甲状腺乳头状癌遗传学标记及超声造影研究

【作者】 杨萌；

【导师】 姜玉新；

【作者基本信息】 北京协和医学院 ， 影像医学与核医学， 2012， 博士

【摘要】 目的1.获得国人散发型PTC全基因组DNA的CNA数据,筛选具有共性和差异的CNA片段内所覆盖的候选基因群组；2通过基因功能富集分析,筛选与PTC发病或生物学表型相关的核心基因。资料和方法对连续性入组的39例手术证实PTC样本进行BRAF(V600E)突变和RAS突变的PCR测序,以及RET的免疫组化检测。其中33例PTC进行array-CGH检查,获得全基因组DNA拷贝数据；使用DNA analytics软件进行原始数据分析筛选,获得33例PTC中具有共性的染色体CNA片段及其候选基因群组,并通过基因功能富集分析构建共性CNA片段中候选基因功能的通路网络。根据33例PTC的CNA变异度差异,比较CNA正常组、低度变异组及显著变异组三组样本临床及生物学表型的差异,同时筛选三组样本有差异的CNA片段及候选基因群,通过功能富集分析,探讨不同CNA类型发病机制的分子水平差异。本组PTC病例的中位年龄41岁(19-71岁),其中男性7例,女性26例。结果1.本组33例散发型PTC样本中,CNA整体变异度不大且以缺失为主；2.本组PTC中BRAF (V600E)突变发生率(58.97%),未发现RAS突变,RET阳性表达5例,BRAF(V600E)突变与RET阳性表达的发生独立存在,总计发生率71.79%,二者与淋巴结转移的发生无显著相关性；3.CNA正常组和CNA显著异常组中BRAF与RET异常发生率高于CNA低度变异组,CNA显著变异组PTC在肿瘤大小、发病年龄分组等临床特点上与CNA非显著变异PTC之间存在差异；4.33例散发型PTC的整体CNA候选基因富集分析结果提示PTC的发病和进展可能有MAPK通路之外的多种信号通路参与,CNA低度变异和CNA显著异常组候选基因富集分析结果提示二者在发病相关通路及功能基因群方面均有差异。结论1.国人散发型PTC不以DNA拷贝数变化为主要的遗传学特征性改变,不属于基因拷贝数高变异度的恶性肿瘤；2. BRAF(V600E)是本组PTC中最常见的MAPK通路基因突变,尤其在CNA正常组中发生率较高,可能是CNA正常组PTC的主要发病机制；CNA低度变异组的BRAF(V600E)发生率低,其发病可能主要受CNA变异片段内功能基因及相关通路的调控：CNA显著变异组发病机制复杂,可能同时受MAPK通路激活及其他多种信号通路的共同调控；3.CNA变异引发的基因功能调控和肿瘤信号通路激活是不同CNA变异组PTC临床及生物学表型差异的潜在遗传学基础；针对上述通路中的相关候选基因进一步筛选,有望发现新的PTC诊断和治疗靶点。4.基因功能富集分析结果提示CNA高变异、低度变异和正常组PTC在候选基因显著关联功能通路数量、候选基因在通路中的功能富集度及关键基因通路等方面均有显著差异目的1.超声微血管显像下定量评估PTC的灌注差异。2.研究PTC微血管灌注差异与CD34微血管密度分级(Microvascular density,MVD、血管内皮生长因子(Vascular endothelial growth factor, VEGF)表达、BRAF突变的相关性。3.探讨PTC灌注模式与CNA及其候选基因群组差异的相关性。资料与方法采用低机械指数实时灰阶谐波超声造影技术,对39例手术及临床诊断证实PTC散发型PTC病例进行超声微血管显像,包括经典型PTC33例,滤泡型PTC6例,采用病灶与甲状腺实质感兴趣区域(region of interest, ROI)时间-强度曲线峰值强度差ΔROI,定量评估PTC微血管显像的灌注特点。术后石蜡标本行CD34,VEGF免疫组织化学染色,比较PTC微血管灌注量化指标ΔROI与CD34,VEGF表达水平及BRAF突变、CNA特点与的相关性。结果1.AROI与CD34呈强正相关(p=0.000),CD34不同分级组间的ΔRO1分布存在显著性差异,且三组间AROI均值的95%置信区间完全没有重叠；2.PTC病灶大小分级与ΔROI及CD34之间均呈正相关,微血管灌注强度随PTC病灶体积增大而增高；3.依据CD34分级组内的ΔROI的阈值范围结合时间-强度曲线达峰时病灶与实质间的回声特点,将PTC的灌注模式定义为低增强17例(43.59%),局部增强10例(25.64%),高增强14例(35.9%),其样本分布特点与CD34一致性较好；BRAF(+)组VEGF以中、高水平表达为主(p=0.017),BRAF突变的多因素logistic回归分析结果提示多变量中仅VEGF的表达分级与BRAF突变独立相关；4.CNA显著变异组PTC以高灌注和局部增强为主,CNA低度变异组以低灌注和局部增强为主要改变,而CNA正常组主要呈低灌注模式；PTC微血管灌注ΔROI的均值随CNA变异度增加而升高。结论1.ΔROI作为一种较为客观的量化指标,可用于评价PTC的微血管灌注模式和强度；2.PTC微血管灌注强度随PTC病灶体积增大而增高,提示PTC发生早期不以大量肿瘤新生血管为特征,血管生成及高灌注状态可能是PTC肿瘤进展的预测指标；3. VEGF的表达分级是BRAF突变的独立相关影响因素,VEGF高表达可能在MAPK通路激活PTC的发生和进展中有调控作用；4.随CNA变异度增加,PTC微血管灌注强度升高,CNA变异度与PTC的微血管灌注强度具有一定的相关性。更多还原

【Abstract】 Objective1. To collect the whole genomic CNA data of Chinese sporadic PTCs, and gather the common chromosomal imbalances with their covered candidate gene groups.2. To select the core functional genes correlated with the pathogenesis and biological phenotype of Chinese sporadic PTC with meta-analysis.Materials and methods39patients were included in this study, total thyroidectomy were taken in PUMCH with clinically and pathologically confirmed sporadic PTC. The mean ages of the39PTCs was40.00±13.82years, with8cases of male and33cases of female. PCR sequencing of BRAF(V600E) and RAS mutation, immune-histochemical examination(IHC) of RET expression were performed. array-CGH was carried out in33of the39PTCs, and the common CNAs of33sporadic PTCs were selected by DNA analytics system from Angilent. Meta-analysis was performed for gene functional grouping and mapping of the pathway network. CNA of the33PTCs in this study was grouped into3types:PTC with normal CNA (N-CNA), PTC with low-variance CNAs (L-CNAs) and PTC with significant abnormal CNAs (A-CNAs), the clinical and biological phenotypes between the3groups were compared. Distinct CNAs with the covered candidate gene groups in the3different CNA types were selected and analyzed by meta-analysis, the difference of the gene type and their function in the pathway networks were compared.Results1. Low variability of chromosomal imbalance is a feature of the33sporadic PTCs;2. The incidence of BRAF(V600E) was (58.97%) in all the39PTCs,5PTCs with RET expression in IHC, the combined incidence of BRAF mutation and RET was71.79%, and no correlation was found between the lymphanode metastasis and the BRAF/RET mutation. No RAS mutation was detected in39PTCs. 3. The incidence of BRAF/RET mutation was higher in N-CNA and A-CNAs groups, comparing with the L-CNAs group. Significant difference was detected between the A-CNAs and L/N-CNAs groups in some clinical features, such as the tumor size and age of the patients.4. Meta-analysis results of the common CNAs in33sporadic PTCs showed that the pathogenesis of sporadic PTC involved several pathways besides classical MAPK pathway; meta-analysis results discovered differences in functional gene groups and related pathway networks between the A-CNAs and L-CNAs group.Conclusion1. Significant copy number variation is not the main genetic event in Chinese sporadic PTCs.2. BRAF (V600E) is the most frequent MAPK pathway related gene mutation in this study group, which might play a key role in the pathogenesis among the N-CNA PTC group. A-CNAs seemed to conceive a more complicated pathogenesis, which might be regulated by several pathways besides the classical MAPK pathway activation. The pathogenesis of L-CNAs is correlated more closely to the functional gene groups in CNAs. Objective1. To evaluate the contrast enhanced perfusion pattern of PTC micro-vascular imaging (MVI) quantitatively.2. To investigate the correlation between PTC MVI features and CD34micro-vascular density (MVD), vascular endothelial growth factor (VEGF), BRAF (V600E) mutation,3. To discuss the correlation of PTC MVI pattern and CNA, together with the candidate gene groups.Method and materials39pathological and clinical confirmed sporadic PTCs were evaluated with real-time gray-scale contrast-enhanced micro-vascular imaging under a low mechanical index. The micro-bubble agent was SoneVue. Of the39PTCs,33were classical PTCs,6were PTC with follicular variant (FVPTC). The AROI,which is the subtraction of peak echo intensity between the lesion region of interest (ROI) and normal thyroid parenchyma ROI, was used to evaluate the perfusion characteristics of PTC MVI quantitatively. The paraffined specimens were selected for immunohistochemical (IHC) staining for CD34and VEGF, and the correlation between△ROI and the CD34, VEGF expression level, the BRAF mutation and CNA groups were evaluated.Results1.△ROI was strongly correlated with the CD34expression(p=0.000), significant differences were detected in the distribution pattern of AROI value among different CD34expression levels, no overlapping of the mean△ROI values and the95%confidence intervals was found among the3CD34expression levels.2. The PTC MVI perfusion was classified into3patterns, low perfusion, focal perfusion and high perfusion, on the basis of combining△ROI values with the peak ehco pattern in time-intensity curve. 3. The median and high VEGF expression level was the mainstay in BRAF (+) PTC. Multi-variance logistic regression analysis showed that VEGF expression level was the independent factor for BRAF mutation.4. The A-CNA PTCs presented mainly with high and focal perfusion pattern, the L-CNA PTCs showed a combination of focal and low perfusion, the N-CNA PTCs mostly demonstrated low perfusion patterns. The mean AROI values rose with the extent of CNAs.Conclusions1. AROI is an objective indicator for the evaluation of PTC MVI pattern and intensity.2. VEGF expression level is the independent effect factor for the BRAF (600E) mutation, the over expression of VEGF might play a key role in the regulation of MAPK pathway activation and other pathways potentially related to the pathogenesis of PTC.3. The MVI perfusion intensity of PTC rose with the extent of CNAs, which showed a positive correlation.更多还原