摘要
目的：
评价四肢肿瘤微结构(微血管密度、血管内皮生长因、增殖细胞核抗原)与肿瘤生物学行为及动态增强磁共振的关系。
材料与方法：
1、一般资料：选择2007.9～2009.12在齐鲁医院行手术治疗的四肢肿瘤患者57例,年龄16～68岁,平均31.3岁。所有患者依次进行常规磁共振及动态增强扫描磁共振检查,穿刺活检,手术治疗和病理诊断。
2、病理取材及免疫组化：在手术切除的标本上对照磁共振动态增强扫描图像明显强化的部分取材,对标本进行常规病理及二步法免疫组化染色。用PBS缓冲液代替一抗作为阴性对照,用正常横纹肌标本代替肿瘤标本组作为正常对照。所使用的主要试剂有①CD34鼠抗人单克隆抗体；②VEGF兔抗人单克隆抗体；③PCNA鼠抗人单克隆抗体；④即用型二步法(非生物素)检测试剂盒。MVD计数方法参照Weidner方法,先在低倍视野(HP×40)下浏览切片,寻找“热点”,再在高倍视野(×400)下计数微血管数目,取5个高倍视野下微血管数目的平均值作为MVD。VEGF着色于细胞浆,统计方法参照Vohn等的评判标准,综合评价高倍视野(×400)下着色细胞百分比和细胞着色强度,分为阴性,弱阳性和强阳性(++)。PCNA主要着色于细胞核,按PCNALI分级,0-25%为Ⅰ级,26～50%为Ⅱ级,51-75%为Ⅲ级,>75%为Ⅳ级。病理及免疫组化评价由一位病理科医师协助完成。
将57例四肢肿瘤按照生物学行为分为良性组、中间性组、恶性组,比较三组间MVD、VEGF分级和PCNALI分级的差异。将57例四肢肿瘤按照VEGF表达分为VEGF弱阳性组和VEGF强阳性组,比较两组间MVD及PCNALI的差异。
3、磁共振动态增强扫描：动态增强磁共振成像(dynamic contrast-enhanced magnetic resonance imaging, DCE-MRI)使用3D FSPGR序列,对比剂使用马根维显(Magnevist,钆喷酸葡胺,0.1mmol/kg),不间断扫描16个时相。应用GE AW4.2软件分析骨骼及软组织肿瘤感兴趣区的时间～信号强度曲线(time-intensity curve,TIC)及动态增强参数(最大上升斜率(MSI)、信号增强幅度(SEE)),测量病变强化达峰值时的信号强度(SIpeak)和达峰值的时间(Tpeak),相对Tpeak,强化早期信号增强幅度(ΔSI)和相对强化早期信号增强幅度(相对ΔSI%)。
将57例四肢肿瘤按照TIC曲线类型分为三组,比较三组间MVD的差别。对磁共振动态增强扫描参数与MVD进行相关性分析。将57例肿瘤按照EGF表达分为VEGF弱阳性组和VEGF强阳性组,比较两组间TIC类型及各动态增强扫描参数的差异。
4、统计方法：
所有数据利用SPSS17.0软件进行处理。对定性资料进行行×列表资料的Fisher精确检验。对各磁共振量化参数及MVD进行正态分布检验,如果参数满足正态分布采用独立样本t检验和单因素方差分析,组间两两比较若方差齐用LSD法,若方差不齐用Dunnett's T3法。如果参数不满足正态分布则采用两个或多个独立样本的非参数检验,对两个独立样本的差异显著性检验用Mann-Whitney U检验,对多个独立样本的差异显著性检验用Kruskal-Wallis H检验。相关性检验用Kendall's tau-b和Spearman分析。检验水准双侧α=0.05。
结果：
1.病理结果来自山东大学齐鲁医院病理科的诊断报告,根据2002年WHO软组织肿瘤分类和WHO骨肿瘤分类,本研究中57例骨骼及软组织肿瘤包括良性肿瘤6例、中间性肿瘤13例、恶性肿瘤38例。
2.所有标本CD34染色均有阳性表达,MVD为24.5±11.6,其中良性肿瘤为8.5±6.2,中间性肿瘤为34.0±9.5,恶性肿瘤为27.8±14.1。MVD良性组、中间性组、恶性组间有统计学差异,两两间比较显示MVD在良性组与中间性组、良性组与恶性组间有统计学差异,在中间性组与恶性组间无统计学差异。VEGF在所有肿瘤中都有表达,积分范围为3-6分,良性肿瘤的VEGF表达呈弱阳性的3例,强阳性的3例,中间性肿瘤的VEGF表达呈弱阳性的5例,强阳性的8例,恶性肿瘤的VEGF表达呈弱阳性的3例,强阳性的35例,良性组与恶性组、中间性组与恶性组间VEGF表达的构成比有统计学差异,良性组与中间性组无统计学差异。PCNA在所有肿瘤细胞的细胞核均有阳性表达,且以Ⅲ级和Ⅳ级为主,PCNA在良性、中间性、恶性组间无统计学显著性差异。VEGF弱阳性组和强阳性组间MVD有统计学差异；PCNA LI II级组、Ⅲ级组和Ⅳ级组间肿瘤MVD无统计学差异。VEGF弱阳性组和强阳性组间PCNALI的构成比无统计学差异。
3.动态增强磁共振检查结果：57例四肢肿瘤的DCE MRI均表现强化。TIC曲线类型包括三型：慢升型,快升型,快升快降型,TIC类型构成在良性组、中间性组、恶性组间有统计学差异。动态增强磁共振半定量参数(Tpeak,相对Tpeak、相对ΔSI%)在良性组、中间性组、恶性组间有统计学差异。
4.动态增强磁共振结果与肿瘤免疫组化指标的相关性：MVD在TIC曲线类型Ⅱ型组、Ⅲ型组和Ⅳ型组间有统计学差异。达峰时间(Tpeak)、相对Tpeak及相对强化早期信号增强幅度(相对ASI%)与MVD有显著相关性。TIC曲线类型、相对Tpeak、Tpeak、相对ΔSI%在VEGF弱阳性组和强阳性组间有统计学差异。各DCE-MRI参数在PCNALIⅡ级组、Ⅲ级组和Ⅳ级组均无统计学差异。
结论：
1、四肢肿瘤的微结构与肿瘤的生物学行为有关,MVD及VEGF可用于判断良恶性肿瘤生物学行为及估计预后,PCNA对肿瘤的生物学行为评价可能没有帮助。
2、动态增强磁共振扫描能反映肿瘤的的形态特征和功能改变,对于四肢肿瘤的鉴别诊断有价值,且与肿瘤血管生成免疫组化指标(MVD、VEGF)有显著相关性,但与细胞增殖指标(PCNALI)无相关性。动态增强磁共振可用来判断肿瘤生物学行为及预后,从而可实现在体、无创、可重复地研究病变的微观结构,追踪肿瘤治疗过程中的生物学变化。
Objectives
To evaluate the correlations of limb's musculoskeletal tumors microstructure (microvessel density, vascular endothelial growth factor, proliferating cell nuclear antigen) and tumor biological behavior by immunohistochemistry, to evaluate the ability of dynamic contrast enhanced MRI in assessing microstructure of limb's tumors.
Materials and Methods
1. Both DCE MRI and immunohistochemistry were abtained in fifty seven patients with musculoskeletal tumors from September 2007 to December 2009.
2. The positive expression of microvessel density (MVD), vascular endothelial growth factor (VEGF), proliferating cell nuclear antigen (PCNA) in tumors were shown by immunohistochemical test (two step method). PBS buffer was used instead of the primary antibody as a negative control and a skeletal muscle specimen was used instead of tumor samples as a normal control. The specimen was observed totally in at a low power lens (×40) to find a 'hot spots' at first, and then at a high power lens (×400) to counting the number of vessels. The VEGF positive stained cells were counted per hundred of cells at a high power lens (×400), the percentage of positive stained cells were classified into 4 degrades, the degree of stain were also classified into 4 degrades, the combination of the positive percentage grade and the staining degree grade was serve as VEGF grade. The PCNA positive stained cells were counted per hundred of cells at a high power lens (×400), and classified into 4 grades serving as PCNALI. The MVD, VEGF grade and PCNALI were compared among the benign, intermediate and malignant limbs' tumors, the correlation of MVD and PCNALI with VEGF grade were assessed.
3. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) was carried out on the 57 patients by using 3.0T MR scanner. DCE-MRI scan with 16 phases was performed consecutively. The contrast material was injected at the rate of 3ml/s with a dose of O.lmmol/kg. The time-intensity curves, the maximum slope of increase, the signal enhancement extent, the signal of peak, the time to peak, the relative time to peak, the signal enhancement extent in early phase and the relative signal enhancement extent in early phase were obtained at AW4.2 workstation.
4. Statistic method:SPSS17.0 was used to analyze the parameters. The Categorical dates were analyzed by using Fisher exact test. The quantitative dates were normally tested. T test and one way ANOVA were used to analyze the normally distributed dates, the rank test was used to analyze the unmorally distributed dates. Kendall's tau-b and spearman rank correlation analysis were used for correlation analysis. P<0.05 was considered to be statistically significant.
Results
1. There were six benign tumors, thirteen intermediate tumors and thirty eight malignant tumors in our study.
2. All the specimen showed positive staining of CD34, VEGF and PCNA. The MVD of all the tumors, benign tumors, intermediate tumors and malignant tumors were 24.5±11.6,8.5±6.2,34.0±9.5,27.8±14.1 correspondingly. There was significant difference among the benign, intermediate and malignant tumors. VEGF staining showed weak positive in 3 benign tumors,5 intermediate tumors and 3 malignant tumors, strong positive in 3 benign tumors,8 strong tumors and 35 malignant tumors, there was significant difference among benign, intermediate and malignant tumors. There was significant difference of MVD between VEGF weak positive group and strong positive group. There was only one benign tumor showing gradeⅡstaining. There were three benign tumors, seven intermediate tumors and 20 malignant tumors showing gradeⅢstaining. There were 2 benign tumors,6 intermediate tumors and 18 malignant tumors showing gradeⅢstaining. There was no significant difference of PCNALI between VEGF weak positive group and VEGF strong positive group.
3. There are three types of TIC could be seen in all of the 57 tumors. There were significant difference of the proportion of TIC types, Tpeak,ΔTpeak ande relative ASI% among benign, intermediate and malignant tumors.
4. There was significant difference of MVD among TIC typeⅡgroup, typeⅢgroup and typeⅣgroup. There were negative correlation between Tpeak, ATpeak and MVD, there was positive correlation between relativeΔSI% and MVD. There were significant differences of proportion of TIC types, Tpeak, ATpeak and relative ASI% between VEGF weak positive group and VEGF strong positive group. There was no significant difference of proportion of TIC types, Tpeak, ATpeak and relative ASI% among PCNA gradeⅡstaining, gradeⅢstaining and gradeⅢstaining group.
Conclusions
1. There were significant differences of MVD and VEGF in the differential diagnosis of benign, intermediate and malignant tumors, while no significant difference of PCNALI. MVD and VEGF can be used to assess the biological behavior of tumors and estimate its prognosis, but not PCNA.
2. There were correlation between dynamic contrast enhanced parameters and the microstructure of the musculoskeletal tumors in limbs.
Objective To investigate the clinical significance of MRI in limb salvage surgery on osteosarcoma around the knee joint. Methods 21 patients with osteosarcoma were examined by X-ray and MRI, and were staged by Enneking surgical staging system. Based on the tumor extent in the medullary cavity and the surrounding soft tissues, the operative plans were formulated and the custom artificial prostheses were made. After chemotherapy, tumors were removed and custom2made prostheses were put on. Postoperatively the histopathological results were compared with the MR images. Followed up for 10 to 55 months (mean 38 months), the function of the involved knee joints was evaluated. Results Of the 21 cases,6 were in stageⅡa and 15 in stageⅡb, all were successfully operated on. The functional recovery rate was 76.7%. MRI was of little diagnostic value in manifesting osteosarcoma, but was of great value in demonstrating intramedullary extension, skip metastasis, the relationship of bone tumor to vessel, nerve, muscle and adjacent joints, bleeding and necrosis in osteosarcoma. Conclusion MRI has great values in making operative plans and ensuring the operative outcome in limb salvage surgery on osteosarcoma.
Background:osteosarcoma is one of the most common primary malignant bone tumors after myeloma. It is characterized by the direct formation of immature bone or osteoid tissue by the tumor cells, occurring with greatest frequency in the long tubular bones. Historically, treatment in patients diagnosed with osteosarcoma consisted of amputation, and with a cure rate of 10%, most patients died within a year after the initial diagnosis. The surgeon's goal was to resect the tumor using a procedure that would maximize patients' cosmetic and functional outcome without jeopardizing the long-term prognosis.
Currently, limb-salvage surgery often is undertaken. In most cases, limb-salvage surgery offers an improved functional outcome without compromising survival and avoids the need for external prosthetic devices, which have their own secondary complications. These techniques have been made possible with the introduction of
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