视网膜母细胞瘤综合治疗及与肿瘤血管生成细胞因子关系的研究
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摘要
研究背景：
视网膜母细胞瘤(retinoblastoma, Rb)是婴幼儿最常见的一种原发性眼内恶性肿瘤,严重危害患儿生命及视功能。随着临床治疗水平的不断提高,治疗的目标也从单纯局限于保护患儿生命转向立足于提高患儿的生存质量,即保留眼球和有用视功能的方向发展,而实现这一目标的最关键问题在于患儿的早期发现和诊断。由于Rb患儿的年龄一般都在3岁以下,肿瘤的早期发现相当困难,这不仅是由于儿童眼底检查配合不佳,更是缺乏清晰、直观的检查成像手段所造成的。广域数字化小儿视网膜图像系统(wide-field digital pediatric retinal imaging system, RetCam Ⅱ)作为目前最先进的儿童眼底成像技术为Rb的防治及持续随访提供了极大地便利。但长期以来仍缺乏对于Rb临床信息的完整收集及整理分析系统,只能采取简单的文字描述或绘图进行记录,不利于患儿临床长期随访观察和持续的综合治疗疗效评估。有鉴于此,我们希望利用计算机技术建立一套收集Rb图像和文字信息的图文一体化系统,为Rb的防治研究提供信息支持。通过吸入麻醉下的眼底成像技术与信息采集系统相结合的Rb图文系统,可以使眼科医师获得全面完整、准确直观的患儿信息资料,促进了Rb的临床诊断、治疗和随访等各个方面的发展,更好的把握Rb的眼底表现特征和病变进展情况,有效的帮助临床医师采取合理干预措施,对于提高患儿的生存质量也具有重要的价值。本研究创建的一整套的检查方式和图文输出,在临床教学和科研中也具有良好的应用前景。
目前,随着化疗为主的综合治疗的兴起,Rb患儿的诊疗模式也发生了巨大的变化,常规外放射治疗渐渐淡出主流治疗范围,原有的Reese-Ellsworth5级分期体系已不再能够适应新的要求,本世纪初产生了新的视网膜母细胞瘤国际分期(International Intraocular Retinoblastoma Classification, IIRC),2005年全球共有27个中心参与,包括我国的同仁眼科中心,证明了IIRC对于Rb化疗的指导作用。但我国Rb患儿就诊时多为晚期,保守治疗效果相对较差。因此,临床治疗仍是目前面临的最主要最现实的问题,我院自2008年开始组建了Rb专业治疗组,开展现代Rb的综合治疗,取得了不错的效果,同时收集了丰富的临床资料。本课题由此点出发,通过对近5年来的Rb综合治疗以及23G微创玻璃体手术联合球内注药化疗的新型治疗方式做一全面的评估与分析,以达到提高Rb治疗水平、探索符合我国国情的个体化治疗手段的目的。
长期以来,防止Rb的复发和转移是其治疗中最为棘手的难点。40年前,Folkman首次提出肿瘤的生长和转移是有血管依赖性的,因此阻断肿瘤的血管化可以作为肿瘤治疗的有效策略。VEGF及其相关的因子作为刺激血管生成的最强大的细胞因子群在肿瘤新生血管化的过程中占据中心地位,是介导新生血管生成的关键因素。有鉴于此,结合以上思路,我们选取综合治疗成功和失败的部分Rb患儿,抽取其血清,运用RayBiotech人血管生成相关细胞因子芯片检测治疗后Rb患儿血清中43种VEGF及其相关细胞因子的表达情况,整体的、动态的评估蛋白质分子水平的变化情况,从VEGF及其相关因子群为核心的肿瘤新生血管化的角度探讨Rb的临床治疗预后和侵袭转移机制。
机体生理机能的真正执行者是蛋白质,因此从蛋白质水平对疾病进行研究显得特别重要。抗体芯片技术作为一种高通量的分子蛋白检测技术,继承了免疫荧光、化学发光和微列阵技术的诸多特点,逐渐发展成为从整体角度定性和定量检测蛋白质分子的研究平台,在各种疾病的相关分子发现、分子诊断和疗效评价方面发挥重要作用,具有良好的应用前景。本研究希望利用这一技术从整体的宏观角度了解肿瘤血管新生化的微环境中复杂的细胞因子网络调控机制,从而探索未来潜在的Rb治疗新的介入手段和干预策略。
研究目的：
1.建立视网膜母细胞瘤临床图文信息采集系统,收集管理Rb患儿所有的临床信息及眼底图片资料,为掌握Rb发病的机制和规律,总结和探索Rb治疗特点奠定基础。
2.探讨视网膜母细胞瘤临床诊治与IIRC分期特点
3.研究视网膜母细胞瘤综合治疗的特点并探讨其相关临床因素,探索适合国情的个体化综合治疗方式；初步总结23G微创玻璃体手术联合球内注药化疗治疗Rb的临床经验和预后效果,摸索晚期Rb治疗的新方法；同时研究眼摘前的化疗与晚期Rb患儿肿瘤转移及死亡率的相关性。
4.利用细胞因子抗体芯片检测技术检测综合治疗保眼成功和失败的相同分期患儿治疗后血清中43种血管生成相关细胞因子的表达变化,从肿瘤新生血管化的角度探讨Rb的临床治疗和侵袭转移机制。
研究方法：
1.使用AVA软件语言,建立视网膜母细胞瘤临床信息采集系统,收集整理近十年的Rb临床资料,为分析Rb的诊疗特点及规律提供信息支持。
2.回顾性分析我院2003年至2012年十年间116例Rb患儿的临床资料。
3.分析我院从2009年至2012年间进行综合治疗的75例Rb患儿的临床资料,综合治疗的方法包括：化疗、局部治疗(激光或冷冻)、眼球摘除、玻璃体手术联合球内注药。其中采取以化疗为主联合局部治疗+眼摘治疗的患儿65例,其余10例为23G微创玻璃体手术联合球内注药化疗的手术干预治疗。
4.利用pTNM分级和特定疾病生存率(Disease-specific survival, DSS)的方法研究眼摘前的化疗与晚期Rb患儿肿瘤转移及死亡率之间的相关性。
5. RayBiotech人血管生成相关细胞因子芯片检测治疗后不同预后的Rb患儿血清中43种细胞因子的表达情况。
6.数据分析采用版本2.15.1的R统计软件中的卡方检验,T检验,Fisher's精确检验,Wilcoxon等级总和检验和生存率分析。P≤0.05认为有显著性差异。
结果：
1.建立了视网膜母细胞瘤临床信息的采集系统,主要有三个模块组成：文字报告模块、影像图片模块及病例查询模块,系统可以生成完整的Rb患儿图文报告资料。
2.分析116例Rb患儿临床资料的IIRC特点,其中单眼患儿为79例(68.1%),双眼患儿为37例(31.9%)；有遗传性家族史的单眼患儿为1例(1.27%),双眼患儿为5例(13.51%),两者经统计分析有显著性差异,P<0.05；在Rb患儿中最常见的初诊症状为白瞳症,比例为78.4%；性别方面男性74例(63.79%),女性42例(36.21%)；患儿总体的初诊时间、诊断滞后时间及随诊时间分别为21.49个月、5.14周及30.72个月,单眼患儿的初诊时间为24.27个月,双眼患儿为15.1个月,单双眼患儿的初诊时间经统计分析有显著性差异,P≤0.05；所有患儿中肿瘤为单个瘤体的占75%,多个瘤体的占25%；Rb患儿的IIRC国际分期方面,所有153只患眼中A期占5只(3.27%),B期占7只(4.58%),C期占11只(7.19%),D期占56只(36.60%),E期占69只(45.10%),自发退变的占4只(2.61%),另有1只眼由于在外院摘除而分期不明；所有患眼中眼球摘除的有97眼,占63.4%,总体保眼率为36.6%；从各分期的保眼率来看A期为5眼(100%),B期为7眼(100%),C期为9眼(81.82%),D期为24眼(42.86%),E期为10眼(14.49%),其余自发退变的4眼中有3眼由于萎缩而摘除。经统计学分析A、B、C期之间无显著性差异,P>0.05；A、B、C期与D、E期之间均有显著性差异,P≤0.05。
3.化疗为主联合局部治疗+眼摘治疗的所有65例92眼(双眼27例,单眼38例)Rb患儿四年的总生存率为92.3%,A期至E期的生存率分别为100%、100%、100%、95%和90.00%,经统计学分析显示各期存活率差异无统计学意义,P>0.05；所有患儿总的保眼率为51%,A期至E期的保眼率分别为100%、100%、85.70%、77.10%和10%,各组间采用两独立样本卡方检验,A、B、C、D各期之间保眼率无显著性差异,P>0.05；但E期和其他各期之间保眼率均有显著性差异,P<0.05；中低风险Rb患儿综合治疗结果：A,B,C期共15只眼,治疗成功14只眼,失败1只眼,治疗成功率为93.3%；高风险Rb患儿综合治疗结果：D、E期Rb患儿共60例,采用化疗联合局部治疗保眼的共42例,分别为D期18例和E期24例,D期的保眼治疗成功率为61.1%(11/18),E期的保眼治疗成功率为16.7%(4/24),其余18例均为单纯眼摘或眼摘后化疗的患儿。进一步分析42例D期和E期的保守治疗与临床因素相关性,治疗成功与失败患儿的初诊时间分别为12和16.1个月,随访时间分别为22.47和28个月,经统计学分析均无显著性差异,P>0.05；与肿瘤位置相关性分析,将42例患儿眼内肿瘤的位置分为3组即：后极部、周边部(包括中周部)和后极部+周边部,三组治疗成功的例数分别为4例,6例,5例；成功率分别为23.50%,85.70%,27.80%,经卡方分析后极部与周边部、周边部与后极部+周边部有显著性差异,P≤0.05；42例患儿中有4例死亡,全部为肿瘤位置处于后极部的患儿,其他两组位置的患儿均无死亡,经统计学分析,后极部与后极部+周边部两组之间有显著性差异,P≤0.05；另外各组间均无显著性差异,P>0.05。
4.23G微创玻璃体手术联合球内注药化疗治疗的Rb患儿10例,平均术后随访时间6.4个月,术中硅油填充的患儿有4例,其中2例已经取出硅油,有7例患儿术后视力有提高,1例同术前,2例下降,所有患儿眼球保存,保眼率为100%,未发现眼外转移,肿瘤灶基本钙化萎缩,1例患儿术后发生玻璃体少量出血,2例术后发生轻度脉络膜脱离,1例发生持续球结膜水肿,后经治疗好转,未发现其他严重并发症。
5.用pTNM分级和特定疾病生存率(DSS)的分析方法对41例的Rb组织病理学报告(26男,15女,双眼16例,单眼25例,摘除的眼球13眼为D期,28眼为E期)进行研究。每位患者仅以一只眼分期纳入统计,双侧Rb患者按病情较重侧患眼的分期参与研究。眼球摘除术作为首选治疗方式的有16只眼,另外25只眼为眼摘前做了化疗治疗的。这两组在性别,单侧与双侧发病,患眼的国际分级或者诊断年龄方面经统计学分析无显著性差异,P>0.05。五名死亡的患儿均为眼摘前化疗组E期患儿,双眼两名,单眼3名。双眼患儿整体的平均诊断年龄为19.3个月,范围2-54个月,单眼患儿整体的平均诊断年龄为25.5个月,范围4-72个月；首选治疗是眼摘的生存患儿的随诊时间(平均31.1个月,范围11-52个月)长于眼摘前化疗的生存患儿随诊时间(平均26.4个月,范围10-50个月)。首选眼球摘除的患儿从诊断到眼球摘除的平均时间是0.9个月,而眼摘前化疗的患儿从诊断到眼球摘除的时间中位数是5.7个月,经统计学分析有显著性差异,P≤0.05。
患儿特定疾病生存率分析：患儿的生存时间从诊断时开始计算,死亡患儿到死亡日期止。由于死亡患儿均为E期的患者,因此比较E期未行化疗直接眼摘患儿与眼摘前行化疗治疗患儿的DSS,经统计学分析两者接近有显著性差异,P=0.05。眼摘时间推迟3个月以上的患儿相对于3个月内眼摘的患儿DSS有显著性差异,P<0.05。
分析41例眼球摘除的病理组织片的pTNM分级,总体来看,眼摘前化疗组患儿与未化疗组患儿pTNM的风险性相比,两者无显著性差异,P>0.05；但在高风险方面,眼摘前化疗组患儿的比例为12%,而眼摘前未化疗组患儿的比例为37.5%,前者的高风险比例明显要少；分期比较,在D期中两组具有显著性差异,P<0.05,即化疗组比未化疗组的风险低；但在E期的患儿中两组的风险性没有显著性差异,P>0.05
6.抽取综合治疗后保眼成功和失败的Rb患儿外周血,进行血清中43种血管生成相关细胞因子的抗体芯片检测,结果发现11种细胞因子的表达发生改变,有显著性差异。其中：发生表达上调的2种,为IFN-γ和Endostatin;发生表达下调的9种,为ENA-78、Angiopoietin-2、VEGF-R2、MCP-1、VEGF、 MMP-9、IL-1β、PIGF和Tie-2。
结论：
1.使用Java语言程序创建了Rb患儿临床信息的图文采集系统,可以完整的搜集、整理各种患儿信息资料,并可将资料数据以EXECL表的形式输出,同时可以形成包含患儿病情和诊疗情况的图文报告。
2.白瞳症是Rb患儿中的初诊症状中比例最高的临床表现,超过患儿总数的3/4；与国外相比,我国患儿的初诊时间和病程普遍偏晚,且肿瘤以单发为主所占比重最大,比率达到75%；分期上也以D期和E期为主的晚期病患为主,超过了80%。治疗上,保眼率为36.6%,且眼摘率和分期密切相关。说明我国的Rb诊断和治疗水平还比较低,患者发现时多病情严重,预后不佳,需要加强对民众的Rb健康教育,提高Rb早期诊断的水平,探索出新的有效且经济的个体化治疗新方法。
3.化疗为主联合局部治疗+眼摘治疗的综合治疗手段提高了Rb患儿的生存率,但患者的生存率和保眼率与其临床分期密切相关,晚期病例的治疗效果不佳,眼摘率较高；同时治疗预后与肿瘤所处的位置有高度相关性。肿瘤的位置对于临床治疗的选择性具有指导意义,也是影响临床Rb综合治疗疗效的重要因素。
4.对于晚期的化疗失败或效果不佳的Rb患儿,玻璃体手术联合球内注药的治疗新方法短期内的观察效果良好,保眼率为100%,且术后未见严重的手术并发症,是一种安全有效的治疗手段,但还需要今后大样本长期的观察评估。
5.如果从诊断到眼球摘除的时间超过3个月,对那些组织病理学上分期较差的严重E期Rb患儿进行眼摘前的化疗治疗,同时没有密切监控和适当的对高风险因素的处理,可能将导致因为转移或复发造成的死亡率升高。研究结果提示在晚期的Rb治疗中要适度掌握治疗的分寸,即如何避免保眼治疗后带来的可能肿瘤转移问题,在何种情况下要及时坚决的摘除眼球或采取保眼治疗,怎样把握两者之间的平衡关系是今后需要深入研究和总结的关键问题。
6.使用细胞因子抗体芯片技术可以检测不同预后的Rb患儿血清中血管生成类细胞因子的表达变化情况,以VEGF/VEGF R2和Ang2/Tie2为核心的肿瘤血管化是Rb综合治疗预后效果的重要影响因素,肿瘤的预后转归是一个以血管生成化为核心的、复杂的涉及免疫、炎症、转移侵袭、凋亡的病理生理过程,VEGF/VEGF R2和Ang2/Tie2可以作为今后Rb治疗的新的靶点,具有巨大的潜在临床应用价值。
创新点：
1.构建了一套行之有效的视网膜母细胞瘤图文信息采集系统。
2.提出了23G微创玻璃体手术联合球内注药化疗治疗晚期视网膜母细胞瘤的新思路,并进行了初步总结,为Rb综合治疗提供了新的手段。
3.提出了过度化疗或保眼治疗可能引起晚期视网膜母细胞瘤患儿死亡率增加或转移风险升高。
4.提出Rb治疗转归的核心为肿瘤血管化,VEGF/VEGF R2和Ang2/Tie2可以作为今后Rb治疗的新的靶点。
Background:
Retinoblastoma (retinoblastoma, Rb) is the most common primary infancy intraocular malignant tumors, with a serious risk of life and visual function of children. With the improving of clinical treatment, the goal of treatment is also shifted from simply limited to the protection of children's life to a new development direction for improving the quality of life of children, that is, the eyeball preservation and keeping useful visual function. Early detection and diagnosis of children is the most critical issue to achieve this goal, because ages of Rb children generally are less than3years old, the early detection of tumors is quite difficult, not only due to the child's poor cooperate with the fundus examination, more is the lack of clear and intuitive means of examination and imaging. Wide-field digital pediatric retinal imaging system (RetCam Ⅱ) as the most advanced children fundus imaging technology, greatly facilitates the prevention and continuous follow-up of Rb. But, for a long time, there is still a lack of collecting and collating analysis system for the complete clinical information of Rb. The recordings can only be taken by simple text description or drawing, which is not conducive to the clinical long-term follow-up observation and ongoing treatment efficacy assessment for children. In view of this, we hope to establish a integration of graphic and text information system for collecting Rb image and text using computer technology, to lay the information support for the prevention and treatment research of Rb. Through Rb graphic and text system, which combining the fundus imaging technology under inhalation anesthesia and information collection system, an ophthalmologist can obtain a full, intuitive and accurate information of children. It can promote the improvement of clinical diagnosis, treatment and follow-up in all aspects of Rb, lead a better grasp the Rb fundus manifestations and lesions progress, help clinicians to take reasonable interventions effectively, and also provide an important value for improving the survival treatment of children.In this study, a set of inspection methods and graphic and text output also has a good prospect in clinical teaching and research.
With the rise of the comprehensive treatment of chemotherapy, the model of diagnosis and treatment for Rb has also undergone a huge change that conventional external radiation therapy has gradually fade out of the range of mainstream treatment, the original of Reese-Ellsworth5level staging system is no longer able to adapt to the new requirements, therefore, a new International Intraocular Retinoblastoma Classification (IIRC) has been established at the beginning of this century. In2005, a total of27centers worldwide, including Tongren Eye Center, proved the directing role of IIRC on Rb chemotherapy. However, most Rb children in China are in advanced stage when visiting hospital, lead to a relatively poor outcome of conservative treatment. Therefore, the clinical treatment is still currently the most important reality. Since January2008, an Rb professional treatment group has been found in our hospital, to carry out a comprehensive modern treatment of Rb, and has achieved good results and collected a wealth of clinical data. Based on this point of view, the topic performed a comprehensive assessment and analysis on the Rb comprehensive treatment in the past five years as well as the new treatment of23G minimally invasive vitreous surgery combined with intraocular injection chemotherapy,and by histopathological pTNM staging of children with Rb in advanced stage, we have studied potential adverse effects for patients with chemotherapy, in order to improve the level of Rb treatment, exploring individualized treatment in line with our national conditions.
For a long time, prevention of relapse and metastasis of Rb is the trickiest difficulty of the treatment.40years ago, Folkman first proposed vascular dependent tumor growth and metastasis, therefore, blocking tumor blood vessels can be considered as effective cancer treatment strategy. VEGF and its related factors, as the most powerful group of cytokines that stimulate angiogenesis, occupy a central position in the process of tumor angiogenesis, and it is a key factor to mediate angiogenesis. In view of this, combining above ideas, we selected some children with successful and unsuccessful chemotherapy, extract the serum, and test the expression of43VEGF and its related cytokines in serum of children after Rb treatment using RayBiotech human angiogenic-related cytokines chip, carry out an overall, dynamic assessment of the changes of level of protein molecules, considering VEGF and its factor group as the core of tumor angiogenesis, we explore the Rb clinical prognosis, invasion and metastasis mechanism.
The body's physiological function is actually executed by protein, thus, the protein levels are particularly important for disease research. Antibody chip technology, as a high-throughput protein molecule detection technology, inherits many characteristics of the Immunofluorescence, chemiluminescence and microarray technology, and gradually develops into a research platform for the overall qualitative and quantitative detection of protein molecules, plays an important role in a variety of disease-related molecular discovery, molecular diagnostics and efficacy evaluation. and has good application prospects. In this study, we hope to take advantage of this technology, from the macro point of view, make an overall understanding of the complex cytokine network regulation mechanisms in new biochemical micro-environment of the tumor vasculature, in order to explore the potential new interventional means and intervention strategies of the Rb treatment in the future.
Objective:
1. Establish the clinical graphic and text information collection system for Rb, collecte and manage all clinical information and fundus images of Rb children, to lay foundations for mastering the pathogenesis and laws of Rb, and summarizing and exploring Rb treatment characteristics.
2. Explore the clinical diagnosis and treatment of retinoblastoma and IIRC staging characteristics
3. Study the characteristics of comprehensive treatment on Rb and to investigate the related clinical factors, to explore the patterns for the individualized treatment; preliminarily summarize the clinical experience and prognostic effect of23G minimally invasive vitreous surgery combined with intraocular injection chemotherapy for Rb treatment, fumble new treatment methods for advanced Rb; and study the correlation between chemotherapy and cancer metastasis and mortality of advanced Rb children.
4. Using cytokine antibody chip technology, for children in the same stage who succeed or failed in eyeball preservation by comprehensive treatment, determine the changes in expression of43angiogenesis-related cytokines in serum after the treatment. Considering tumor angiogenesis, we explore the Rb clinical treatment, and invasion and metastasis mechanism.
Methods:
1. Use the JAVA software or language, establish the Rb clinical information acquisition system, and collect Rb clinical data over the past ten years, to provide information support for the analysis of clinic characteristics and laws of Rb.
2. Perform a retrospective analysis on the clinical data of116cases of Rb children in our hospital over the past ten years from2003to2012.
3. Analyze75cases of Rb children's clinical data with comprehensive treatment in our hospital from2009to2012, the comprehensive treatment methods include: chemotherapy, local treatment (laser or cryotherapy), enucleation, vitreous surgery combined with injection. In which,65cases have taken chemotherapy-based joint topical treatment+enucleation, while the remaining10cases have taken surgical intervention by23G minimally invasive vitreous surgery combined with intraocular infusion chemotherapy.
4. By pTNM staging and disease-specific survival (DSS) methods, study on the correlation between chemotherapy before enucleation and cancer metastasis and mortality of the children with advanced Rb.
5. Using RayBiotech angiogenesis-related cytokine chip, determine the changes in expression of43angiogenesis-related cytokines in serum of children with different prognosis after the treatment.
6. Data analyses were performed with R2.15.1version software. Chi-square test, T test, Fisher's exact test, Wilcoxon rank sum test and survival analysis were performed. A P-value≤0.05was chosen to define statistical significance.
Results:
1. The Rb clinical information collection system has been established, mainly consisting of three modules:the text report module, graphic image module and case query module, and the system can generate a complete graphic and text report information for Rb children.
2. Analyze on clinical information of116cases of Rb children, in which,79 cases with unilateral Rb (68.1%),37cases with bilateral Rb (31.9%); in children with a genetic family history,1case was unilateral (1.27%),5cases were bilateral (13.51%), there were significant differences in statistical analysis (P<0.05); most common symptoms in initial diagnosed Rb children were leukocoria (78.4%); On gender aspect,74males (63.79%) and42females (36.21%); for those children, overall initial diagnosed time, diagnostic delay time and follow-up time were21.49months,5.14weeks and30.72months, respectively. The initial diagnosed time of unilateral Rbs in children were24.27months, bilateral Rbs were15.1months, there were significant differences in statistical analysis between the two groups, P≤0.05; In all children,75%had single tumor,25%had more than one tumor; For IIRC of Rb children, of all153affected eyes,5in group A (3.27%),7in group B (4.58%),11in group C (7.19%),56in group D (36.6%),69in group E (45.10%), and4with spontaneous degeneration (2.61%), another1eye removed from the other hospital and the stage was unknown; in all eyes with Rb,97performed enucleation (63.4%), the overall eyeball preservation rate was36.6%; considering the eyeball preservation rate of different staging,5in group A (100%),7in group B (100%),9in group C (81.82%),24in group D (42.86%),10in group E (14.49%),3of4spontaneous degenerative eyeballs were removed due to atrophy. The statistical analysis of the groups A, B, C showed no statistically significant difference (P>0.05); Group A, B, C and Group D, E showed a significant difference (P<0.05).
3. With chemotherapy combined topical treatment+enucleation, in all65cases (92eyes,27bilateral cases,38unilateral cases) of Rb children,4-year total survival rate was92.3%, in groups A-E, the survival rates were100%,100%,100%,95%and90.00%, the statistical analysis showed that survival rate in different groups was not statistically significant (P>0.05); total eyeball preservation rate of all children was51%, in group A to E, eyeball preservation rates were100%,100%,85.70%,77.10% and10%, respectively, using the chi-square test of two independent samples among the groups, the eyeball preservation rates among A, B, C, D groups showed no significant difference, P>0.05; but eyeball preservation rates between group E and other groups showed significant difference, P<0.05; the comprehensive treatment outcomes in low-risk Rb children:with15eyes in groups A, B, C,14eyes successful and1eye failed, success rate was93.3%; the comprehensive treatment outcomes in high-risk Rb children:with a total of60cases of Rb children in groups D, E, a total of42cases were treated with chemotherapy combined local therapy,18in group D and24in group E, the eyeball preservation treatment success rate of group D was61.1%(11/18), in group E was16.7%(4/24), the remaining18cases were simple eyeball enucleation or eyeball enucleation after chemotherapy. Perform further analysis on the correlation of conservative treatment and clinical factors of42cases in groups D and E, in children with successful and failed treatment, respectively, the initial diagnosed time were12and16.1months, follow-up time was22.47and28months, there were no statistical significant difference, P>0.05; perform relevance analysis to the location of the tumor,42patients were divided into three groups according to the location of the tumors in the eye:the posterior pole retina, peripheral retina(include the mid-peripheral retina) and the posterior pole+peripheral retina, the number of patients with successful treatment in the three groups were4cases,6cases,5cases; success rate of23.50%,85.70%,27.80%, using chi-square analysis on posterior pole and the peripheral portion, the peripheral group and posterior pole+peripheral group, there were significant differences, P<0.05;4of42patients have died, all with the location of the tumors in the posterior pole retina, groups of the other two locations, no deaths, there were statistical significant differences between posterior pole group and posterior pole+peripheral group, P<0.05; for other groups, there was no significant difference, P>0.05.
4.10cases of Rb children were treated with23G minimally invasive vitreous surgery combined with injection chemotherapy, an average postoperative follow-up time was6.4months,4cases performed surgery with silicone oil tamponade, the silicone oil removed from2cases, for visual acuity,7cases improved,1case the same as preoperative,2cases declined, eyeballs of all children were preserved, and the eyeball preservation rate was100%, no extraocular transfer was found, the tumor focus was almost calcification and atrophy,1case had a small amount of postoperative vitreous hemorrhage,2cases had mild choroidal detachment,1case had persistent chemosis, improved after treatment and found no other serious complications.
5. By pTNM staging and disease-specific survival (DSS) analysis methods, study on the Rb histopathology report of41cases (26males,15females,16bilateral cases,25unilateral cases,13group D eyes and28group E eyes performed enucleation). Only count the phase of one eye for each patient into statistical, for patients with bilateral Rb, count worse side in the study.16eyes used enucleation as the preferred treatment,25eyes was carried out chemotherapy treatment before enucleation. There were no significant statistical difference between two groups in the incidence of gender, unilateral and bilateral, international staging or diagnostic age, P>0.05.5deaths were all in the group E with chemotherapy before enucleation,2were bilateral and3were unilateral. Overall average diagnosis age of bilateral children was19.3months (range2-54months), overall average diagnosis age of unilateral children was25.5months (range4-72months); Followed up time of the survival children with enucleation as the first choice treatment (average time31.1months, range11-52months) was longer than the followed up time of the ones with chemotherapy before enucleation (average time26.4months, range10-50months). Average time from diagnosis to enucleation of the children with enucleation as the first choice treatment was0.9months, while median time from diagnosis to enucleation of the children with chemotherapy before enucleation was5.7months, There are significant differences in the statistical analysis, P≤0.05.
Disease-specific survival analysis of children:children's survival time was calculated from the time of diagnosis, up to the date of death of the death of children. Since the deaths are all the group E children, therefore, comparing the DSS between the enucleation children with no chemotherapy and the children with chemotherapy before enucleation, there are near significant differences by statistical analysis, P=0.05. There was significant difference between DDS of children whose enucleation delayed for more than3months and children whose enucleation performed within3months (P≤0.05).
Analyze the PTNM staging of the pathological tissue pieces from enucleation of41cases. Overall, in pTNM risk comparison of children with and without chemotherapy before enucleation, there was no significant difference (P>0.05); in the high risk comparison, the proportion of children with chemotherapy before enucleation was12%, while the proportion of children without chemotherapy before enucleation was37.5%, the high risk was significantly less in the former; in the staging comparison, the groups in group D had a significant difference, P≤0.05, i.e. the risk of chemotherapy group is lower than no chemotherapy group; but no significant difference in the risk of the children in the two groups in group E, P>0.05
6. Drawing peripheral blood of children who succeed or failed in eyeball preservation by comprehensive treatment, determine the43angiogenesis-related cytokines in serum by antibody chip, and found that the expression of11cytokines change, there was a significant difference. In which:2kinds were upregulated:IFN-y and Endostatin;9kinds were downregulated:ENA-78, Angiopoietin-2, VEGFR2, MCP-1, VEGF, MMP-9, IL-1β, PIGF and Tie-2.
Conclusion:
1. A graphic and text collection system for clinical information of Rb children were created using the Java language, it can collect and collate information of children completely, and output data in the form of EXECL table, at the same time, form a graphic and text report containing the disease and diagnosis and treat of children.
2. The leukocoria is the most common clinical manifestations of the symptoms of the Rb children with initial diagnosed, accounting for more than3/4of the total number of children; compared with foreign countries, the time and cause of Chinese children with initial diagnosed Rb when first visiting hospital were generally late, and single tumor accounted for main proportion, the ratio reached75%; For staging, mainly were the advanced patients in groups D and E, which was more than80%.For treatment, eyeball preservation rate was36.6%, and closely related to enucleation rate and staging. It suggested that Rb diagnosis and treatment is still relatively low in our country, patients are found with more serious illness, the prognosis is poor, and need to strengthen the public awareness on the Rb health education, to raise the level of Rb early diagnosis, improve the treatment of disease, and to explore a new effective and economic individualized treatment method.
3. Comprehensive treatment of chemotherapy combined with local treatment+enucleation improved the survival and eyeball preservation rate of children with Rb, but the survival rate of patients and eyeball preservation rate was closely related to its clinical stage, the treatment of advanced cases was ineffective with a higher rate of enucleation; at the same time, there is a high degree of correlation between treatment prognosis and tumor location. The location of the tumor has a guiding significance for the choice of clinical treatment; it is also an important factor affecting the clinical Rb comprehensive treatment efficacy.
4. To Rb advanced children with failed or ineffective chemotherapy, vitreous surgery combined with intraocular injection of drugs, as a new treatment method with observed effects in the short term, had an eyeball preservation rate was100%, and postoperative no serious complications. It is a safe and effective treatment, but also need long-term future observation and assessment on large sample.
5. If the time from diagnosis to enucleation is more than three months, chemotherapy treatment before enucleation in serious group E of those histopathological poor staging Rb children, and there is no close monitoring and appropriate high-risk factors processing, and which will likely lead to increased mortality caused by metastasis or recurrence. The findings suggest that, the advanced Rb treatment should be in appropriate measure, that is, how to avoid the problem of possible tumor metastasis brought by eyeball preservation treatment, and under what circumstances should we resolute to carry out enucleation or eyeball preservation treatment timely. How to grasp the balance between the two is the key issue requiring in-depth study and summarize.
6. Using cytokine antibody chip, we can determine the changes in expression of43angiogenesis-related cytokines in serum of children with different prognosis after the comprehensive treatment. The tumor vascularization with a core of VEGF/VEGFR2and Ang2/Tie2is important influencing factor to prognosis of Rb comprehensive treatment, the tumor prognosis outcome is a complex pathophysiological process, vascular generated at the core, involved in immunity, inflammation, metastasis invasion and apoptosis. VEGF/VEGFR2and the Ang2/Tie2, as Rb new treatment targets, have tremendous potential clinical value.
Innovations:
1. Established a set of effective retinoblastoma graphic and text information collection system.
2. Proposed a new idea of23G minimally invasive vitreous surgery combined with injection chemotherapy for advanced retinoblastoma, and made a preliminary summary, provided a new means for the comprehensive treatment of Rb.
3. Proposed that the excessive chemotherapy or eyeball preservation treatment may cause an increase in mortality or elevated risk in transference in children with advanced retinoblastoma.
4. Proposed that the core of prognosis on Rb is tumor angiogenesis, and the VEGF/VEGF R2and Ang2/Tie2can be considered as a new target for future Rb treatment.
视网膜母细胞瘤(retinoblastoma, Rb)是婴幼儿最常见的一种原发性眼内恶性肿瘤,严重危害患儿生命及视功能。随着临床治疗水平的不断提高,治疗的目标也从单纯局限于保护患儿生命转向立足于提高患儿的生存质量,即保留眼球和有用视功能的方向发展,而实现这一目标的最关键问题在于患儿的早期发现和诊断。由于Rb患儿的年龄一般都在3岁以下,肿瘤的早期发现相当困难,这不仅是由于儿童眼底检查配合不佳,更是缺乏清晰、直观的检查成像手段所造成的。广域数字化小儿视网膜图像系统(wide-field digital pediatric retinal imaging system, RetCam Ⅱ)作为目前最先进的儿童眼底成像技术为Rb的防治及持续随访提供了极大地便利。但长期以来仍缺乏对于Rb临床信息的完整收集及整理分析系统,只能采取简单的文字描述或绘图进行记录,不利于患儿临床长期随访观察和持续的综合治疗疗效评估。有鉴于此,我们希望利用计算机技术建立一套收集Rb图像和文字信息的图文一体化系统,为Rb的防治研究提供信息支持。通过吸入麻醉下的眼底成像技术与信息采集系统相结合的Rb图文系统,可以使眼科医师获得全面完整、准确直观的患儿信息资料,促进了Rb的临床诊断、治疗和随访等各个方面的发展,更好的把握Rb的眼底表现特征和病变进展情况,有效的帮助临床医师采取合理干预措施,对于提高患儿的生存质量也具有重要的价值。本研究创建的一整套的检查方式和图文输出,在临床教学和科研中也具有良好的应用前景。
目前,随着化疗为主的综合治疗的兴起,Rb患儿的诊疗模式也发生了巨大的变化,常规外放射治疗渐渐淡出主流治疗范围,原有的Reese-Ellsworth5级分期体系已不再能够适应新的要求,本世纪初产生了新的视网膜母细胞瘤国际分期(International Intraocular Retinoblastoma Classification, IIRC),2005年全球共有27个中心参与,包括我国的同仁眼科中心,证明了IIRC对于Rb化疗的指导作用。但我国Rb患儿就诊时多为晚期,保守治疗效果相对较差。因此,临床治疗仍是目前面临的最主要最现实的问题,我院自2008年开始组建了Rb专业治疗组,开展现代Rb的综合治疗,取得了不错的效果,同时收集了丰富的临床资料。本课题由此点出发,通过对近5年来的Rb综合治疗以及23G微创玻璃体手术联合球内注药化疗的新型治疗方式做一全面的评估与分析,以达到提高Rb治疗水平、探索符合我国国情的个体化治疗手段的目的。
长期以来,防止Rb的复发和转移是其治疗中最为棘手的难点。40年前,Folkman首次提出肿瘤的生长和转移是有血管依赖性的,因此阻断肿瘤的血管化可以作为肿瘤治疗的有效策略。VEGF及其相关的因子作为刺激血管生成的最强大的细胞因子群在肿瘤新生血管化的过程中占据中心地位,是介导新生血管生成的关键因素。有鉴于此,结合以上思路,我们选取综合治疗成功和失败的部分Rb患儿,抽取其血清,运用RayBiotech人血管生成相关细胞因子芯片检测治疗后Rb患儿血清中43种VEGF及其相关细胞因子的表达情况,整体的、动态的评估蛋白质分子水平的变化情况,从VEGF及其相关因子群为核心的肿瘤新生血管化的角度探讨Rb的临床治疗预后和侵袭转移机制。
机体生理机能的真正执行者是蛋白质,因此从蛋白质水平对疾病进行研究显得特别重要。抗体芯片技术作为一种高通量的分子蛋白检测技术,继承了免疫荧光、化学发光和微列阵技术的诸多特点,逐渐发展成为从整体角度定性和定量检测蛋白质分子的研究平台,在各种疾病的相关分子发现、分子诊断和疗效评价方面发挥重要作用,具有良好的应用前景。本研究希望利用这一技术从整体的宏观角度了解肿瘤血管新生化的微环境中复杂的细胞因子网络调控机制,从而探索未来潜在的Rb治疗新的介入手段和干预策略。
研究目的：
1.建立视网膜母细胞瘤临床图文信息采集系统,收集管理Rb患儿所有的临床信息及眼底图片资料,为掌握Rb发病的机制和规律,总结和探索Rb治疗特点奠定基础。
2.探讨视网膜母细胞瘤临床诊治与IIRC分期特点
3.研究视网膜母细胞瘤综合治疗的特点并探讨其相关临床因素,探索适合国情的个体化综合治疗方式；初步总结23G微创玻璃体手术联合球内注药化疗治疗Rb的临床经验和预后效果,摸索晚期Rb治疗的新方法；同时研究眼摘前的化疗与晚期Rb患儿肿瘤转移及死亡率的相关性。
4.利用细胞因子抗体芯片检测技术检测综合治疗保眼成功和失败的相同分期患儿治疗后血清中43种血管生成相关细胞因子的表达变化,从肿瘤新生血管化的角度探讨Rb的临床治疗和侵袭转移机制。
研究方法：
1.使用AVA软件语言,建立视网膜母细胞瘤临床信息采集系统,收集整理近十年的Rb临床资料,为分析Rb的诊疗特点及规律提供信息支持。
2.回顾性分析我院2003年至2012年十年间116例Rb患儿的临床资料。
3.分析我院从2009年至2012年间进行综合治疗的75例Rb患儿的临床资料,综合治疗的方法包括：化疗、局部治疗(激光或冷冻)、眼球摘除、玻璃体手术联合球内注药。其中采取以化疗为主联合局部治疗+眼摘治疗的患儿65例,其余10例为23G微创玻璃体手术联合球内注药化疗的手术干预治疗。
4.利用pTNM分级和特定疾病生存率(Disease-specific survival, DSS)的方法研究眼摘前的化疗与晚期Rb患儿肿瘤转移及死亡率之间的相关性。
5. RayBiotech人血管生成相关细胞因子芯片检测治疗后不同预后的Rb患儿血清中43种细胞因子的表达情况。
6.数据分析采用版本2.15.1的R统计软件中的卡方检验,T检验,Fisher's精确检验,Wilcoxon等级总和检验和生存率分析。P≤0.05认为有显著性差异。
结果：
1.建立了视网膜母细胞瘤临床信息的采集系统,主要有三个模块组成：文字报告模块、影像图片模块及病例查询模块,系统可以生成完整的Rb患儿图文报告资料。
2.分析116例Rb患儿临床资料的IIRC特点,其中单眼患儿为79例(68.1%),双眼患儿为37例(31.9%)；有遗传性家族史的单眼患儿为1例(1.27%),双眼患儿为5例(13.51%),两者经统计分析有显著性差异,P<0.05；在Rb患儿中最常见的初诊症状为白瞳症,比例为78.4%；性别方面男性74例(63.79%),女性42例(36.21%)；患儿总体的初诊时间、诊断滞后时间及随诊时间分别为21.49个月、5.14周及30.72个月,单眼患儿的初诊时间为24.27个月,双眼患儿为15.1个月,单双眼患儿的初诊时间经统计分析有显著性差异,P≤0.05；所有患儿中肿瘤为单个瘤体的占75%,多个瘤体的占25%；Rb患儿的IIRC国际分期方面,所有153只患眼中A期占5只(3.27%),B期占7只(4.58%),C期占11只(7.19%),D期占56只(36.60%),E期占69只(45.10%),自发退变的占4只(2.61%),另有1只眼由于在外院摘除而分期不明；所有患眼中眼球摘除的有97眼,占63.4%,总体保眼率为36.6%；从各分期的保眼率来看A期为5眼(100%),B期为7眼(100%),C期为9眼(81.82%),D期为24眼(42.86%),E期为10眼(14.49%),其余自发退变的4眼中有3眼由于萎缩而摘除。经统计学分析A、B、C期之间无显著性差异,P>0.05；A、B、C期与D、E期之间均有显著性差异,P≤0.05。
3.化疗为主联合局部治疗+眼摘治疗的所有65例92眼(双眼27例,单眼38例)Rb患儿四年的总生存率为92.3%,A期至E期的生存率分别为100%、100%、100%、95%和90.00%,经统计学分析显示各期存活率差异无统计学意义,P>0.05；所有患儿总的保眼率为51%,A期至E期的保眼率分别为100%、100%、85.70%、77.10%和10%,各组间采用两独立样本卡方检验,A、B、C、D各期之间保眼率无显著性差异,P>0.05；但E期和其他各期之间保眼率均有显著性差异,P<0.05；中低风险Rb患儿综合治疗结果：A,B,C期共15只眼,治疗成功14只眼,失败1只眼,治疗成功率为93.3%；高风险Rb患儿综合治疗结果：D、E期Rb患儿共60例,采用化疗联合局部治疗保眼的共42例,分别为D期18例和E期24例,D期的保眼治疗成功率为61.1%(11/18),E期的保眼治疗成功率为16.7%(4/24),其余18例均为单纯眼摘或眼摘后化疗的患儿。进一步分析42例D期和E期的保守治疗与临床因素相关性,治疗成功与失败患儿的初诊时间分别为12和16.1个月,随访时间分别为22.47和28个月,经统计学分析均无显著性差异,P>0.05；与肿瘤位置相关性分析,将42例患儿眼内肿瘤的位置分为3组即：后极部、周边部(包括中周部)和后极部+周边部,三组治疗成功的例数分别为4例,6例,5例；成功率分别为23.50%,85.70%,27.80%,经卡方分析后极部与周边部、周边部与后极部+周边部有显著性差异,P≤0.05；42例患儿中有4例死亡,全部为肿瘤位置处于后极部的患儿,其他两组位置的患儿均无死亡,经统计学分析,后极部与后极部+周边部两组之间有显著性差异,P≤0.05；另外各组间均无显著性差异,P>0.05。
4.23G微创玻璃体手术联合球内注药化疗治疗的Rb患儿10例,平均术后随访时间6.4个月,术中硅油填充的患儿有4例,其中2例已经取出硅油,有7例患儿术后视力有提高,1例同术前,2例下降,所有患儿眼球保存,保眼率为100%,未发现眼外转移,肿瘤灶基本钙化萎缩,1例患儿术后发生玻璃体少量出血,2例术后发生轻度脉络膜脱离,1例发生持续球结膜水肿,后经治疗好转,未发现其他严重并发症。
5.用pTNM分级和特定疾病生存率(DSS)的分析方法对41例的Rb组织病理学报告(26男,15女,双眼16例,单眼25例,摘除的眼球13眼为D期,28眼为E期)进行研究。每位患者仅以一只眼分期纳入统计,双侧Rb患者按病情较重侧患眼的分期参与研究。眼球摘除术作为首选治疗方式的有16只眼,另外25只眼为眼摘前做了化疗治疗的。这两组在性别,单侧与双侧发病,患眼的国际分级或者诊断年龄方面经统计学分析无显著性差异,P>0.05。五名死亡的患儿均为眼摘前化疗组E期患儿,双眼两名,单眼3名。双眼患儿整体的平均诊断年龄为19.3个月,范围2-54个月,单眼患儿整体的平均诊断年龄为25.5个月,范围4-72个月；首选治疗是眼摘的生存患儿的随诊时间(平均31.1个月,范围11-52个月)长于眼摘前化疗的生存患儿随诊时间(平均26.4个月,范围10-50个月)。首选眼球摘除的患儿从诊断到眼球摘除的平均时间是0.9个月,而眼摘前化疗的患儿从诊断到眼球摘除的时间中位数是5.7个月,经统计学分析有显著性差异,P≤0.05。
患儿特定疾病生存率分析：患儿的生存时间从诊断时开始计算,死亡患儿到死亡日期止。由于死亡患儿均为E期的患者,因此比较E期未行化疗直接眼摘患儿与眼摘前行化疗治疗患儿的DSS,经统计学分析两者接近有显著性差异,P=0.05。眼摘时间推迟3个月以上的患儿相对于3个月内眼摘的患儿DSS有显著性差异,P<0.05。
分析41例眼球摘除的病理组织片的pTNM分级,总体来看,眼摘前化疗组患儿与未化疗组患儿pTNM的风险性相比,两者无显著性差异,P>0.05；但在高风险方面,眼摘前化疗组患儿的比例为12%,而眼摘前未化疗组患儿的比例为37.5%,前者的高风险比例明显要少；分期比较,在D期中两组具有显著性差异,P<0.05,即化疗组比未化疗组的风险低；但在E期的患儿中两组的风险性没有显著性差异,P>0.05
6.抽取综合治疗后保眼成功和失败的Rb患儿外周血,进行血清中43种血管生成相关细胞因子的抗体芯片检测,结果发现11种细胞因子的表达发生改变,有显著性差异。其中：发生表达上调的2种,为IFN-γ和Endostatin;发生表达下调的9种,为ENA-78、Angiopoietin-2、VEGF-R2、MCP-1、VEGF、 MMP-9、IL-1β、PIGF和Tie-2。
结论：
1.使用Java语言程序创建了Rb患儿临床信息的图文采集系统,可以完整的搜集、整理各种患儿信息资料,并可将资料数据以EXECL表的形式输出,同时可以形成包含患儿病情和诊疗情况的图文报告。
2.白瞳症是Rb患儿中的初诊症状中比例最高的临床表现,超过患儿总数的3/4；与国外相比,我国患儿的初诊时间和病程普遍偏晚,且肿瘤以单发为主所占比重最大,比率达到75%；分期上也以D期和E期为主的晚期病患为主,超过了80%。治疗上,保眼率为36.6%,且眼摘率和分期密切相关。说明我国的Rb诊断和治疗水平还比较低,患者发现时多病情严重,预后不佳,需要加强对民众的Rb健康教育,提高Rb早期诊断的水平,探索出新的有效且经济的个体化治疗新方法。
3.化疗为主联合局部治疗+眼摘治疗的综合治疗手段提高了Rb患儿的生存率,但患者的生存率和保眼率与其临床分期密切相关,晚期病例的治疗效果不佳,眼摘率较高；同时治疗预后与肿瘤所处的位置有高度相关性。肿瘤的位置对于临床治疗的选择性具有指导意义,也是影响临床Rb综合治疗疗效的重要因素。
4.对于晚期的化疗失败或效果不佳的Rb患儿,玻璃体手术联合球内注药的治疗新方法短期内的观察效果良好,保眼率为100%,且术后未见严重的手术并发症,是一种安全有效的治疗手段,但还需要今后大样本长期的观察评估。
5.如果从诊断到眼球摘除的时间超过3个月,对那些组织病理学上分期较差的严重E期Rb患儿进行眼摘前的化疗治疗,同时没有密切监控和适当的对高风险因素的处理,可能将导致因为转移或复发造成的死亡率升高。研究结果提示在晚期的Rb治疗中要适度掌握治疗的分寸,即如何避免保眼治疗后带来的可能肿瘤转移问题,在何种情况下要及时坚决的摘除眼球或采取保眼治疗,怎样把握两者之间的平衡关系是今后需要深入研究和总结的关键问题。
6.使用细胞因子抗体芯片技术可以检测不同预后的Rb患儿血清中血管生成类细胞因子的表达变化情况,以VEGF/VEGF R2和Ang2/Tie2为核心的肿瘤血管化是Rb综合治疗预后效果的重要影响因素,肿瘤的预后转归是一个以血管生成化为核心的、复杂的涉及免疫、炎症、转移侵袭、凋亡的病理生理过程,VEGF/VEGF R2和Ang2/Tie2可以作为今后Rb治疗的新的靶点,具有巨大的潜在临床应用价值。
创新点：
1.构建了一套行之有效的视网膜母细胞瘤图文信息采集系统。
2.提出了23G微创玻璃体手术联合球内注药化疗治疗晚期视网膜母细胞瘤的新思路,并进行了初步总结,为Rb综合治疗提供了新的手段。
3.提出了过度化疗或保眼治疗可能引起晚期视网膜母细胞瘤患儿死亡率增加或转移风险升高。
4.提出Rb治疗转归的核心为肿瘤血管化,VEGF/VEGF R2和Ang2/Tie2可以作为今后Rb治疗的新的靶点。
Background:
Retinoblastoma (retinoblastoma, Rb) is the most common primary infancy intraocular malignant tumors, with a serious risk of life and visual function of children. With the improving of clinical treatment, the goal of treatment is also shifted from simply limited to the protection of children's life to a new development direction for improving the quality of life of children, that is, the eyeball preservation and keeping useful visual function. Early detection and diagnosis of children is the most critical issue to achieve this goal, because ages of Rb children generally are less than3years old, the early detection of tumors is quite difficult, not only due to the child's poor cooperate with the fundus examination, more is the lack of clear and intuitive means of examination and imaging. Wide-field digital pediatric retinal imaging system (RetCam Ⅱ) as the most advanced children fundus imaging technology, greatly facilitates the prevention and continuous follow-up of Rb. But, for a long time, there is still a lack of collecting and collating analysis system for the complete clinical information of Rb. The recordings can only be taken by simple text description or drawing, which is not conducive to the clinical long-term follow-up observation and ongoing treatment efficacy assessment for children. In view of this, we hope to establish a integration of graphic and text information system for collecting Rb image and text using computer technology, to lay the information support for the prevention and treatment research of Rb. Through Rb graphic and text system, which combining the fundus imaging technology under inhalation anesthesia and information collection system, an ophthalmologist can obtain a full, intuitive and accurate information of children. It can promote the improvement of clinical diagnosis, treatment and follow-up in all aspects of Rb, lead a better grasp the Rb fundus manifestations and lesions progress, help clinicians to take reasonable interventions effectively, and also provide an important value for improving the survival treatment of children.In this study, a set of inspection methods and graphic and text output also has a good prospect in clinical teaching and research.
With the rise of the comprehensive treatment of chemotherapy, the model of diagnosis and treatment for Rb has also undergone a huge change that conventional external radiation therapy has gradually fade out of the range of mainstream treatment, the original of Reese-Ellsworth5level staging system is no longer able to adapt to the new requirements, therefore, a new International Intraocular Retinoblastoma Classification (IIRC) has been established at the beginning of this century. In2005, a total of27centers worldwide, including Tongren Eye Center, proved the directing role of IIRC on Rb chemotherapy. However, most Rb children in China are in advanced stage when visiting hospital, lead to a relatively poor outcome of conservative treatment. Therefore, the clinical treatment is still currently the most important reality. Since January2008, an Rb professional treatment group has been found in our hospital, to carry out a comprehensive modern treatment of Rb, and has achieved good results and collected a wealth of clinical data. Based on this point of view, the topic performed a comprehensive assessment and analysis on the Rb comprehensive treatment in the past five years as well as the new treatment of23G minimally invasive vitreous surgery combined with intraocular injection chemotherapy,and by histopathological pTNM staging of children with Rb in advanced stage, we have studied potential adverse effects for patients with chemotherapy, in order to improve the level of Rb treatment, exploring individualized treatment in line with our national conditions.
For a long time, prevention of relapse and metastasis of Rb is the trickiest difficulty of the treatment.40years ago, Folkman first proposed vascular dependent tumor growth and metastasis, therefore, blocking tumor blood vessels can be considered as effective cancer treatment strategy. VEGF and its related factors, as the most powerful group of cytokines that stimulate angiogenesis, occupy a central position in the process of tumor angiogenesis, and it is a key factor to mediate angiogenesis. In view of this, combining above ideas, we selected some children with successful and unsuccessful chemotherapy, extract the serum, and test the expression of43VEGF and its related cytokines in serum of children after Rb treatment using RayBiotech human angiogenic-related cytokines chip, carry out an overall, dynamic assessment of the changes of level of protein molecules, considering VEGF and its factor group as the core of tumor angiogenesis, we explore the Rb clinical prognosis, invasion and metastasis mechanism.
The body's physiological function is actually executed by protein, thus, the protein levels are particularly important for disease research. Antibody chip technology, as a high-throughput protein molecule detection technology, inherits many characteristics of the Immunofluorescence, chemiluminescence and microarray technology, and gradually develops into a research platform for the overall qualitative and quantitative detection of protein molecules, plays an important role in a variety of disease-related molecular discovery, molecular diagnostics and efficacy evaluation. and has good application prospects. In this study, we hope to take advantage of this technology, from the macro point of view, make an overall understanding of the complex cytokine network regulation mechanisms in new biochemical micro-environment of the tumor vasculature, in order to explore the potential new interventional means and intervention strategies of the Rb treatment in the future.
Objective:
1. Establish the clinical graphic and text information collection system for Rb, collecte and manage all clinical information and fundus images of Rb children, to lay foundations for mastering the pathogenesis and laws of Rb, and summarizing and exploring Rb treatment characteristics.
2. Explore the clinical diagnosis and treatment of retinoblastoma and IIRC staging characteristics
3. Study the characteristics of comprehensive treatment on Rb and to investigate the related clinical factors, to explore the patterns for the individualized treatment; preliminarily summarize the clinical experience and prognostic effect of23G minimally invasive vitreous surgery combined with intraocular injection chemotherapy for Rb treatment, fumble new treatment methods for advanced Rb; and study the correlation between chemotherapy and cancer metastasis and mortality of advanced Rb children.
4. Using cytokine antibody chip technology, for children in the same stage who succeed or failed in eyeball preservation by comprehensive treatment, determine the changes in expression of43angiogenesis-related cytokines in serum after the treatment. Considering tumor angiogenesis, we explore the Rb clinical treatment, and invasion and metastasis mechanism.
Methods:
1. Use the JAVA software or language, establish the Rb clinical information acquisition system, and collect Rb clinical data over the past ten years, to provide information support for the analysis of clinic characteristics and laws of Rb.
2. Perform a retrospective analysis on the clinical data of116cases of Rb children in our hospital over the past ten years from2003to2012.
3. Analyze75cases of Rb children's clinical data with comprehensive treatment in our hospital from2009to2012, the comprehensive treatment methods include: chemotherapy, local treatment (laser or cryotherapy), enucleation, vitreous surgery combined with injection. In which,65cases have taken chemotherapy-based joint topical treatment+enucleation, while the remaining10cases have taken surgical intervention by23G minimally invasive vitreous surgery combined with intraocular infusion chemotherapy.
4. By pTNM staging and disease-specific survival (DSS) methods, study on the correlation between chemotherapy before enucleation and cancer metastasis and mortality of the children with advanced Rb.
5. Using RayBiotech angiogenesis-related cytokine chip, determine the changes in expression of43angiogenesis-related cytokines in serum of children with different prognosis after the treatment.
6. Data analyses were performed with R2.15.1version software. Chi-square test, T test, Fisher's exact test, Wilcoxon rank sum test and survival analysis were performed. A P-value≤0.05was chosen to define statistical significance.
Results:
1. The Rb clinical information collection system has been established, mainly consisting of three modules:the text report module, graphic image module and case query module, and the system can generate a complete graphic and text report information for Rb children.
2. Analyze on clinical information of116cases of Rb children, in which,79 cases with unilateral Rb (68.1%),37cases with bilateral Rb (31.9%); in children with a genetic family history,1case was unilateral (1.27%),5cases were bilateral (13.51%), there were significant differences in statistical analysis (P<0.05); most common symptoms in initial diagnosed Rb children were leukocoria (78.4%); On gender aspect,74males (63.79%) and42females (36.21%); for those children, overall initial diagnosed time, diagnostic delay time and follow-up time were21.49months,5.14weeks and30.72months, respectively. The initial diagnosed time of unilateral Rbs in children were24.27months, bilateral Rbs were15.1months, there were significant differences in statistical analysis between the two groups, P≤0.05; In all children,75%had single tumor,25%had more than one tumor; For IIRC of Rb children, of all153affected eyes,5in group A (3.27%),7in group B (4.58%),11in group C (7.19%),56in group D (36.6%),69in group E (45.10%), and4with spontaneous degeneration (2.61%), another1eye removed from the other hospital and the stage was unknown; in all eyes with Rb,97performed enucleation (63.4%), the overall eyeball preservation rate was36.6%; considering the eyeball preservation rate of different staging,5in group A (100%),7in group B (100%),9in group C (81.82%),24in group D (42.86%),10in group E (14.49%),3of4spontaneous degenerative eyeballs were removed due to atrophy. The statistical analysis of the groups A, B, C showed no statistically significant difference (P>0.05); Group A, B, C and Group D, E showed a significant difference (P<0.05).
3. With chemotherapy combined topical treatment+enucleation, in all65cases (92eyes,27bilateral cases,38unilateral cases) of Rb children,4-year total survival rate was92.3%, in groups A-E, the survival rates were100%,100%,100%,95%and90.00%, the statistical analysis showed that survival rate in different groups was not statistically significant (P>0.05); total eyeball preservation rate of all children was51%, in group A to E, eyeball preservation rates were100%,100%,85.70%,77.10% and10%, respectively, using the chi-square test of two independent samples among the groups, the eyeball preservation rates among A, B, C, D groups showed no significant difference, P>0.05; but eyeball preservation rates between group E and other groups showed significant difference, P<0.05; the comprehensive treatment outcomes in low-risk Rb children:with15eyes in groups A, B, C,14eyes successful and1eye failed, success rate was93.3%; the comprehensive treatment outcomes in high-risk Rb children:with a total of60cases of Rb children in groups D, E, a total of42cases were treated with chemotherapy combined local therapy,18in group D and24in group E, the eyeball preservation treatment success rate of group D was61.1%(11/18), in group E was16.7%(4/24), the remaining18cases were simple eyeball enucleation or eyeball enucleation after chemotherapy. Perform further analysis on the correlation of conservative treatment and clinical factors of42cases in groups D and E, in children with successful and failed treatment, respectively, the initial diagnosed time were12and16.1months, follow-up time was22.47and28months, there were no statistical significant difference, P>0.05; perform relevance analysis to the location of the tumor,42patients were divided into three groups according to the location of the tumors in the eye:the posterior pole retina, peripheral retina(include the mid-peripheral retina) and the posterior pole+peripheral retina, the number of patients with successful treatment in the three groups were4cases,6cases,5cases; success rate of23.50%,85.70%,27.80%, using chi-square analysis on posterior pole and the peripheral portion, the peripheral group and posterior pole+peripheral group, there were significant differences, P<0.05;4of42patients have died, all with the location of the tumors in the posterior pole retina, groups of the other two locations, no deaths, there were statistical significant differences between posterior pole group and posterior pole+peripheral group, P<0.05; for other groups, there was no significant difference, P>0.05.
4.10cases of Rb children were treated with23G minimally invasive vitreous surgery combined with injection chemotherapy, an average postoperative follow-up time was6.4months,4cases performed surgery with silicone oil tamponade, the silicone oil removed from2cases, for visual acuity,7cases improved,1case the same as preoperative,2cases declined, eyeballs of all children were preserved, and the eyeball preservation rate was100%, no extraocular transfer was found, the tumor focus was almost calcification and atrophy,1case had a small amount of postoperative vitreous hemorrhage,2cases had mild choroidal detachment,1case had persistent chemosis, improved after treatment and found no other serious complications.
5. By pTNM staging and disease-specific survival (DSS) analysis methods, study on the Rb histopathology report of41cases (26males,15females,16bilateral cases,25unilateral cases,13group D eyes and28group E eyes performed enucleation). Only count the phase of one eye for each patient into statistical, for patients with bilateral Rb, count worse side in the study.16eyes used enucleation as the preferred treatment,25eyes was carried out chemotherapy treatment before enucleation. There were no significant statistical difference between two groups in the incidence of gender, unilateral and bilateral, international staging or diagnostic age, P>0.05.5deaths were all in the group E with chemotherapy before enucleation,2were bilateral and3were unilateral. Overall average diagnosis age of bilateral children was19.3months (range2-54months), overall average diagnosis age of unilateral children was25.5months (range4-72months); Followed up time of the survival children with enucleation as the first choice treatment (average time31.1months, range11-52months) was longer than the followed up time of the ones with chemotherapy before enucleation (average time26.4months, range10-50months). Average time from diagnosis to enucleation of the children with enucleation as the first choice treatment was0.9months, while median time from diagnosis to enucleation of the children with chemotherapy before enucleation was5.7months, There are significant differences in the statistical analysis, P≤0.05.
Disease-specific survival analysis of children:children's survival time was calculated from the time of diagnosis, up to the date of death of the death of children. Since the deaths are all the group E children, therefore, comparing the DSS between the enucleation children with no chemotherapy and the children with chemotherapy before enucleation, there are near significant differences by statistical analysis, P=0.05. There was significant difference between DDS of children whose enucleation delayed for more than3months and children whose enucleation performed within3months (P≤0.05).
Analyze the PTNM staging of the pathological tissue pieces from enucleation of41cases. Overall, in pTNM risk comparison of children with and without chemotherapy before enucleation, there was no significant difference (P>0.05); in the high risk comparison, the proportion of children with chemotherapy before enucleation was12%, while the proportion of children without chemotherapy before enucleation was37.5%, the high risk was significantly less in the former; in the staging comparison, the groups in group D had a significant difference, P≤0.05, i.e. the risk of chemotherapy group is lower than no chemotherapy group; but no significant difference in the risk of the children in the two groups in group E, P>0.05
6. Drawing peripheral blood of children who succeed or failed in eyeball preservation by comprehensive treatment, determine the43angiogenesis-related cytokines in serum by antibody chip, and found that the expression of11cytokines change, there was a significant difference. In which:2kinds were upregulated:IFN-y and Endostatin;9kinds were downregulated:ENA-78, Angiopoietin-2, VEGFR2, MCP-1, VEGF, MMP-9, IL-1β, PIGF and Tie-2.
Conclusion:
1. A graphic and text collection system for clinical information of Rb children were created using the Java language, it can collect and collate information of children completely, and output data in the form of EXECL table, at the same time, form a graphic and text report containing the disease and diagnosis and treat of children.
2. The leukocoria is the most common clinical manifestations of the symptoms of the Rb children with initial diagnosed, accounting for more than3/4of the total number of children; compared with foreign countries, the time and cause of Chinese children with initial diagnosed Rb when first visiting hospital were generally late, and single tumor accounted for main proportion, the ratio reached75%; For staging, mainly were the advanced patients in groups D and E, which was more than80%.For treatment, eyeball preservation rate was36.6%, and closely related to enucleation rate and staging. It suggested that Rb diagnosis and treatment is still relatively low in our country, patients are found with more serious illness, the prognosis is poor, and need to strengthen the public awareness on the Rb health education, to raise the level of Rb early diagnosis, improve the treatment of disease, and to explore a new effective and economic individualized treatment method.
3. Comprehensive treatment of chemotherapy combined with local treatment+enucleation improved the survival and eyeball preservation rate of children with Rb, but the survival rate of patients and eyeball preservation rate was closely related to its clinical stage, the treatment of advanced cases was ineffective with a higher rate of enucleation; at the same time, there is a high degree of correlation between treatment prognosis and tumor location. The location of the tumor has a guiding significance for the choice of clinical treatment; it is also an important factor affecting the clinical Rb comprehensive treatment efficacy.
4. To Rb advanced children with failed or ineffective chemotherapy, vitreous surgery combined with intraocular injection of drugs, as a new treatment method with observed effects in the short term, had an eyeball preservation rate was100%, and postoperative no serious complications. It is a safe and effective treatment, but also need long-term future observation and assessment on large sample.
5. If the time from diagnosis to enucleation is more than three months, chemotherapy treatment before enucleation in serious group E of those histopathological poor staging Rb children, and there is no close monitoring and appropriate high-risk factors processing, and which will likely lead to increased mortality caused by metastasis or recurrence. The findings suggest that, the advanced Rb treatment should be in appropriate measure, that is, how to avoid the problem of possible tumor metastasis brought by eyeball preservation treatment, and under what circumstances should we resolute to carry out enucleation or eyeball preservation treatment timely. How to grasp the balance between the two is the key issue requiring in-depth study and summarize.
6. Using cytokine antibody chip, we can determine the changes in expression of43angiogenesis-related cytokines in serum of children with different prognosis after the comprehensive treatment. The tumor vascularization with a core of VEGF/VEGFR2and Ang2/Tie2is important influencing factor to prognosis of Rb comprehensive treatment, the tumor prognosis outcome is a complex pathophysiological process, vascular generated at the core, involved in immunity, inflammation, metastasis invasion and apoptosis. VEGF/VEGFR2and the Ang2/Tie2, as Rb new treatment targets, have tremendous potential clinical value.
Innovations:
1. Established a set of effective retinoblastoma graphic and text information collection system.
2. Proposed a new idea of23G minimally invasive vitreous surgery combined with injection chemotherapy for advanced retinoblastoma, and made a preliminary summary, provided a new means for the comprehensive treatment of Rb.
3. Proposed that the excessive chemotherapy or eyeball preservation treatment may cause an increase in mortality or elevated risk in transference in children with advanced retinoblastoma.
4. Proposed that the core of prognosis on Rb is tumor angiogenesis, and the VEGF/VEGF R2and Ang2/Tie2can be considered as a new target for future Rb treatment.
引文
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