tumor malignancy grade and clinical stage stepped up. The mRNA expression quantity of KLK11 stepped down when Gleason score, tumor malignancy grade and clinical stage stepped up. The ratio of TMPRSS2/KLK11 Ct value can act as a clinical index for the prostate cancer progression-free survival time.
3. The ratio of TMPRSS2/KLK11 Ct value helped to indicate the prostate cancer progression-free survival time. The prostate cancer progression-free survival time will be longer when the ratio less than 2. While the time will be shorter when the ratio exceed of equal to 2.
引文
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[2]Jemal A, Siegel R, Ward E, Murray T, Xu J, Smigal C, and Thun MJ (2006). Cancer statistics,2006. CA Cancer J Clin 56,106-130.
[3]Peyromaure M, Debre B, Mao K, etal. Management of prostate cancer in China: a multicenter report of 6 institutions. J Urol.2005; 174:1794-1797.
[4]顾方六.现代前列腺病学.北京：人民军医出版社,2003：276-278.
[5]Black MH, Diamandis EP. The diagnostic and prognostic utility of prostate specific antigen for diseases of the breast. Breast Cancer Res Treat 2000;59: 1-14.
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