结直肠癌CT灌注成像与肿瘤血管生成及侵袭转移的相关性研究
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摘要
前言
结直肠癌是人类常见恶性肿瘤之一,居恶性肿瘤死亡原因的第二位,近年来发病率呈明显上升趋势。常规CT扫描具有良好的空间分辨率和密度分辨率,尤其是MSCT的应用,灵活运用多平面成像(MPR)可以较好的显示结直肠癌的肿瘤形态,可以判断对结直肠周围的侵犯情况及有否转移,但不能评价器官功能的改变。CT灌注成像的作用不同于常规CT扫描,可以评价器官、组织的血供情况及血流动力学的状态,并能对灌注过程做出定量及半定量的分析,由此反映肿瘤的恶性程度、预后及复发情况,从病变形态学的观察过度到微观的代谢和功能状态分析,在病变的诊断、临床分期及疗效的预测和评价方面均可发挥作用。随着多层螺旋CT的应用,使得灌注成像更快捷,时间分辨率更高,CT灌注技术已经被广泛运用于腹部脏器。
侵袭、转移是恶性肿瘤最重要也是最本质的生物学特征,是恶性肿瘤患者死亡的主要原因,结直肠癌也不例外。恶性肿瘤的生长、转移和播散离不开自身血供的建立,这个过程的建立与血管生成因子(如血管内皮细胞生长因子,vascularendothelial growth factor,VEGF)释放增加及随之而来的微血管密度增加有关,而基质金属蛋白酶2(matrix metalloproteinases-2,MMP-2)可通过对细胞外基质降解,促进癌细胞对周围组织浸润和转移的发生,在肿瘤侵袭和转移过程中起关键作用。本实验就结直肠癌CT灌注成像参数与MMP-2和VEGF表达情况及MVD计数相互间的相关性进行研究,从而为应用CT灌注成像预测结直肠癌侵袭转移能力提供依据。
材料与方法
1、临床资料
2008年6月～2008年12月在中国医科大学附属第一医院就诊,临床诊断结直肠癌的61例患者行多层螺旋CT结直肠灌注成像。其中在检查后2周内经手术证实为结直肠癌,并且获取了肿瘤大体标本及完整病理报告的41例纳入本研究。男性30例,女性11例。年龄36～81岁,平均62.05±12.13岁。肿瘤位于直肠20例,直肠、乙状结肠交界处4例,乙状结肠4例,降结肠3例,横结肠3例,盲升结肠7例。41例均为腺癌,其中高分化4例,中分化34例,低分化3例。临床病理分期采用Dukes分期法,标准为:A期:癌肿侵犯粘膜或粘膜下层、浅肌层、深肌层;B期:侵犯浆膜层、浆膜外;C期:淋巴结转移;D期:远处转移或腹膜转移。本组41例中A期7例,B期19例,C期10例,D期5例。
2、CT灌注扫描方法、图像后处理及分析
(1)CT灌注扫描方法
确认被检查者CT扫描前一周之内未做过消化道造影,检查前6小时禁食,确保肠道清洁。扫描前对患者进行呼吸训练,用腹带固定。检查前60～90min口服1%泛影葡胺500ml充盈消化道并嘱患者充盈膀胱,直肠内充气50～100ml。检查者仰卧于检查床上,先行腹盆部CT平扫。平扫后,选择病变最大层面做为靶层面进行灌注扫描。选用18G静脉留置针经肘前静脉注入造影剂欧乃派克(剂量50ml,注射速率6ml/s)后,延迟10秒后启动灌注扫描模式,持续时间50s,管电压120kv,管电流50mAs,层厚7～10mm,得到200幅图像。灌注扫描结束后,即刻经高压注射器继续注入50ml欧乃派克,速度3ml/s,再行腹部常规增强扫描,延迟时间分别为30s及70s,作为诊断图像。
(2)CT灌注图像后处理及分析
将CT灌注图像传送到Siemens图像后处理工作站,采用预装的Body Perfusion软件处理数据。西门子公司的CT灌注后处理软件采用的是非去卷积算法。经图像后处理获得病灶、靶动脉的时间密度曲线(time-density curve,TDC),通过TDC可计算出血流量(blood flow,BF)、血容量(blood volume,BV)、灌注起始时间(time to start,TTS)、灌注峰值时间(time to peak,TTP)、血管表面通透性(permeability surface,PS)和Patlak血容量(Patlak blood volume,PBV)等参数,同时得到BF、BV、TTS、TTP、PS、PBV伪彩图。ROI区域要选择明确的实性部分,多点采集取平均值。依结直肠癌病变水平高低的不同,流入动脉ROI取腹主动脉、髂外动脉(或股动脉)轴位图像的中心区域。
3、病理标本处理及免疫组织化学方法
病理学切片制备、染色及读片结果均在病理科老师指导下完成。
(1)切片制作
对所选病例的石蜡包埋组织行连续切片,切片厚4μ,连续切片4张,第一张切片行HE染色,其余3张用于免疫组化染色。
(2)免疫组织化学实验方法
免疫组织化学方法采用过氧化物酶标记的链霉素卵白素法(SP)染色。CD34、VEGF、MMP-2单克隆抗体及SP免疫组化试剂盒均购于北京中杉金桥生物技术有限公司。已知的阳性结直肠癌切片作为阳性对照,阴性对照用PBS缓冲液代替一抗染色。
(3)免疫组织化学结果判断
微血管密度(microvascular density,MVD)计数:MVD定义为单位面积组织切片内CD34染色阳性的微血管细胞数。肿瘤内孤立的棕黄色血管内皮细胞或细胞簇代表1条单独的微血管。先在低倍镜下(100×)浏览全片,找出微血管密度大的“热点”(hot spot),换高倍镜(200×),每张切片记录5个“热点”,取其平均数作为该病例的MVD。
VEGF判定标准:以胞浆或胞膜呈均一棕黄色着色的细胞为阳性,无着色的细胞为阴性。判定标准参照Park等的方法:先用低倍镜(40×)全面观察切面,选定待测组织VEGF染色最强处,然后在200倍镜下计数100个细胞,阳性染色细胞＜5%为阴性(-);5～15%为弱阳性(+);16～50%为阳性(++);＞50%为强阳性(+++)。
MMP-2判定标准:阳性主要表现为显微镜下肿瘤细胞胞质、血管内皮、间质中出现棕、黄色颗粒或团块。判定标准:先用低倍镜(40×)全面观察切面,选定待测组织MMP-2染色最强处,然后在200倍镜下计数100个细胞,阳性染色细胞＜5%为阴性(-);5～15%为弱阳性(+);16～50%为阳性(++);＞50%为强阳性(+++)。
4、统计学处理
所有数据用SPSS 13.0统计学软件包做分析处理。计量资料以均数±标准差表示,对于正态分布、方差齐性的计量资料,两组间比较采用t检验法或配对t检验法,多组间比较采用单因素方差分析的方法。对于偏态分布的计量资料或等级资料,采用非参数检验。参数间相关分析采用Person法或Spearman秩相关分析。以P＜0.05为差异有统计学意义。
结果
1、本研究获得结直肠癌病灶、靶动脉的时间密度曲线,及一组完整的MIP图和彩色编码图包括BF、BV、TTS、TTP、PS、PBV图的资料和CT灌注参数值。MIP图可以观察肿瘤增强情况,类似于传统的增强扫描;而灌注图红色、黄色区域代表肿瘤内部血流灌注丰富的区域,绿色代表中度灌注区域,蓝色代表血流平乏的低灌注区域,因而彩色编码图可直接显示肿瘤内部的血流灌注状态(图1-8)。
2、结直肠癌CT灌注参数的平均值
41例结直肠癌的BF值为(65.65±27.37 ml/100ml/min),BV值为[85.11±37.14(1/1000)],TTS值为[26.91±13.98(1/10s)],TTP值为[97.70～288.50(1/10s)],PS值为[16.50～174.60(0.5ml/100ml/min)],PBV值为[17.10～291.10(1/1000)]。
3、CT灌注参数值与MVD计数,VEGF和MMP-2表达间的相关性分析
Person相关分析及Spearman秩相关分析表明:肿瘤实质区CT灌注参数BF、BV、PS与VEGF表达呈正相关(P＜0.05);肿瘤实质区CT灌注参数BF、BV、PS与MVD计数呈正相关(P＜0.05);MVD计数与VEGF表达亦呈正相关(P＜0.05);肿瘤实质区PS值与MMP-2表达呈正相关(P＜0.05)。
结论
1、BF、BV值在浆膜浸润组高于无浆膜浸润组,肝转移组的BF、BV值高于无肝转移组(P值＜0.05)。PS值在浆膜浸润、肝转移、淋巴结转移各组中均高于无浆膜浸润、无肝转移、无淋巴结转移组(P值＜0.05)。
2、CT灌注成像某些参数可以反映血流灌注特点,这为进一步研究肿瘤微血管,找出肿瘤灌注参数与免疫组化指标间VEGF、MMP-2间的相关性,评价肿瘤侵袭转移能力奠定基础。
3、肿瘤实质区CT灌注参数BF、BV和PS值与VEGF表达呈正相关,肿瘤实质区CT灌注参数BF、BV和PS与MVD表达呈正相关,MVD表达与VEGF表达亦呈正相关。CT灌注成像参数PS与MMP-2的表达显著相关。
4、CT灌注参数不仅能反映结直肠癌微血管生成情况的MVD,而且可反映VEGF和MMP-2表达情况,表明CT灌注参数BF、BV和PS可作为一种活体评价肿瘤微血管生成和侵袭转移能力的指标。
Preface
Human colorectal cancer is one of the commonest malignant tumors,ranking second only to lung cancer as the leading cause of mortality in malignant tumors.There has been a marked increase in the incidence in recent years.Conventional CT scanning has a good spatial resolution and density resolution,in particular the application of MSCT multiplanar reformation(MPR) image is flexible which can better show the tumor form of colorectal cancer and determine violations of the surrounding circumstances and metastasis,but can not evaluate the changes in organ function.CT perfusion imaging is different from the conventional CT scan.It can evaluate organs, tissues and the blood supply of the hemodynamic status,and make a quantitative and semi-quantitative analysis of perfusion process to reflect the malignancy,the prognosis and recurrence of the tumor.It has brought about the change from morphological observation to the micro-analysis of metabolic and functional status and played an important role in diagnosis,the determination of clinical stage and the prediction and evaluation of curative effects.With the application of multi-slice spiral CT,perfusion imaging allows a more efficient,higher temporal resolution,and it has been widely used in abdominal organs.
Invasion and metastasis are the most important and most essential biological characteristics of malignant tumors,and remain the main causes of death in patients with malignant tumors,and colorectal cancer has no exception.The growth,metastasis and dissemination of malignant tumors can't do without its own blood supply,and the process of setting up blood supply is related to the increasing release of angiogenic factors(such as vascular endothelial growth factor,vascular endothelial growth factor, VEGF) and the subsequent increase in the density of capiliaries.Matrix metalloproteinase-2(MMP-2) plays a crucial role during the course of tumor invasion and metastasis by promoting cancer cells to invade the surrounding tissues and the occurrence of metastasis by way of degrading the extracellular matrix.This experiment aims to provide a basis for the prediction of invasion and metastatic ability in colorectal cancer with CT perfusion imaging by studying the correlation between CT perfusion imaging parameters,MMP-2 and VEGF expression,and MVD count of colorectal cancer.
Materials and Methods
1.Clinical Data
From June 2008 to December 2008,among treatments at the First Affiliated Hospital of China Medical University,61 cases were clinically diagnosed with colorectal cancer by colorectal multi-slice spiral CT perfusion imaging.Forty-one cases which were confirmed by surgery within 2 weeks after diagnosis and had general access to the tumor specimens,as well as complete reports were involved in this study.There are 30 male cases and 11 female cases.Age ranges from 36 to 81 years old,with an average age of 62.05±12.13. Twenty cases of tumors were located in the rectum,4 cases at the junction of rectum and sigmoid colon,4 cases of sigmoid colon,3 cases in descending colon,3 cases of transverse colon,and 7 cases of cecal-ascending colon.All 41 cases were adenocarcinoma,including 4 well-differentiated, 34 differentiated,and 3 poorly differentiated cases.Clinicopathological stage was estimated by the Modified Duke Staging System.The standard is as follows:Modified Duke A,the tumor penetrates into the mucosa of the bowel wall but no further.Modified Duke B,B1:tumor penetrates into,but not through the muscularis propria(the muscular layer) of the bowel wall.B2:tumor penetrates into and through the muscularis propria of the bowel wall.Modified Duke C,C1:tumor penetrates into, but not through the muscularis propria of the bowel wall;there is pathologic evidence of colon cancer in the lymph nodes.C2:tumor penetrates into and through the muscularis propria of the bowel wall;there is pathologic evidence of colon cancer in the lymph nodes.Modified Duke D,the tumor,which has spread beyond the confines of the lymph nodes(to organs such as the liver,lung or bone).Among the 41 cases,there are 7 Duke A,19 Duke B,10 Duke C,and 5 Duke D cases.
2.CT perfusion imaging,image post-processing and analysis
(1)CT perfusion scanning
Make sure of no digestive tract imaging a week before CT scan.Fast for six hours and perform respiratory training in patients with fixed supporter before scanning.Fill in the patient's digestive tract with one percent of oral diatrizoate 500ml,ask to expand bladder and rectum with rectal inflation of 50～100ml gas.
Make patients lie on back to perfom abdominal pelvic CT scan.After that,choose the maximum level of disease as the target level of perfusion imaging.18G intravenous catheter system was used to inject contrast agent Omnipaque(dosage 50ml,injection rate is 6ml/s),then start perfusion scan mode after 10 seconds delayed period,with 50s duration,120kv tube voltage,50mAs tube Current,7～10mm slice thickness,and 200 images.Inject 50ml omnipaque with the high-pressure syringe(the speed is 3ml/s) the instant the perfusion imaging is done,and perform conventional abdominal CT again with 30s and 70s delayed periods as a diagnostic image.
(2)CT perfusion image post-processing and analysis
CT perfusion images will be sent to the Siemens image post-processing workstations,and processed by pre-installed software for data processing Body Perfusion.Siemens pose perfusion CT software is non-deconvolution algorithm.CT perfusion image post-processing can shed light on lesions,the target artery time density curve(time-density curve,TDC),the flow through the TDC calculation hemorrhage (blood flow,BF),blood volume(blood volume,BV),the initial reperfusion period (time to start,TTS),time to peak perfusion(time to peak,TTP),the surface of vascular permeability(permeability surface,PS),Patlak volume(Patlak blood volume,PBV) and other parameters,as well as BF,BV,TTS,TTP,PS,PBV pseudo-color at the same time.ROI region needs to select specific solid part with multi-point averaging acquisition.According to different lesion levels of colorectal cancer,ROI is attribute to the abdominal aortic artery check,external iliac artery(or femoral artery) axial images of the central region.
2.Pathology specimens processing method and immunohistochemical analysis.
Preparation of histopathological sections,staining were done under the guidance of teachers from Department of Pathology.
(1)slice production
Make 4 serial slices of the selected cases of paraffin-embedded tissues with 4μm each slice.The first slice was proceed with the HE staining,and the remaining three were for immunohistochemical staining.
(2)immunohistochemical analysis
Peroxidase labeled streptomycin-avidin method(SP) was used.CD34,VEGF, MMP-2 monoclonal antibody and SP immunohistochemical kit were purchased from the Jinqiao Biotechnology Ltd,Beijing.Make known colorectal cancer-positive biopsy as positive control,PBS buffer as negative control.
(3)Judgement of immunohistochemical results
MVD(microvascular density,MVD) count:MVD unit area is defined as tissue stained with CD34-positive microvascular cells.Cancer isolated brown with vascular endothelial cells or cell clusters on behalf of an individual microvessel.First view the whole film at low magnification(100×),larger microvessel density to identify“hot spots”(hot spot) with high-power microscope(200×),each slice records five“hot spots”.Choose the average number of cases as the MVD.
criterion for VEGF:cytoplasmic membrane with homogeneous or brown coloring was taken positive,non-staining cell was negative.Park,such as criterion-referenced to determine[19]method:First observe a comprehensive cross-section with the low-fold mirror(40×) to select the strongest VEGF staining,then count 100 cells at 200 times magnification.Positive staining cells＜5%is for the negative(--);5～15%for weakly positive(+);16 ~ 50%of(++);＞50%positive for the strong positive(+++)。
criterion for MMP-2:Mainly positive for microscopic tumor cells,vascular endothelial,mesenchymal appear brown,yellow or particle mass.Criteria:First observe a comprehensive cross-section with the low-fold mirror(40×) to select the strongest MMP-2 staining,then count 100 cells at 200 times magnification.Positive staining cells＜5%is for the negative(--);5～15%for weakly positive(+);16～50% of(++);＞50%) positive for the strong positive(+++)。
4.Statistical treatment of experimental data
All data were analysed with SPSS 13.0 statistical software.Measurement data to both the standard deviation of the number of people said that for the normal distribution, homogeneity of variance of the measurement data,to compare the two groups using t test or paired t test,comparison between groups using one-way ANOVA analysis,then the use of SNK-q test for multiple comparisons are a few.Skewed distribution of the information or Level measurement data was analysed by non-parametric test, comparison between two groups was analysed by Wilcoxon test,and multi-group comparison was analysed by Kruskal Wallis test.If the p-value＜0.05,the difference has statistical significance.
Results
1.In this study,lesions of colorectal cancer was the target arterial time density curve,and a complete set of MIP maps and color-coded map,including BF,BV,TTS, TTP,PS,PBV map information and CT perfusion parameter values.MIP map shows the enhancement of tumor,which is similar to the conventional CT;The red and yellow regions on the perfusion map stand for rich blood flow,green for medium blood flow, and blue for poor blood flow in the low level of regional perfusion.Such color-coded map shows the tumor blood perfusion status directly.
2.the average of CT perfusion parameters in colorectal cancer
Among forty-one cases of colorectal cancer,BF values(61.9 persons 31.2 ml/100ml/min),BV values[83.9 disabilities 39.2(a ratio of 1/1000)],TTS values [30.0 disabilities 25.2(1/10s)]),TTP values[145.7 disabilities 38.9(1/10s)],PS values [56.1 disabilities 34.4(0.5ml/100ml/min)],and PBV values[84.7 disabilities 59.6(a ratio of 1/1000)].
3.the correlation analysis between CT perfusion parameter values,the MVD counts,VEGF and MMP-2 expression
Pearson correlation analysis and Spearman rank correlation analysis showed that: tumor parenchyma CT perfusion parameters of BF,BV,PS and VEGF expression were positively correlated(P＜0.05);tumor parenchyma CT perfusion parameters of BF,BV, PS and MVD count were positively correlated(P＜0.05);MVD count and VEGF expression were also positively correlated(P＜0.05);and tumor parenchyma PS values and MMP-2 expression were positively correlated(P＜0.05).
Conclusion
1.BF,BV values of serosal invasion group were higher than those of without serosal invasion group,and liver metastasis group were higher than those of without liver metastasis group(P value＜0.05).PS values of serosal invasion,liver metastasis, and lymph node metastasis groups were higher than those of without serosal invasion, no liver metastasis,no lymph node metastasis groups(P value＜0.05).
2.some parameters of CT Perfusion imaging can reflect the characteristics of blood perfusion,which further lays the foundation for studying tumor microvascular in order to identify the correlation between tumor perfusion and immunohistochemical parameters of VEGF,MMP-2 as well as evaluating metastasis.
3.CT perfusion parameters of BF,BV and PS values were positively correlated to VEGF expression in Parenchyma tumor.CT perfusion parameters of BF,BV and PS values were positively correlated to MVD expression in Parenchyma tumor,MVD expression and VEGF expression were also positively correlated.CT perfusion imaging parameters of PS and MMP-2 expression were significantly correlated.
4.CT perfusion parameters can reflect not only the angiogenesis of colorectal cancer of the MVD,but also the VEGF and MMP-2 expression,indicating that CT perfusion parameters of BF,BV and PS can be used as an in vivo assessment of angiogenesis,tumor invasion and metastatic ability.
结直肠癌是人类常见恶性肿瘤之一,居恶性肿瘤死亡原因的第二位,近年来发病率呈明显上升趋势。常规CT扫描具有良好的空间分辨率和密度分辨率,尤其是MSCT的应用,灵活运用多平面成像(MPR)可以较好的显示结直肠癌的肿瘤形态,可以判断对结直肠周围的侵犯情况及有否转移,但不能评价器官功能的改变。CT灌注成像的作用不同于常规CT扫描,可以评价器官、组织的血供情况及血流动力学的状态,并能对灌注过程做出定量及半定量的分析,由此反映肿瘤的恶性程度、预后及复发情况,从病变形态学的观察过度到微观的代谢和功能状态分析,在病变的诊断、临床分期及疗效的预测和评价方面均可发挥作用。随着多层螺旋CT的应用,使得灌注成像更快捷,时间分辨率更高,CT灌注技术已经被广泛运用于腹部脏器。
侵袭、转移是恶性肿瘤最重要也是最本质的生物学特征,是恶性肿瘤患者死亡的主要原因,结直肠癌也不例外。恶性肿瘤的生长、转移和播散离不开自身血供的建立,这个过程的建立与血管生成因子(如血管内皮细胞生长因子,vascularendothelial growth factor,VEGF)释放增加及随之而来的微血管密度增加有关,而基质金属蛋白酶2(matrix metalloproteinases-2,MMP-2)可通过对细胞外基质降解,促进癌细胞对周围组织浸润和转移的发生,在肿瘤侵袭和转移过程中起关键作用。本实验就结直肠癌CT灌注成像参数与MMP-2和VEGF表达情况及MVD计数相互间的相关性进行研究,从而为应用CT灌注成像预测结直肠癌侵袭转移能力提供依据。
材料与方法
1、临床资料
2008年6月～2008年12月在中国医科大学附属第一医院就诊,临床诊断结直肠癌的61例患者行多层螺旋CT结直肠灌注成像。其中在检查后2周内经手术证实为结直肠癌,并且获取了肿瘤大体标本及完整病理报告的41例纳入本研究。男性30例,女性11例。年龄36～81岁,平均62.05±12.13岁。肿瘤位于直肠20例,直肠、乙状结肠交界处4例,乙状结肠4例,降结肠3例,横结肠3例,盲升结肠7例。41例均为腺癌,其中高分化4例,中分化34例,低分化3例。临床病理分期采用Dukes分期法,标准为:A期:癌肿侵犯粘膜或粘膜下层、浅肌层、深肌层;B期:侵犯浆膜层、浆膜外;C期:淋巴结转移;D期:远处转移或腹膜转移。本组41例中A期7例,B期19例,C期10例,D期5例。
2、CT灌注扫描方法、图像后处理及分析
(1)CT灌注扫描方法
确认被检查者CT扫描前一周之内未做过消化道造影,检查前6小时禁食,确保肠道清洁。扫描前对患者进行呼吸训练,用腹带固定。检查前60～90min口服1%泛影葡胺500ml充盈消化道并嘱患者充盈膀胱,直肠内充气50～100ml。检查者仰卧于检查床上,先行腹盆部CT平扫。平扫后,选择病变最大层面做为靶层面进行灌注扫描。选用18G静脉留置针经肘前静脉注入造影剂欧乃派克(剂量50ml,注射速率6ml/s)后,延迟10秒后启动灌注扫描模式,持续时间50s,管电压120kv,管电流50mAs,层厚7～10mm,得到200幅图像。灌注扫描结束后,即刻经高压注射器继续注入50ml欧乃派克,速度3ml/s,再行腹部常规增强扫描,延迟时间分别为30s及70s,作为诊断图像。
(2)CT灌注图像后处理及分析
将CT灌注图像传送到Siemens图像后处理工作站,采用预装的Body Perfusion软件处理数据。西门子公司的CT灌注后处理软件采用的是非去卷积算法。经图像后处理获得病灶、靶动脉的时间密度曲线(time-density curve,TDC),通过TDC可计算出血流量(blood flow,BF)、血容量(blood volume,BV)、灌注起始时间(time to start,TTS)、灌注峰值时间(time to peak,TTP)、血管表面通透性(permeability surface,PS)和Patlak血容量(Patlak blood volume,PBV)等参数,同时得到BF、BV、TTS、TTP、PS、PBV伪彩图。ROI区域要选择明确的实性部分,多点采集取平均值。依结直肠癌病变水平高低的不同,流入动脉ROI取腹主动脉、髂外动脉(或股动脉)轴位图像的中心区域。
3、病理标本处理及免疫组织化学方法
病理学切片制备、染色及读片结果均在病理科老师指导下完成。
(1)切片制作
对所选病例的石蜡包埋组织行连续切片,切片厚4μ,连续切片4张,第一张切片行HE染色,其余3张用于免疫组化染色。
(2)免疫组织化学实验方法
免疫组织化学方法采用过氧化物酶标记的链霉素卵白素法(SP)染色。CD34、VEGF、MMP-2单克隆抗体及SP免疫组化试剂盒均购于北京中杉金桥生物技术有限公司。已知的阳性结直肠癌切片作为阳性对照,阴性对照用PBS缓冲液代替一抗染色。
(3)免疫组织化学结果判断
微血管密度(microvascular density,MVD)计数:MVD定义为单位面积组织切片内CD34染色阳性的微血管细胞数。肿瘤内孤立的棕黄色血管内皮细胞或细胞簇代表1条单独的微血管。先在低倍镜下(100×)浏览全片,找出微血管密度大的“热点”(hot spot),换高倍镜(200×),每张切片记录5个“热点”,取其平均数作为该病例的MVD。
VEGF判定标准:以胞浆或胞膜呈均一棕黄色着色的细胞为阳性,无着色的细胞为阴性。判定标准参照Park等的方法:先用低倍镜(40×)全面观察切面,选定待测组织VEGF染色最强处,然后在200倍镜下计数100个细胞,阳性染色细胞＜5%为阴性(-);5～15%为弱阳性(+);16～50%为阳性(++);＞50%为强阳性(+++)。
MMP-2判定标准:阳性主要表现为显微镜下肿瘤细胞胞质、血管内皮、间质中出现棕、黄色颗粒或团块。判定标准:先用低倍镜(40×)全面观察切面,选定待测组织MMP-2染色最强处,然后在200倍镜下计数100个细胞,阳性染色细胞＜5%为阴性(-);5～15%为弱阳性(+);16～50%为阳性(++);＞50%为强阳性(+++)。
4、统计学处理
所有数据用SPSS 13.0统计学软件包做分析处理。计量资料以均数±标准差表示,对于正态分布、方差齐性的计量资料,两组间比较采用t检验法或配对t检验法,多组间比较采用单因素方差分析的方法。对于偏态分布的计量资料或等级资料,采用非参数检验。参数间相关分析采用Person法或Spearman秩相关分析。以P＜0.05为差异有统计学意义。
结果
1、本研究获得结直肠癌病灶、靶动脉的时间密度曲线,及一组完整的MIP图和彩色编码图包括BF、BV、TTS、TTP、PS、PBV图的资料和CT灌注参数值。MIP图可以观察肿瘤增强情况,类似于传统的增强扫描;而灌注图红色、黄色区域代表肿瘤内部血流灌注丰富的区域,绿色代表中度灌注区域,蓝色代表血流平乏的低灌注区域,因而彩色编码图可直接显示肿瘤内部的血流灌注状态(图1-8)。
2、结直肠癌CT灌注参数的平均值
41例结直肠癌的BF值为(65.65±27.37 ml/100ml/min),BV值为[85.11±37.14(1/1000)],TTS值为[26.91±13.98(1/10s)],TTP值为[97.70～288.50(1/10s)],PS值为[16.50～174.60(0.5ml/100ml/min)],PBV值为[17.10～291.10(1/1000)]。
3、CT灌注参数值与MVD计数,VEGF和MMP-2表达间的相关性分析
Person相关分析及Spearman秩相关分析表明:肿瘤实质区CT灌注参数BF、BV、PS与VEGF表达呈正相关(P＜0.05);肿瘤实质区CT灌注参数BF、BV、PS与MVD计数呈正相关(P＜0.05);MVD计数与VEGF表达亦呈正相关(P＜0.05);肿瘤实质区PS值与MMP-2表达呈正相关(P＜0.05)。
结论
1、BF、BV值在浆膜浸润组高于无浆膜浸润组,肝转移组的BF、BV值高于无肝转移组(P值＜0.05)。PS值在浆膜浸润、肝转移、淋巴结转移各组中均高于无浆膜浸润、无肝转移、无淋巴结转移组(P值＜0.05)。
2、CT灌注成像某些参数可以反映血流灌注特点,这为进一步研究肿瘤微血管,找出肿瘤灌注参数与免疫组化指标间VEGF、MMP-2间的相关性,评价肿瘤侵袭转移能力奠定基础。
3、肿瘤实质区CT灌注参数BF、BV和PS值与VEGF表达呈正相关,肿瘤实质区CT灌注参数BF、BV和PS与MVD表达呈正相关,MVD表达与VEGF表达亦呈正相关。CT灌注成像参数PS与MMP-2的表达显著相关。
4、CT灌注参数不仅能反映结直肠癌微血管生成情况的MVD,而且可反映VEGF和MMP-2表达情况,表明CT灌注参数BF、BV和PS可作为一种活体评价肿瘤微血管生成和侵袭转移能力的指标。
Preface
Human colorectal cancer is one of the commonest malignant tumors,ranking second only to lung cancer as the leading cause of mortality in malignant tumors.There has been a marked increase in the incidence in recent years.Conventional CT scanning has a good spatial resolution and density resolution,in particular the application of MSCT multiplanar reformation(MPR) image is flexible which can better show the tumor form of colorectal cancer and determine violations of the surrounding circumstances and metastasis,but can not evaluate the changes in organ function.CT perfusion imaging is different from the conventional CT scan.It can evaluate organs, tissues and the blood supply of the hemodynamic status,and make a quantitative and semi-quantitative analysis of perfusion process to reflect the malignancy,the prognosis and recurrence of the tumor.It has brought about the change from morphological observation to the micro-analysis of metabolic and functional status and played an important role in diagnosis,the determination of clinical stage and the prediction and evaluation of curative effects.With the application of multi-slice spiral CT,perfusion imaging allows a more efficient,higher temporal resolution,and it has been widely used in abdominal organs.
Invasion and metastasis are the most important and most essential biological characteristics of malignant tumors,and remain the main causes of death in patients with malignant tumors,and colorectal cancer has no exception.The growth,metastasis and dissemination of malignant tumors can't do without its own blood supply,and the process of setting up blood supply is related to the increasing release of angiogenic factors(such as vascular endothelial growth factor,vascular endothelial growth factor, VEGF) and the subsequent increase in the density of capiliaries.Matrix metalloproteinase-2(MMP-2) plays a crucial role during the course of tumor invasion and metastasis by promoting cancer cells to invade the surrounding tissues and the occurrence of metastasis by way of degrading the extracellular matrix.This experiment aims to provide a basis for the prediction of invasion and metastatic ability in colorectal cancer with CT perfusion imaging by studying the correlation between CT perfusion imaging parameters,MMP-2 and VEGF expression,and MVD count of colorectal cancer.
Materials and Methods
1.Clinical Data
From June 2008 to December 2008,among treatments at the First Affiliated Hospital of China Medical University,61 cases were clinically diagnosed with colorectal cancer by colorectal multi-slice spiral CT perfusion imaging.Forty-one cases which were confirmed by surgery within 2 weeks after diagnosis and had general access to the tumor specimens,as well as complete reports were involved in this study.There are 30 male cases and 11 female cases.Age ranges from 36 to 81 years old,with an average age of 62.05±12.13. Twenty cases of tumors were located in the rectum,4 cases at the junction of rectum and sigmoid colon,4 cases of sigmoid colon,3 cases in descending colon,3 cases of transverse colon,and 7 cases of cecal-ascending colon.All 41 cases were adenocarcinoma,including 4 well-differentiated, 34 differentiated,and 3 poorly differentiated cases.Clinicopathological stage was estimated by the Modified Duke Staging System.The standard is as follows:Modified Duke A,the tumor penetrates into the mucosa of the bowel wall but no further.Modified Duke B,B1:tumor penetrates into,but not through the muscularis propria(the muscular layer) of the bowel wall.B2:tumor penetrates into and through the muscularis propria of the bowel wall.Modified Duke C,C1:tumor penetrates into, but not through the muscularis propria of the bowel wall;there is pathologic evidence of colon cancer in the lymph nodes.C2:tumor penetrates into and through the muscularis propria of the bowel wall;there is pathologic evidence of colon cancer in the lymph nodes.Modified Duke D,the tumor,which has spread beyond the confines of the lymph nodes(to organs such as the liver,lung or bone).Among the 41 cases,there are 7 Duke A,19 Duke B,10 Duke C,and 5 Duke D cases.
2.CT perfusion imaging,image post-processing and analysis
(1)CT perfusion scanning
Make sure of no digestive tract imaging a week before CT scan.Fast for six hours and perform respiratory training in patients with fixed supporter before scanning.Fill in the patient's digestive tract with one percent of oral diatrizoate 500ml,ask to expand bladder and rectum with rectal inflation of 50～100ml gas.
Make patients lie on back to perfom abdominal pelvic CT scan.After that,choose the maximum level of disease as the target level of perfusion imaging.18G intravenous catheter system was used to inject contrast agent Omnipaque(dosage 50ml,injection rate is 6ml/s),then start perfusion scan mode after 10 seconds delayed period,with 50s duration,120kv tube voltage,50mAs tube Current,7～10mm slice thickness,and 200 images.Inject 50ml omnipaque with the high-pressure syringe(the speed is 3ml/s) the instant the perfusion imaging is done,and perform conventional abdominal CT again with 30s and 70s delayed periods as a diagnostic image.
(2)CT perfusion image post-processing and analysis
CT perfusion images will be sent to the Siemens image post-processing workstations,and processed by pre-installed software for data processing Body Perfusion.Siemens pose perfusion CT software is non-deconvolution algorithm.CT perfusion image post-processing can shed light on lesions,the target artery time density curve(time-density curve,TDC),the flow through the TDC calculation hemorrhage (blood flow,BF),blood volume(blood volume,BV),the initial reperfusion period (time to start,TTS),time to peak perfusion(time to peak,TTP),the surface of vascular permeability(permeability surface,PS),Patlak volume(Patlak blood volume,PBV) and other parameters,as well as BF,BV,TTS,TTP,PS,PBV pseudo-color at the same time.ROI region needs to select specific solid part with multi-point averaging acquisition.According to different lesion levels of colorectal cancer,ROI is attribute to the abdominal aortic artery check,external iliac artery(or femoral artery) axial images of the central region.
2.Pathology specimens processing method and immunohistochemical analysis.
Preparation of histopathological sections,staining were done under the guidance of teachers from Department of Pathology.
(1)slice production
Make 4 serial slices of the selected cases of paraffin-embedded tissues with 4μm each slice.The first slice was proceed with the HE staining,and the remaining three were for immunohistochemical staining.
(2)immunohistochemical analysis
Peroxidase labeled streptomycin-avidin method(SP) was used.CD34,VEGF, MMP-2 monoclonal antibody and SP immunohistochemical kit were purchased from the Jinqiao Biotechnology Ltd,Beijing.Make known colorectal cancer-positive biopsy as positive control,PBS buffer as negative control.
(3)Judgement of immunohistochemical results
MVD(microvascular density,MVD) count:MVD unit area is defined as tissue stained with CD34-positive microvascular cells.Cancer isolated brown with vascular endothelial cells or cell clusters on behalf of an individual microvessel.First view the whole film at low magnification(100×),larger microvessel density to identify“hot spots”(hot spot) with high-power microscope(200×),each slice records five“hot spots”.Choose the average number of cases as the MVD.
criterion for VEGF:cytoplasmic membrane with homogeneous or brown coloring was taken positive,non-staining cell was negative.Park,such as criterion-referenced to determine[19]method:First observe a comprehensive cross-section with the low-fold mirror(40×) to select the strongest VEGF staining,then count 100 cells at 200 times magnification.Positive staining cells＜5%is for the negative(--);5～15%for weakly positive(+);16 ~ 50%of(++);＞50%positive for the strong positive(+++)。
criterion for MMP-2:Mainly positive for microscopic tumor cells,vascular endothelial,mesenchymal appear brown,yellow or particle mass.Criteria:First observe a comprehensive cross-section with the low-fold mirror(40×) to select the strongest MMP-2 staining,then count 100 cells at 200 times magnification.Positive staining cells＜5%is for the negative(--);5～15%for weakly positive(+);16～50% of(++);＞50%) positive for the strong positive(+++)。
4.Statistical treatment of experimental data
All data were analysed with SPSS 13.0 statistical software.Measurement data to both the standard deviation of the number of people said that for the normal distribution, homogeneity of variance of the measurement data,to compare the two groups using t test or paired t test,comparison between groups using one-way ANOVA analysis,then the use of SNK-q test for multiple comparisons are a few.Skewed distribution of the information or Level measurement data was analysed by non-parametric test, comparison between two groups was analysed by Wilcoxon test,and multi-group comparison was analysed by Kruskal Wallis test.If the p-value＜0.05,the difference has statistical significance.
Results
1.In this study,lesions of colorectal cancer was the target arterial time density curve,and a complete set of MIP maps and color-coded map,including BF,BV,TTS, TTP,PS,PBV map information and CT perfusion parameter values.MIP map shows the enhancement of tumor,which is similar to the conventional CT;The red and yellow regions on the perfusion map stand for rich blood flow,green for medium blood flow, and blue for poor blood flow in the low level of regional perfusion.Such color-coded map shows the tumor blood perfusion status directly.
2.the average of CT perfusion parameters in colorectal cancer
Among forty-one cases of colorectal cancer,BF values(61.9 persons 31.2 ml/100ml/min),BV values[83.9 disabilities 39.2(a ratio of 1/1000)],TTS values [30.0 disabilities 25.2(1/10s)]),TTP values[145.7 disabilities 38.9(1/10s)],PS values [56.1 disabilities 34.4(0.5ml/100ml/min)],and PBV values[84.7 disabilities 59.6(a ratio of 1/1000)].
3.the correlation analysis between CT perfusion parameter values,the MVD counts,VEGF and MMP-2 expression
Pearson correlation analysis and Spearman rank correlation analysis showed that: tumor parenchyma CT perfusion parameters of BF,BV,PS and VEGF expression were positively correlated(P＜0.05);tumor parenchyma CT perfusion parameters of BF,BV, PS and MVD count were positively correlated(P＜0.05);MVD count and VEGF expression were also positively correlated(P＜0.05);and tumor parenchyma PS values and MMP-2 expression were positively correlated(P＜0.05).
Conclusion
1.BF,BV values of serosal invasion group were higher than those of without serosal invasion group,and liver metastasis group were higher than those of without liver metastasis group(P value＜0.05).PS values of serosal invasion,liver metastasis, and lymph node metastasis groups were higher than those of without serosal invasion, no liver metastasis,no lymph node metastasis groups(P value＜0.05).
2.some parameters of CT Perfusion imaging can reflect the characteristics of blood perfusion,which further lays the foundation for studying tumor microvascular in order to identify the correlation between tumor perfusion and immunohistochemical parameters of VEGF,MMP-2 as well as evaluating metastasis.
3.CT perfusion parameters of BF,BV and PS values were positively correlated to VEGF expression in Parenchyma tumor.CT perfusion parameters of BF,BV and PS values were positively correlated to MVD expression in Parenchyma tumor,MVD expression and VEGF expression were also positively correlated.CT perfusion imaging parameters of PS and MMP-2 expression were significantly correlated.
4.CT perfusion parameters can reflect not only the angiogenesis of colorectal cancer of the MVD,but also the VEGF and MMP-2 expression,indicating that CT perfusion parameters of BF,BV and PS can be used as an in vivo assessment of angiogenesis,tumor invasion and metastatic ability.
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38 Chen CN,Cheng YM,Liang JT,et al.Color Doppler vascularity index can predict distant metastasis and survival in colon cancer patientsl Cancer Res,2000,60:2892-2897.
39 Soker S,Takashima S,Miao HQ,et al.Neuroplin-I is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor [J].Cell,1998,92(6):735-745.
1 孙灿辉,孟悛非,李子平等.结直肠癌微血管密度与螺旋 CT 灌注成像的相关性.中华放射学杂志.2006,40(1):77-80
2 Axel L.Cerebral blood flow determination by rapid sequence computed tomography:theoretical analysis.Radiology,1980,137(3):679-686
3 Miles KA,Griffiths MR.Perfusion CT:a worthwhile enhancement.B JR,2003,76,220-231
4 Miles KA.Tumor angiogenesis and its relation to contrast enhancement on computer tomography:a review.Eur J Radiol,1999,30(3):198-205
5 Miles KA,Measurement of tissue perfusion by dynamic computed tomography.B JR,1991,64:409-412
6 Axel L.Cerebral blood flow determination by rapid sequence computed tomography.Radiology,1980,137:679-686
7 Miles KA.Measurement of tissue perfusion by dynamic computed tomography.B JR,1991,64:409-412
8 Kenneth AM,Michael PH,Adrian KD.Functional images hepatic perfusion obtained with dynamic CT.Radiology,1993,188(2):405-411
9 Miles KA,Hayball MP,Dixon AK.Measurement of human pancreatic perfusion using dynamic computed tomography with perfusion imaging.BJR,1995,68(809):471-475
10 Cenic A,Nabavi DG,Craen RA,et al.Dynamic CT measurement of cerebral blood flow:A validation study.AJNR,1999,20(1):63-73
11 Peters AM,Gunasekera RD,Henderson BL,et al Non-invasive measurements of blood flow and extraction fraction Nucl Med Commun,1987,8:823
12 Cenic A,Nabavi DG.Graen RA,et al.Dynamic CT measurement of cerebral blood flow:a validation study,AJNR,1999,20(1):63-73
13 Blomley MJ,Coulden R,Dawson P,et al.Liver perfusion studied with ultrafast CT.Journal of Computer Assisted Tomography,1995,19(3):424-433
14 Miles KA.Perfusion CT for the assessment of tumour vascularity:which protocol?BJR,2003,76:S36-S42
15 Bell SD,Peters AM.Measurement of blood flow from first-pass radionuclide angiography:influence of bolus volume.Eur J Nucl Med,1991,18(11):885
16 熊正文,李春光,丁华野,等.肿瘤血管生成及微循环形态学研究技术.中国组织化学与细胞化学杂志,1998,7(4):499-500
17 Syviasta A,Laidler P,Davies R,et al.The prognostic significance of tumor vascularity in intermediate thick-ness kin melanoma.Am J Pathol,1998,133:419
18 Yancopoulos G.D,Davis S,Gale N.W.,R,Wiegand S.J.,Holash J.Vascular-specific growth factors and blood-vessel formation.Nature(Lond.),2000,407:242-248
19 Carmeliet P.,Jain R.K.Angiogenesis in cancer and other diseases.Nature(Lond),2000,407:249-257
20 Uzzan B,Nicolas P,Cucherat M,et al.Microvessel density as a prognostic factor in women with breast cancer:a systematic review of the literature and met-analysis.Cancer Res,2004,64(9):2941-2955
21 Obermair A,Wanner C,Bilgi S,Speiser P,Kaider A,Reinthaller A,et al.Tumor angiogenesis in stage IB cervical cancer:correlation of microvessel density with survival.Am j Obstet Gynecol,1998;178:314-319
22 Bosari S,Lee AK,Delellis RA,et al.Microvessel quantitation and prognosis in invasive breast carcinoma.Hum pathol,1992,23(7):755-761
23 Camphausen K,Menard C.Angiogenesis inhibitors and radiotherapy of primary tumours.Expert Opin Biol Ther,2002,2(5):477-481
24 Vora HH,Shah NG,Patel DD,et al.Prognostic significance of biomarkers in squamous cell carcinoma of the tongue:multivariate analysis.J Surg Oncol,2003,82(1):34-50
25 熊正文,朱光君,李春光,等.肿瘤血管及非小细胞肺癌检测技术研究进展.中华现代医学杂志,2001,1(3):12-15
26 Ward NL,Dumont DJ.The angiopoietins and Tie2/Tek:adding to the complexity of cardiovascular development.Semin Cell Dev Biol,2002,13(1):19-27
27 Yancopoulos G.D.,Davis S.,Gale N.W.,R.,Wiegand S.J.,Holash J.Vascular-specific growth factors and blood vessel formation.Nature(Lond),2000,407:242-248
28 Carmeliet P,Jain RK.Angiogenesis in cancer and other diseases.Nature(Lond),2000,407:249-257
29 吴泰璜,吴莹光,粱欣荣,等。血管内皮生长因子瘤内微血件密度与大肠癌生物学行为及预后的关系.中国肛肠病杂志,2001,21:6
30 谭江平,校宏兵.血管内皮生长因子表达与结肠癌预后的关系.中国癌症杂志,2001,11:17
31 Takahashi Y.Kitadai Y.Bucana CD.Et al.Expression of vascular endothelial growth factor and its receptor.KDR,correlates with vascularity,metastasis,and proliferation of human colon cancer.Cancer Res,1995.55:3964
32 White JD.Hewett PW.Kosuge D,et al.Vascular endothelial growth factor-D expression is an independent prognostic marker for survival in colorectal carcinoma.Cancer Res,2002,62:1669
33 MLES KA,LEGGETTD A,KELLEYB B,et al.In vivo measurements of neovascularization of liver matastases using perfusion CT[J].Br J Radiol,1998,71(843):276-281.
34 MLES K A.Functional CT imaging in oncology[J].Eur J Radiol,2003,13(Supp 15):134-138.
35 TATEISH IU,NISH IHARA H,WATANABE S,et al.Tumor angiogenesis and dynamic CT in lung adenocarcinoma:radiologic-pathologic correlation[J].Comput Assist Tomogr,2001,25(1):23-27.
36 KWAKB K,SHM H J,PARK E S,et al.Hepatocellular carcinoma:correlation between vascular endothelial growth factor level and degree of enhancement by multiphase contrast enhanced computed tomography[J].Invest Radiol,2001,36(8):487-492.
37 WANG Z Q,LU GM,L I J S,et al.The comparative study of tumor angiogenesis and CT enhancement in pancreatic carcinoma[J].European Journal of Radiology,2004,49(3):274-280.
38 WANG J H,Q U M P,WANG P J,et al.Dynamic CT evaluation of tumor vascularity in renal cell carcinoma[J].American Journal of Roentgenology,2006,186(5):1423-1430.
39 CEN IC A,NABAV ID G,CRAEN R A.A CT method to measure hemodynamics in brain tumors:validation and application of cerebral blood flow maps[J].AJNR,2000,21(3):462-470.
40 SWENSEN S J,VIGGIANO R W,MDTHUN D E,et al.Lung nodule enhancement at CT:multicenter study[J].Radiology,2000,214(1):73-80
41 周华,张敏鸣,肖圣祥,等.动态增强 CT 功能成像评价肺癌肿瘤血管生成的研究[J].中华放射学杂志,2006,40(2):171-175
42 张敏鸣,周华,邹煜.动态增强 CT 对孤立性肺结节的定量研究[J].中华放射学杂志,2004,38(3):263-267
43 文利,丁仕义,牟伟,等.肝细胞癌 CT 灌注参数与微血管密度的相关性研究[J].中华放射学杂志,2005,39(3):280-284
44 Dushyant V.Sahani,Sanjeeva P.Kalva,Leena M.Hamberg,I Dushyant V.Sahani,Sanjeeva P.Kalva,Leena M.Hamberg,Assessing Tumor Perfusion and Treatment Response in Rectal Cancer with Multisection CT:Initial Observations.Radiology 2005,234:785-792
2 Sahani DV,Klava SP,Hamberg LM,et al.Assessing tumor perfusion and treatment response in rectal cancer with multisection CT:initial observation.Radiology,2005,234(3):785-792.
3 Miles KA.Functional computed tomography in oncology.Eur J Cancer,2001,38(16):2079-2084.
4 Henderson E,Milosevic MA,et al.Functional CT imaging of prostate cancer.Phys Med Biol 2003,48:3085-3100.
5 Harvey CJ,Amin Z,Hare CM,et al.Helical CT pneumocolon to assess colonic tumors:radiologic-pathologic correlation.AJR,1998,170:1439-1443.
6 Pandraripande PV,Krinsky GA,Rusinek H,et at.Perfusion imaging of the liver:current challenges and future goals[J].Radiology,2005,234(3):661-673.
7 Miles KA.Functional computed tomography in oncology.Eur J cancer,2002,38(16):2079-2084.
8 Miles KA.Tumour angiogenesis and its relation to contrast enhancement on computed tomography:a review.Eur J Radiol,1999,30(3):198-205.
9 Miles KA,Blomley M,Tsushima Y,et al.Ideal contrast medium bolus for perfusion measurement in dynamic lung.Radiology,1998,209:583-585.
10 Axel L.Cerebral blood flow determination by rapid-sequence computed tomography:theoretical analysis[J].Radiology,1980,137(3):679-686.
11 李智勇,伍建林,宁殿秀.多层螺旋 CT 灌注成像在全身肿瘤性病变的初步应用[J].放射学实践,2003,18(4):297-300.
12 丁毅,张镭,钱晓军.64层螺旋 CT 灌注成像在孤立性肺结节鉴别诊断中的应用[J].中国医学影像技术,2007,23(2):214-218.
13 Miles KA,Griffiths MR,Fuentes MA.Standardized perfusion value:universal CT contrast enhancement scale that correlates with FDG PET in lung nodules[J].Radiology,2001,220(2):548-553.
14 Delorme S,Knopp MV.Non-invasive vascular imaging:assessing tumor vascularity[J].Eur Radiol,1998,8(4):517-527.
15 孙灿辉,孟悛非,李子平,等.结直肠癌血管内皮生长因子与螺旋 CT 灌注成像的相关性[J].临床放射学杂志,2006,25(2):138-142.
16 Goh V,Halliqan S,Gartner L,et al.Quantitative colorectal cancer perfusion measurement by multidetector-row CT:does greater tumour coverage improve measurement reproducibility [J].Br JR adiol,2006,79(943):578-583.
17 Goh V,Halligan S,Hugill JA,et al.Quantitative assessment of tissue perfusion using MDCT:comparison of colorectal cancer and skeletal muscle measurement reproducibility[J].AJR AmJ Roentgenol,2006,187 (1):164-169.
18 Goh V,Halligan S,Taylor SA,et al.Differentiation between diverticulitis and colorectal cancer:quantitative CT perfusion measurements versus morphologic criteria initial experience [J].Radiology,2007,242 (2):456-462.
19 Liotta LA,Steeg PS,Stetler Stevenson WG.Cancer metastasis and angiogenesis:an imbalance of positive and negative regulation [J].Cell,1991,64(2):32.
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21 Vamashita K,Upadhay S,Mimori K,et al.Clinical significance of secreted protein acidic and rich in cystein in esophageal carcinoma and its relation to carcinoma progression [J].Cancer,2003,97:2412-2419.
22 Xie TX,Wei D,Liu M,et al.Stat3 activation regulates the expression of matrix metalloproteinase-2 and tumor invasion and metastasis [J].Oncogene,2004,23:3550-3560.
23 Ross J S,Kaur P,Fisher H A,et al.Prognostic significance of matrix metalloproteinase 2 and tissue inhibitor of metalloproteinase 2 expression in prostate cancer [J].Mod Pathol,2003,16:198-205.
24 Bodey B,GrogerAM,Siegel SE,et al.Invasion and metastasis:the expression and significance of matrix metalloproteinases in carcinomas of the lung [J].In Vivo,2001,15:175-180.
25 Chan C C,MengesM.Increased matrix metalloproteinase-2 concentration and transcript expression in advanced colorectal carcinoma [J].Int J Colorectal Dis,2001,16 (3):133.
26 Mstsuyama Y,Takao S,Aikou T.Comparision of matrix metalloproteinase expression between primary tumors with or without liver metastasis in pancreatic and colorectal carcinoma [J].J Surg Oncol,2002,80 (2):105.
27 Eastwood JD,Lev MH,Azhari T,et al.CT perfusion scanning with deconvolution analysis:pilot study in patients with acute middle cerebral artery stroke [J].Radiology,2002,222(1):227-236.
28 赵振军,梁长虹,谢淑飞,等．多层螺旋CT肺灌注对肺肿瘤的诊断价值．中国医学影像技术,2004,20(2)：232-236．
29 Weir HK,Thun MJ,Hankey BF,et al.Annual report to t he nation on t he status of cancer 1975-2000,featuring t he uses of surveillance data for cancer prevention and cont rol [J].J N at I Cancer Inst,2003,95 (17):1276-1299.
30 Park YN,Kim YB,Yang KM,et al.Increased expression of vascular endothelial growth factor and angiogenesis in the early stage of multistep hepatocarcinogenesis.Arch Pathol Lab Med, 2000,124(7):1061-1065.
31 Weidrner N.Tumoural vascularity as a prognostic factor in cancer patients:the evidence continues to grow[J].J Pathol,1998,184(4):119-122.
32 吴泰璜,吴莹光,粱欣荣,等.血管内皮生长因子及微血管密度与大肠癌生物学行为及预后的关系.中国肛肠病杂志.2001,21:6.
33 谭江平,校宏兵.血管内皮生长因子表达与结肠癌预后的关系.中国癌症杂志,2001,11:17.
34 Takahashi Y,Kitadai Y,Bucana CD,et al.Expression of vascular endothelial growth factor and its receptor.KDR,correlates with vascularity,metastasis,and proliferation of human colon cancer.Cancer Res,1995,55:3964.
35 White JD,Hewett PW,Kosuge D,et al.Vascular endothelial factor-D expression is an independent prognostic marker for survival in colorectal carcinoma.Cancer Res,2002,62:1669.
36 Takebayashi Y,Aklyama S,Yamada K,et al.Angiogenesis as an unfavorable prognostic factor in human colorectal carcinoma.Cancer,1996,78:226-231.
37 Bossi P,Viale G,Lee AK,et al.Angiogenesis in colorectal tumors:microvessel quantitation in adenomas and carcinomas with clinicopathological correlationsl Cancer Res,1995,55:5049-5053.
38 Chen CN,Cheng YM,Liang JT,et al.Color Doppler vascularity index can predict distant metastasis and survival in colon cancer patientsl Cancer Res,2000,60:2892-2897.
39 Soker S,Takashima S,Miao HQ,et al.Neuroplin-I is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor [J].Cell,1998,92(6):735-745.
1 孙灿辉,孟悛非,李子平等.结直肠癌微血管密度与螺旋 CT 灌注成像的相关性.中华放射学杂志.2006,40(1):77-80
2 Axel L.Cerebral blood flow determination by rapid sequence computed tomography:theoretical analysis.Radiology,1980,137(3):679-686
3 Miles KA,Griffiths MR.Perfusion CT:a worthwhile enhancement.B JR,2003,76,220-231
4 Miles KA.Tumor angiogenesis and its relation to contrast enhancement on computer tomography:a review.Eur J Radiol,1999,30(3):198-205
5 Miles KA,Measurement of tissue perfusion by dynamic computed tomography.B JR,1991,64:409-412
6 Axel L.Cerebral blood flow determination by rapid sequence computed tomography.Radiology,1980,137:679-686
7 Miles KA.Measurement of tissue perfusion by dynamic computed tomography.B JR,1991,64:409-412
8 Kenneth AM,Michael PH,Adrian KD.Functional images hepatic perfusion obtained with dynamic CT.Radiology,1993,188(2):405-411
9 Miles KA,Hayball MP,Dixon AK.Measurement of human pancreatic perfusion using dynamic computed tomography with perfusion imaging.BJR,1995,68(809):471-475
10 Cenic A,Nabavi DG,Craen RA,et al.Dynamic CT measurement of cerebral blood flow:A validation study.AJNR,1999,20(1):63-73
11 Peters AM,Gunasekera RD,Henderson BL,et al Non-invasive measurements of blood flow and extraction fraction Nucl Med Commun,1987,8:823
12 Cenic A,Nabavi DG.Graen RA,et al.Dynamic CT measurement of cerebral blood flow:a validation study,AJNR,1999,20(1):63-73
13 Blomley MJ,Coulden R,Dawson P,et al.Liver perfusion studied with ultrafast CT.Journal of Computer Assisted Tomography,1995,19(3):424-433
14 Miles KA.Perfusion CT for the assessment of tumour vascularity:which protocol?BJR,2003,76:S36-S42
15 Bell SD,Peters AM.Measurement of blood flow from first-pass radionuclide angiography:influence of bolus volume.Eur J Nucl Med,1991,18(11):885
16 熊正文,李春光,丁华野,等.肿瘤血管生成及微循环形态学研究技术.中国组织化学与细胞化学杂志,1998,7(4):499-500
17 Syviasta A,Laidler P,Davies R,et al.The prognostic significance of tumor vascularity in intermediate thick-ness kin melanoma.Am J Pathol,1998,133:419
18 Yancopoulos G.D,Davis S,Gale N.W.,R,Wiegand S.J.,Holash J.Vascular-specific growth factors and blood-vessel formation.Nature(Lond.),2000,407:242-248
19 Carmeliet P.,Jain R.K.Angiogenesis in cancer and other diseases.Nature(Lond),2000,407:249-257
20 Uzzan B,Nicolas P,Cucherat M,et al.Microvessel density as a prognostic factor in women with breast cancer:a systematic review of the literature and met-analysis.Cancer Res,2004,64(9):2941-2955
21 Obermair A,Wanner C,Bilgi S,Speiser P,Kaider A,Reinthaller A,et al.Tumor angiogenesis in stage IB cervical cancer:correlation of microvessel density with survival.Am j Obstet Gynecol,1998;178:314-319
22 Bosari S,Lee AK,Delellis RA,et al.Microvessel quantitation and prognosis in invasive breast carcinoma.Hum pathol,1992,23(7):755-761
23 Camphausen K,Menard C.Angiogenesis inhibitors and radiotherapy of primary tumours.Expert Opin Biol Ther,2002,2(5):477-481
24 Vora HH,Shah NG,Patel DD,et al.Prognostic significance of biomarkers in squamous cell carcinoma of the tongue:multivariate analysis.J Surg Oncol,2003,82(1):34-50
25 熊正文,朱光君,李春光,等.肿瘤血管及非小细胞肺癌检测技术研究进展.中华现代医学杂志,2001,1(3):12-15
26 Ward NL,Dumont DJ.The angiopoietins and Tie2/Tek:adding to the complexity of cardiovascular development.Semin Cell Dev Biol,2002,13(1):19-27
27 Yancopoulos G.D.,Davis S.,Gale N.W.,R.,Wiegand S.J.,Holash J.Vascular-specific growth factors and blood vessel formation.Nature(Lond),2000,407:242-248
28 Carmeliet P,Jain RK.Angiogenesis in cancer and other diseases.Nature(Lond),2000,407:249-257
29 吴泰璜,吴莹光,粱欣荣,等。血管内皮生长因子瘤内微血件密度与大肠癌生物学行为及预后的关系.中国肛肠病杂志,2001,21:6
30 谭江平,校宏兵.血管内皮生长因子表达与结肠癌预后的关系.中国癌症杂志,2001,11:17
31 Takahashi Y.Kitadai Y.Bucana CD.Et al.Expression of vascular endothelial growth factor and its receptor.KDR,correlates with vascularity,metastasis,and proliferation of human colon cancer.Cancer Res,1995.55:3964
32 White JD.Hewett PW.Kosuge D,et al.Vascular endothelial growth factor-D expression is an independent prognostic marker for survival in colorectal carcinoma.Cancer Res,2002,62:1669
33 MLES KA,LEGGETTD A,KELLEYB B,et al.In vivo measurements of neovascularization of liver matastases using perfusion CT[J].Br J Radiol,1998,71(843):276-281.
34 MLES K A.Functional CT imaging in oncology[J].Eur J Radiol,2003,13(Supp 15):134-138.
35 TATEISH IU,NISH IHARA H,WATANABE S,et al.Tumor angiogenesis and dynamic CT in lung adenocarcinoma:radiologic-pathologic correlation[J].Comput Assist Tomogr,2001,25(1):23-27.
36 KWAKB K,SHM H J,PARK E S,et al.Hepatocellular carcinoma:correlation between vascular endothelial growth factor level and degree of enhancement by multiphase contrast enhanced computed tomography[J].Invest Radiol,2001,36(8):487-492.
37 WANG Z Q,LU GM,L I J S,et al.The comparative study of tumor angiogenesis and CT enhancement in pancreatic carcinoma[J].European Journal of Radiology,2004,49(3):274-280.
38 WANG J H,Q U M P,WANG P J,et al.Dynamic CT evaluation of tumor vascularity in renal cell carcinoma[J].American Journal of Roentgenology,2006,186(5):1423-1430.
39 CEN IC A,NABAV ID G,CRAEN R A.A CT method to measure hemodynamics in brain tumors:validation and application of cerebral blood flow maps[J].AJNR,2000,21(3):462-470.
40 SWENSEN S J,VIGGIANO R W,MDTHUN D E,et al.Lung nodule enhancement at CT:multicenter study[J].Radiology,2000,214(1):73-80
41 周华,张敏鸣,肖圣祥,等.动态增强 CT 功能成像评价肺癌肿瘤血管生成的研究[J].中华放射学杂志,2006,40(2):171-175
42 张敏鸣,周华,邹煜.动态增强 CT 对孤立性肺结节的定量研究[J].中华放射学杂志,2004,38(3):263-267
43 文利,丁仕义,牟伟,等.肝细胞癌 CT 灌注参数与微血管密度的相关性研究[J].中华放射学杂志,2005,39(3):280-284
44 Dushyant V.Sahani,Sanjeeva P.Kalva,Leena M.Hamberg,I Dushyant V.Sahani,Sanjeeva P.Kalva,Leena M.Hamberg,Assessing Tumor Perfusion and Treatment Response in Rectal Cancer with Multisection CT:Initial Observations.Radiology 2005,234:785-792