【作者】 王辉；

【导师】 吕申；

【作者基本信息】 大连医科大学 ， 病理学与病理生理学， 2009， 博士

【摘要】 目前,超声影像学诊断前列腺癌主要依靠这些征象:回声以低回声为主、外形不规则,有毛刺、高血运分级、高收缩期峰值血流速度(Vs)、高阻力指数(RI)。依靠这些征象,大部分前列腺癌可以确诊。此外,在前列腺癌内,还常常可以看到周边声晕、微钙化、后方回声衰减等超声征象。这些征象对甲状腺癌及乳腺癌的诊断很重要,但对前列腺癌诊断率的提高有无帮助,研究的却很少。另外低回声征象在前列腺癌的诊断中很重要,但良性增生结节也有很多呈低回声;各与血流有关的征象对前列腺癌的诊断也很重要,但有学者认为前列腺内外腺血流特点有差异,研究内腺低回声结节良恶性的鉴别及内外腺前列腺癌血流灌注特点,对我们认识这些征象对前列腺癌的诊断价值有一定的意义。前列腺癌任何影像学的改变都以微观病理的改变为基础,都与分子生物学因子的表达有密切的关系。这些超声征象与癌灶的生物学行为的关系,我们觉得值得进一步研究。如果知道癌灶影像学征象与某些分子生物学因子表达的相关性,就可以通过影像学表现无创的判断该癌灶微观分子生物学特性,为判断癌灶的发生发展及预后,提供有力的证据。目前,在放射线领域,肺癌、肝癌这类的研究较多。但超声领域,这方面的研究国内外罕见报道。目的:我们首先筛选出对前列腺癌有诊断价值的超声征象,再研究前列腺癌组织中与肿瘤增殖分化、浸润转移、促血管生成密切相关因子的蛋白表达情况,最后分析各超声征象与这些因子蛋白表达的相关性。探讨前列腺癌超声征象的分子生物学基础,为无创判断前列腺癌的发展预后提供理论依据。方法:一、对前列腺癌有诊断价值超声征象的筛选1、前列腺癌灶与良性增生结节超声特征的比较选取2005年至2009年来我院行经直肠彩超检查的患者,前列腺癌及增生患者均经穿刺活检病理确诊。前列腺癌74例,均为腺癌。增生51例,做为对照。回顾分析所有病例的经直肠彩超资料,比较两组病灶在回声情况(低回声)、外形(不规则有毛刺)、后方衰减、声晕、微钙化出现率,血运分级、收缩期峰值血流速度(Vs)及阻力指数(RI)大小方面的差异。2、内腺低回声结节良恶性的鉴别上述74例前列腺癌中选取癌灶位于内腺的,且呈低回声的患者18例,51例良性增生组中选取内腺有低回声良性增生结节者31例。回顾分析两组病例的经直肠彩超资料,比较两组病灶在外形(不规则有毛刺)、后方衰减、微钙化出现率,Vs及RI大小方面的差异。3、正常前列腺、前列腺癌血流灌注的研究从上述74例前列腺癌患者中选取19例,癌灶位于外腺者11例,位于内腺者8例。另外选取2005年至2008年来我院就诊的怀疑前列腺疾病,最终诊断为正常者16例。分别行经直肠超声造影检查。回顾性分析他们造影检查资料,分别比较正常者前列腺外腺与内腺、前列腺癌患者外腺癌灶与内腺癌灶造影增强起始时间、显影终止时间有无差别。二、各因子蛋白表达特点及与临床病理的关系上述74例前列腺癌进行分类。组织分化:中高分化癌23例,低分化癌51例;临床分期:A期+B期40例,C期+D期34例。增生51例为对照。将所收集的穿刺活检标本制成点阵切片。通过免疫组化的方法研究前列腺癌组织EGFR、k-ras、Braf、hMSH2、hMSH6、MMP2、MMP9、VEGF蛋白表达特点,并计数MVD,分析它们与临床病理的关系。三、各超声特征与各因子蛋白表达相关性分析总结上述74例前列腺癌病例的超声征象:回声情况、外形(不规则有毛刺)、后方衰减、微钙化有无、血运分级、Vs及RI,结合各病例EGFR、k-ras、Braf、hMSH2、hMSH6、MMP2、MMP9、VEGF蛋白表达特点及MVD,利用统计学方法分析各超声特点与各因子蛋白表达的关系。结果:一、对前列腺癌有诊断价值超声征象的筛选1、前列腺癌灶与良性增生结节超声特征的比较前列腺癌组与良性增生组中低回声病灶所占比例分别为56.76%,35.90% (p<0.05),外形不规则有毛刺者所占比例分别为85.14%,15.38%(p<0.05),微钙化者所占比例分别为39.19%,10.26%(p<0.05),有后方衰减者所占比例分别为41.89%,12.82%(p<0.05),Vs分别为44.00±15.30,17.32±4.6(5p<0.05), RI分别为0.76±0.10, 0.51±0.03(p<0.05)。前列腺癌组与良性增生组与血运分级有明显相关性(r=-0.388,p<0.01),前列腺癌组较良性增生组血运分级要高。有声晕者所占比例分别为35.14%,38.46%(p>0.05)。2、内腺低回声结节良恶性的鉴别低回声癌结节组与低回声良性增生结节组中外形不规则有毛刺者所占比例分别为88.89%,12.90%(p<0.05),有微钙化者所占比例分别为38.89%,19.68%(p<0.05),有后方衰减者所占比例分别为44.44%,16.13%(p<0.05)。Vs分别为55.5±3.67, 19.42±1.61(p<0.05), RI分别1.14±0.13,0.45±0.04(p<0.05)。3、正常前列腺、前列腺癌血流灌注的研究正常内腺组与外腺组造影增强起始时间、显影终止时间无明显差别(P>0.05),癌灶组与外腺癌灶组增强起始时间、显影终止时间也无明显差别(P>0.05)。二、前列腺癌组织各因子蛋白表达特点及与临床病理的关系(一)各因子蛋白表达特点前列腺癌与增生EGFR蛋白表达阳性率分别为59.46%,10.26%( P<0.05 )。K-ras79.73% , 23.08% ( P<0.05 )。Braf64.86% , 10.26%(P<0.05)。hMSH2 55.41%、23.08%(P<0.05)。hMSH6 72.97%、15.38%(P<0.05)。MMP2 67.57%,15.38%(P<0.05)。MMP9 67.57%,15.38%(P<0.05)。VEGF 70.27%,12.82%(P<0.05)。MVD分别为36.41±9.14, 10.68±4.29 (P<0.05)。(二)与临床病理的关系1、与年龄的关系≤60岁前列腺癌病例与>60岁病例EGFR蛋白表达阳性率分别为47.62%、75.00%( P<0.05)。K-ras45.24%、93.75%( P<0.05)。两组间Braf、hMSH2、hMSH6、MMP2、MMP9、VEGF、MVD均无明显差异,(P>0.05)。2、与组织分化的关系中高分化前列腺癌与低分化前列腺癌Braf蛋白表达阳性率分别为43.48%、74.51%(P<0.05)。MMP2 47.83%、76.47%(P<0.05)。MMP9 43.48%、92.16%( P<0.05)。VEGF47.83%、80.39%( P<0.05)。MVD分别为29.70±6.30、39.43±8.63(P<0.05)。两组间EGFR、K-ras、hMSH2、hMSH6蛋白表达阳性率无明显差异(P>0.05)。3、与临床分期的关系A+B期前列腺癌与C+D期前列腺癌Braf蛋白表达阳性率分别为42.50%、91.18%( P<0.05)。VEGF 50.00%、94.12%( P<0.05)。MMP2 45.00%、94.12%。MMP9 45.00%、94.12%(P<0.05)。MVD 32.98±8.57、40.44±8.20(P<0.05)。两组间EGFR、K-ras。、hMSH2、hMSH6蛋白表达阳性率无明显差异(P>0.05)。4、EGFR、K-ras、Braf蛋白表达彼此相关性K-ras蛋白表达与Braf蛋白表达之间呈正相关, r=0.272,p<0.05;K-ras与EGFR正相关, r=0.615,p<0.001;EGFR与Braf之间有明正相关,r=0.269,p<0.05。5、MMP2、MMP9、VEGF蛋白表达、MVD彼此相关性前列腺癌VEGF蛋白表达与MVD之间呈正相关,r=0.436,p<0.001;VEGF与MMP2正相关, r=0.468,p<0.001;VEGF与MMP9正相关, r=0.534,p<0.001;MVD与MMP2正相关,r=0.445,p<0.001;MVD与MMP9正相关,r=0.570,p<0.001;MMP2与MMP9之间有正相关,r=0.777,p<0.001。三、各超声征象与各因子表达的关系1、癌灶等回声9例,低回声42例,高回声5例,混合回声18例。前列腺癌灶回声不同与VEGF(r=0.305,p=0.008)、MMP2(r=0.325,p=0.005)、MMP9(r=0.340,p=0.003)蛋白表达有明显的关系。其中混合回声癌灶各蛋白表达阳性率均最高,等回声癌灶各蛋白表达阳性率均最低。2、无外形(不规则、有毛刺)征象癌灶11例,有征象癌灶63例。前列腺癌灶外形(不规则、有毛刺)征象与MMP2(r=0.331,p=0.004)、MMP9(r=0.283,p=0.015)、VEGF(r=0.547、p<0.01)、MVD计数(r=0.279,p=0.016),蛋白表达有明显相关性。有声晕癌灶各分子蛋白表达更高。3、无后方衰减征象的癌灶43例,有后方回声衰减征象的癌灶31例。后方回声衰减征象与MMP2蛋白表达呈明显相关性,r=0.237,p=0.042。有后方回声衰减征像的癌灶MMP2蛋白表达更高。4、无微钙化癌灶45例,有微钙化癌灶29例。前列腺癌灶微钙化征象与k-ras(r=0.398,p<0.01)、EGFR(r=0.268,p=0.021)蛋白表达有明显相关性。有微钙化癌灶蛋白表达更高。5、癌灶血运分级0级16例, 1级38例, 2级16例, 3级4例。前列腺癌血运分级与MVD(r=0.587,p<0.01)、VEGF(r=0.432,p<0.01)、Braf (r=0.284,p=0.014)、MMP2(r=0.547,p<0.01)、MMP9(r=0.833,p<0.01)蛋白表达呈明显正相关。6、收缩期峰值血流速度(Vs)≤50cm/s癌灶43例,>50cm/s癌灶31例。前列腺癌收缩期峰值血流速度(Vs)与MVD(r=0.508,p<0.01)、VEGF(r=0.354,p=0.002)、EGFR(r=0.231,p=0.035)、MMP2(r=0.307,p=0.014)蛋白表达有明显相关性。血流速度越高蛋白表达越高。7、阻力指数(RI)≤0.8的癌灶32例, RI>0.8的癌灶42例。前列腺癌灶阻力指数(RI)与MVD计数、VEGF、Braf、k-ras、EGFR、MMP2、MMP9、hMSH2、hMSH6蛋白表达都没有明显相关性。结论:1、前列腺癌灶回声与MMP2、MMP9、VEGF蛋白表达呈明显的相关性。可以反映前列腺癌的浸润转移能力及预后。混合回声癌灶浸润转移性较强,预后较差,等回声癌灶浸润转移性较弱,预后相对较好。2、外形(不规则、有毛刺)征象与MMP2、MMP9、VEGF蛋白表达及MVD有明显相关性。可以反映前列腺癌的浸润转移能力及预后。3、后方衰减征象与MMP2蛋白表达有明显相关性。可以反映前列腺癌的浸润转移能力及预后。4、微钙化征象与EGFR、k-ras蛋白表达有明显相关性。可以反映前列腺癌的增殖分化能力。5、血运分级与Braf、MMP2、MMP9、VEGF蛋白表达及MVD呈明显的正相关,主要反映前列腺癌的浸润转移能力及预后。6、Vs大小与EGFR、MMP2、VEGF、蛋白表达及MVD有明显相关性。可以反映前列腺癌的浸润转移能力及预后。7、RI与各因子蛋白表达都没有明显关系。究竟能反映前列腺癌的哪些生物学特性,还需进一步研究。更多还原

【Abstract】 By using transrectal ultrasound (TRUS), prostate cancer (PCa) could be found out by certain signs, such as hypoechoic lesion, morphous (irregular, thorn-like), high grade in blood flow classification, increased peak velocity in systolic (Vs), high resistant index (RI). According to these sings, most prostate cancer can be diagnosed.Meanwhile, some other signs could also be detected in prostate cancer, such as echo halo marginal, micro-calcification and posterior acoustic attenuation. Those sings are of grate importance in thyroid and breast cancer diagnosis. Whether these signs are useful in diagnosis of prostate cancer is remain unclear.Because hypoechoic lesion and blood flow are important for prostate cancer diagnosis,it is important to study the hypoechoic lesion in prostate inner gland and the character of blood flow in normal prostate and prostate cancer.Changes in ultrasonic images were based on the changes of pathology, and such changes were also related to changes in molecular expression. But the relationship between these changes was indefinite.Once the relationship between ultrasound manifestation and molecular basis have been clarified, the micro molecular behavior could be judged by noninvasive ultrasound, and could provide a proof in prognosis of prostate cancer and predicting the process of prostate cancer. Similar researches have been in progress in radiation field, while the studies with ultrasound are rare.Objectives:After screening the ultrasonic manifestation which may be helpful to prostate cancer diagnosis and expression of proteins which related to tumor proliferation and differentiation, invasion and metastasis, blood vessel proliferation factors, we analyzed the relationship between those factors protein expression and ultrasonic manifestations to discuss the micro- biology basis of ultrasonic manifestation and to provide theoretical basis for predict prognosis noninvasively.Methods:1. screening the ultrasonic manifestation in prostate cancer(1) Ultrasonic features compared between prostate cancer and hyper- plasia of prostate:Patients who had undergone TRUS exam and TRUS guided biopsy proved to be prostate cancer and benign prostate hyperplasia (BPH) were selected from 2005 to 2009. 74 cases were PCa, and 51 cases were BPH. Retrospective analysis TRUS data in all cases, the differences between two groups were compared, including ultrasonic manifestation (hypoechoic), morphous (irregular, thorn-like), posterior acoustic attenuation, micro- calcification, blood flow classification, Vs, RI.(2) ultrasound manifestation of hypoechoic lesion in prostate inner cancer and BPH were compared18 hypoechoic lesion case in prostate inner cancer were selected from 74 PCa cases,31 hypoechoic lesion case in BPH were selected from 51 BPH cases, the differences between two groups were compared, including morphous (irregular, thorn-like), posterior acoustic attenuation, micro- calcification, Vs, RI.(3) the character of blood flow in normal prostate and prostate cancer.11 prostate outer gland cancer cases and 8 inner gland cancer cases were selected from above 74 PCa cases . 16 normal cases were selected from the patients who had undergone TRUS exam 2005 to 2009. Observed the starting time and the ending time of Transrectal contrast enhanced ultrasonography in the prostate inner gland(16 cases)、the prostate outer gland(16 cases)、the prostate inner gland cancer lesion(8 cases)、and prostate outer gland cancer Lesion.(11 cases).2. The relationship between features of factors expression and clinical pathology in prostate cancer 74 cases were classified in groups. According to histodifferentiation, 23 cases were middle-well differentiated and 51 poorly differentiated. According to the Whitmore-Jewett clinical staging system, all cases were divided into A+B stage (40 cases) and C+D stage (34 cases). 51 BPH cases were include as comparison group. The TRUS guided biopsy tissue were collected and be made as dot matrix slices. Immunohistochemical characters of EGFR,k-ras ,Braf ,hMSH2 ,hMSH6 , MMP2, MMP9,VEGF and MVD expression and the their relationship with clinical pathology were studied.3. Relationship between utrasonic features and factorsWe summerize TRUS manifestations characters of 74 PCa, including echotexture, morphous (irregular, thorn-like), posterior acoustic attenuation, with or without micro-calcification, blood flow classification, Vs, RI, combined with expression characters of EGFR,k-ras, Braf, hMSH2, hMSH6, MMP2, MMP9, VEGF and MVD, to find out the relationship between the TRUS manifestation and expression of factors protein by statistical methods.Results:1. screening the ultrasonic manifestation in prostate cancer(1) Ultrasonic features compared between prostate cancer and hyperplasia of prostate:The percentage of hypoechoic lesion in prostate cancer and hyperplasia of prostate was 56.76% and 35.90% respectively (P<0.05), morphous of irregular and thorn-like in prostate cancer and hyperplasia of prostate was 85.14% and 15.38% respectively(p<0.05), micro-calcification was 39.19% and 10.26% respectively( p<0.05) , posterior acoustic attenuation was 41.89% and 12.82% respectively ( p<0.05 ) , Vs was 44.00±15.30 and19.42±1.61 respectively ( p<0.05 ) and RI was1.14±0.13 and 0.45±0.04respectively(p<0.05).(2) ultrasound manifestation of hypoechoic lesion in prostate inner cancer and BPH were compared.morphous of irregular and thorn-like in hypoechoic lesion in prostate inner cancer and BPH was 88.89% and 12.90% respectively(p<0.05), micro-calcification was 38.89% and 19.68% respectively ( p<0.05 ) , posterior acoustic attenuation was 44.44% and 16.13% respectively(p< 0.05), Vs was 55.50±3.67 and 17.32±4.65 respectively(p<0.05)and RI was 0.76±0.10 and 0.51±0.03 respectively(p<0.05). Flow grade was related significantly to prostate cancer and hyperplasia of prostate(r=-0.388,p<0.01) and the grade of prostate cancer were higher than hyperplasia of prostate. The percentage of echo halo marginal in prostate cancer and hyperplasia of prostate was 35.14% and 38.46% respectively(p>0.05).(3) the character of blood flow in normal prostate and prostate cancer. there was no significant difference between the starting time of the prostate inner gland(27.36±3.05s)and the prostate outer gland (25.13±2.54s),no significant difference between the starting time of the inner gland cancer lesion (14.72±1.81s) and the outer gland cancer lesion (16.32±1.98s) too. the ending time of the prostate inner gland ,the prostate outer gland , the inner gland cancer lesion and the outer gland cancer Lesion had no difference.2. The relationship between features of factors expression and clinical pathology in prostate cancer(1) Features of factors expression:EGFR positive expression in prostate cancer and hyperplasia of prostate was 59.46% and 10.26% respectively(P<0.05), Braf was 64.86% and 10.26% respectively( P<0.05) , hMSH2 was 55.41% and 23.08% respectively ( P<0.05 ) , hMSH6 was 72.97% and 15.38% respectively(P<0.05), MMP2 was 67.57% and 15.38% respectively(P<0.05), MMP9 was 67.57% and 15.38% respectively(P<0.05), VEGF was 70.27% and 12.82% respectively(P<0.05) and MVD was36.41±9.14 and 10.68±4.29 respectively(P<0.05).(2) Relationship of clinical pathology1) Relationship of ages:Percentage of EGFR positive expression in prostate cancer with age not older than 60 and older than 60 was 47.62% and 75.00% respectively(P<0.05), K-ras was 45.24% and 93.75% respectively(P<0.05). There was no significant difference between two age groups in Braf , hMSH2, hMSH6, MMP2, MMP9, VEGF and MVD expression(P>0.05).2) Relationship of histological differentiation:Percentage of Braf positive expression in middle-well differentiation and poor differentiation was 43.48% and 74.51% respectively(P<0.05), MMP2 was 47.83% and 76.47% respectively(P<0.05), MMP9 was 43.48% and 92.16% respectively ( P<0.05 ) , VEGF was 47.83% and 80.39% respectively ( P<0.05 ) and MVD was 29.70±6.30 and 39.43±8.63 respectively(P<0.05).There was no significant difference between two groups in positive expression of EGFR,K-ras,hMSH2,hMSH6(P>0.05).3) Relationship of clinical stages:Percentage of Braf positive expression in A+B stages and C+D stages was 42.50% and 91.18% respectively(P<0.05), VEGF was 50.00% and 94.12% respectively(P<0.05), MMP2 was 45.00% and 94.12% respectively(P<0.05), MMP9 was 45.00% and 94.12% respectively(P<0.05) and MVD was 32.98±8.57 and 40.44±8.20 respectively(P<0.05)。There was no significant difference between two groups in positive expression of EGFR, K-ras, hMSH2 and hMSH6(P>0.05)4) Relationship between EGFR, K-ras and BrafThere was a positive correlation between expression of K-ras and Braf ( r=0.272,p<0.05) , K-ras and EGFR (r=0.615,p<0.001) and EGFR was positively correlated with Braf significantly (r=0.269,p<0.05).5) Relationship between expression of MMP2, MMP9, VEGF and MVD.There was a positive correlation between expression of VEGF and MVD (r=0.436, p<0.001) , VEGF and MMP2 (r=0.468, p<0.001) , VEGF and MMP9 (r=0.534, p<0.001), MVD and MMP2 (r=0.445, p<0.001), MVD and MMP9 (r=0.570, p<0.001) and MMP2 and MMP9 (r=0.777, p<0.001) respectively.3. Relationship between utrasonic features and factors:(1) There were 9 cases of isoechogenicity, 42 cases of hypo- echogenicity, 5 cases of hyperechogenicity and 18 cases of mixed- echogenicity. The difference of echogenicity were related significantly to expression of VEGF(r=0.305, p=0.008), MMP2(r=0.325,p=0.005) and MMP9(r=0.340,p=0.003). The expression of these factors was of the highest in mixed echogenicity and lowest in isoechogenicity.(2) There were 11 cases without features in morphous (irregular and thorn-like) and 63 cases with these features. The features in morphous were related significantly to expression of MMP2(r=0.331, p=0.004), MMP9(r=0.283, p=0.015), VEGF(r=0.547, p<0.01) and MVD(r=0.279,p=0.016). The expression of these factors was higher in PCa with echo halo marginal than those of without.(3) There were 43 cases without posterior acoustic attenuation and 31 cases with posterior acoustic attenuation. The posterior acoustic attenuation was related to expression of MMP2 significantly (r=0.237, p=0.042), it expressed higher in prostate cancer with posterior acoustic attenuation.(4) There were 45 cases without micro-calcification and 29 cases with micro-calcification. The micro-calcification were related to expression of k-ras(r=0.398, p<0.01)and EGFR(r=0.268,p=0.021)significantly. Those expressed higher in prostate cancer with micro-calcification.(5) There were 16 cases of 0 grade, 38 cases of 1 grade, 16 cases of 2 grade and 4 cases of 3 grade in blood flow classification. The grade of flow classification in prostate cancer were related to expression of VEGF(r=0.432,p<0.01), Braf (r=0.284,p=0.014), MMP2(r=0.547,p<0.01), MMP9(r=0.833,p<0.01)and MVD(r=0.587,p<0.01),.(6) There were 43 cases with Vs not higher than 50 cm/s and 31 cases with Vs higer than 50 cm/s. The Vs were related to expression of VEGF(r=0.354,p=0.002), EGFR(r=0.231,p=0.035), MMP2(r=0.307,p=0.014)and MVD(r=0.508,p<0.01). With higher blood flow speed, the protein expression increased.(7) There was 32 cases with RI less than 0.8 and 42 cases with RI higher than 0.8. The RI were not related to expression of VEGF, Braf, k-ras, EGFR, MMP2, MMP9, hMSH2, hMSH6 and MVD.Conclusions1. There was a significant relationship between PCa echo features and MMP2, MMP9, VEGF positive expression. It can reflect the infiltration and metastasis ability of PCa, and its prognosis.2. The features in morphous (irregular and thorn-like) were related significantly to expression of MMP2, MMP9, VEGF and MVD. It can reflect the infiltration and metastasis ability of PCa and its prognosis.3. The posterior acoustic attenuation was related to expression of MMP2 significantly. It can reflect the infiltration and metastasis ability of PCa, and its prognosis.4. The micro-calcification was significantly related to the expression of k-ras and EGFR. It can reflect the proliferation and differentiation ability of PCa.5. There was a positive correlation between blood flow classification and Braf, MMP2, MMP9, VEGF protein expression and MVD. It mainly reflected the infiltration and metastasis ability, and prognosis.6. There was a significant relationship between Vs and EGFR, MMP2, VEGF protein expression and MVD. It can reflect the infiltration and metastasis ability, and prognosis.7. There was no significant relationship between RI and any of the factors protein expression. What can reflect the biological characters of PCa needs further study.更多还原