共聚焦显微内镜在体诊断胃黏膜上皮内瘤变和胃癌分子成像的研究
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摘要
背景和目的
目前胃癌仍是世界上病死率排名第二位的恶性肿瘤。1988年,Correa提出的慢性萎缩性胃炎-胃黏膜肠上皮化生-胃黏膜不典型增生-肠型胃癌的多阶段胃癌发展模型已被较多临床随访研究、胃癌手术切除标本及基础实验等所验证。故萎缩性胃炎、胃黏膜肠上皮化生及不典型增生(Dysplasia)均为公认的胃黏膜癌前病变。尽管如此,胃黏膜不典型增生作为肠型胃癌形成的倒数第二步演变步骤,是胃腺癌最为直接的癌前病变,其早期诊断对防治胃癌具有十分重要的临床意义。2010年,世界卫生组织正式提出用“上皮内瘤变(Intraepithelial Neoplasia)"取代“不典型增生”。
内镜检查是早期发现胃黏膜瘤变的最有效手段。尤其是近年来应用于临床的先进的内镜技术如高清晰内镜、放大内镜、色素内镜及窄带成像(Narrow-band Imaging, NBI)等,均可提供胃黏膜表面颜色及细微结构变化的高清晰图像。然而,早期胃黏膜瘤变的内镜下宏观改变缺乏特异性,故虽然上述内镜技术可以-定程度提高早期胃癌的检出率,但仍无法观察到胃黏膜上皮细胞、腺体和微血管等显微结构的改变。因此,为确诊病变,内镜医师仍需留取多块活检标本,由此造成的活检瘢痕对胃黏膜上皮内瘤变(Gastric Intraepithelial Neoplasia, GIN)患者短期内实行内镜治疗亦有诸多不良影响。此外,活检标本的组织病理学检查需耗费2-3天时间,对于确诊为高级别上皮内瘤变(High-grade Intraepithelial Neoplasia, HGIN)和早期胃癌的患者来说,不但有可能延误治疗时机,而且增加了患者的精神与经济负担。
共聚焦激光显微内镜(Confocal Laser Endomicroscopy, CLE)是一种新型的内镜技术,可以在白光内镜观察的同时,通过静脉注射或黏膜表面喷洒荧光造影剂,实时观察消化道黏膜上皮细胞、腺体及血管等显微结构,获取类似病理组织学的显微内镜图像。CLE的问世为胃黏膜癌前病变及早期胃癌的诊断提供了新的有力工具。然而,目前尚无研究将其应用于胃黏膜上皮内瘤变的诊断。
早期诊断是改善胃癌患者预后的关键。内镜活检虽为目前公认的诊断胃癌的金标准,但仍存在致使病变形态改变、引发出血、穿孔等并发症的潜在缺陷。此外,常规内镜活检的病理组织学确诊病变后再行内镜下治疗还会延后治疗时间,增添患者的心理和医疗负担。因此,目前亟需一种能够实现实时显微组织观察的内镜技术,使医师在普通内镜检查的同时观察到病变的显微结构改变并立即做出诊断,采取相应的治疗措施,从而提高胃癌的检出率和诊断、治疗水平。近年来,已有多项研究致力于将CLE应用于在体诊断胃癌；研究结果显示,根据已制定的CLE形态学诊断标准,共聚焦激光显微内镜能够较为准确的鉴别胃黏膜癌变病变与非癌变病变。但上述研究均采用对患者静脉注射荧光素钠作为对比剂,获取胃黏膜显微结构的非特异性荧光成像,无法达到对胃肠道黏膜疾病功能性和分子水平改变进行观察的目的。近期几项研究表明,利用荧光物质标记的特异性抗体,CLE能够实现活体内分子水平的观察。
因此,本课题拟围绕胃黏膜上皮内瘤变和胃癌的显微内镜诊断方面展开研究,主要研究目的为：(1)探讨利用CLE对胃黏膜非瘤变和瘤变病变进行鉴别诊断的可行性,前瞻性评估CLE对胃黏膜上皮内瘤变进行实时诊断和分级诊断的效能；(2)系统研究CLE对胃癌活体分子成像的可行性。
方法
第一部分：共聚焦激光显微内镜在体诊断胃黏膜上皮内瘤变的可行性研究
研究过程分为两个阶段,第一阶段采用共聚焦激光显微内镜对已知正常胃黏膜、非瘤变和胃黏膜上皮内瘤变的病变进行检查,将所扫描部位的共聚焦激光显微内镜图像与相应部位横切面的组织病理学图像进行对比,制定共聚焦激光显微内镜对胃黏膜病变的鉴别诊断标准和GIN分级诊断的标准体系。第二阶段,前瞻性的纳入2009年7月至2010年1月间于齐鲁医院胃镜室预约行CLE检查且符合相应纳入标准的患者为研究对象。由CLE操作者对胃黏膜病变进行实时CLE诊断；由另外一名未参与实时诊断的有经验的内镜医师在检查结束后对所留取的图片进行盲法诊断。以病理组织学诊断结果为金标准,验证上述诊断标准。为了评估观察者间和观察者内的一致性,我们还邀请了3名内镜医师和1名病理医师独立地对前瞻性研究中的50处病变的共聚焦图像按本研究制定的诊断标准进行分类诊断。其中一名内镜医师间隔一周后再次对这50处病变的CLE图像进行诊断。
第二部分：共聚焦激光显微内镜对胃癌活体分子成像的研究
研究分为3个步骤,分别为细胞学研究、胃癌荷瘤鼠动物模型体内分子成像和人体新鲜组织体外研究。
(1)将BGC823和SGC7901这2种人源性胃癌细胞系分别与AF488-MG7抗体,同型对照抗体,或PBS体外共孵育,首先进行流式细胞仪分析,然后利用CLE (FIVEl, Optiscan)对未固定的活细胞进行显微荧光成像,荧光显微镜观察；最后进行MG7抗原细胞学免疫组织化学染色。
(2)建立上述两种胃癌细胞系的荷瘤鼠动物模型,心内注射AF680-MG7抗体或AF680-同型对照抗体,利用小动物活体成像系统(Kodak2000MM, Kodak,USA)对其进行整体动态观察,总结肿瘤特异性荧光成像的最佳抗体剂量及时间。然后根据上述结果进行CLE荷瘤鼠体内显微水平的分子成像。对CLE观察部位的组织进行MG7抗原免疫组织化学染色,以相同荷瘤鼠的肝脏组织为阴性对照。
(3)选取新鲜胃癌组织及相同患者的非癌组织的手术标本(5-10mm)或内镜活检标本为研究对象,与AF488-MG7抗体溶液共孵育,然后立即用CLE (FIVEl, Optiscan)进行观察。全部CLE操作均由同一研究者完成,该研究者在进行CLE观察时不知晓标本的病理组织学诊断和位置来源。将CLE图像利用半定量方法分为阴性(0),弱阳性(+),阳性(++)和强阳性(+++)4个等级,由另外3位有经验的CLE研究者分别盲法独立对CLE图片进行半定量分析,从而评估该CLE分子成像半定量评估体系的可靠性。CLE扫描结束后将标本行HE染色和MG7抗原免疫组化实验。以病理组织学诊断作为本实验的金标准。对所得实验结果利用Spearman相关系数评估CLE半定量结果与相应的免疫组化半定量结果的相关性。
结果
第一部分：
前瞻性研究共纳入75例患者,包括91处可见病变,留取CLE图像15093张。利用本研究中制定的CLE胃黏膜病变鉴别诊断标准,CLE最终诊断GIN的灵敏度、特异度、阳性预测值、阴性预测值、阳性似然比和阴性似然比分别为77.8%(95%CI,63.7%-87.5%),81.8%(95%CI,68.0%-90.5%),81.4%(95%CI,67.4%-90.3%),78.3%(95%CI,64.4%-87.7%),4.28(95%CI,2.24-8.16)口0.27(95%CI,0.16-0.48)。 CLE实时诊断与最终诊断的准确度比较无统计学差异(P=0.549)。
观察者间一致性的平均Kappa值为0.70(内镜医师之间)和0.71(内镜医师与病理医师之间)。观察者内一致性亦较高(kappa=0.78)。上皮内瘤变评分≥5用于区分高级别和低级别上皮内瘤变的灵敏度和特异度分别为66.7%和88.0%。
第二部分：
(1)流式细胞术检测结果显示本研究中所使用的BGC823和SGC7901胃癌细胞系均存在MG7-Ag的表达；CLE (FIVE1, Optiscan)图像显示细胞膜和胞浆的特异性荧光信号；即刻台式荧光显微镜观察结果与CLE相似。细胞免疫组化结果也验证了上述两种细胞系的细胞膜和胞浆中存在MG7-Ag的表达。
(2)荷瘤鼠整体动态荧光成像观察结果显示,心内注射AF680-MG7抗体(1μg/g体重)避光饲养24小时后,小动物活体成像仪摄取的图像中即可见肿瘤部位出现特异性荧光信号。该肿瘤部位的特异性荧光信号在48小时达最强。CLE观察肿瘤表面均可见特异性荧光信号；体外免疫组化结果和台式荧光显微镜观察冰冻切片的图像与体内CLE图像相似。
(3)共前瞻性纳入了46例新鲜的人组织标本,包括18例手术标本和28例内镜活检标本(来自23例胃癌患者)。23例胃癌组织标本中有22例在CLE实验中观察到了特异性荧光信号(+～+++),而非癌组织标本则无或仅有弱荧光信号(0或+,n=18, n=5)。CLE半定量分析结果与不同等级的免疫组化结果的相关性较好(Spearman r=0.87, P<0.001)。CLE图像半定量分析方法的观察者间一致性较高(ICC=0.883,95%可信区间：0.819～0.929)。
结论
1.通过识别共聚焦显微图像中腺体结构、细胞形态和血管结构的特征性改变,可以区分正常胃黏膜、非瘤变病变及胃黏膜上皮内瘤变病变。
2.通过制定共聚焦显微图像瘤变改变的评分系统,可初步区分低级别和高级别上皮内瘤变。
3.CLE诊断胃黏膜上皮内瘤变的观察者间和观察者内一致性较高,具有较好的可靠性。
4.利用荧光标记的MG7抗体,CLE可以对胃癌荷瘤鼠进行活体内显微水平的靶向分子成像；CLE对胃癌患者的新鲜黏膜组织体外荧光分子成像的结果与传统的体外免疫组织化学染色结果相关性好。
研究意义
本研究证实CLE可清晰识别GIN特征性的细胞异常、结构紊乱和微血管变化,建立的CLE下胃黏膜病变的诊断标准可实时区分GIN病变与非瘤变病变。CLE对GIN的分类诊断标准的观察者间及观察者内一致性均较高,说明该诊断标准的可靠性较好。利用CLE图像中GIN的改变的严重程度的差异可以对GIN进行分级诊断,高特异度的实时鉴别诊断HGIN病变,从而使内镜医师对病变部位即刻进行内镜下治疗,避免治疗时机的延误、活检疤痕对内镜治疗的不良影响以及医疗资源的重复消耗。本研究首次利用荧光标记的MG7抗体,以CLE为观察手段,实现了对胃癌荷瘤鼠的活体内靶向分子成像和人体活组织的体外分子成像。CLE作为一种新的在体显微分子成像技术,有助于提高胃癌的诊断效能,准确筛选MG7-Ag阳性的患者。尽管该项技术的临床价值尚待进一步人体内研究的验证,我们目前的研究结果已初步确定了CLE辅助下的对胃癌患者进行活体免疫学诊断的可行性。
Backgrounds and aims
Gastric cancer remains the world's second leading cause of cancer-related death. In1988, the multistep carcinogenesis process of gastric carcinoma was proposed by Correa, and it was described as atrophic gastritis-intestinal metaplasia-dysplasia-intestinal type gastric cancer. This theory has been validated by many follow-up clinical trials todate. Therefore, atrophic gastritis, intestinal metaplasia, and dysplasia are all widely considered as premalinant lesions of the gastric mucosa. Nevertheless, as the penultimate step for the development of intestinal type gastric cancer, gastric dysplasia is the most threatening precancerous changes. Thus the early diagnosis of gastric dysplasia is significant for the prevention and treatment of gastric cancer. In2010, the World Health Organization suggested to use the nomenclature of "intraepithelial neoplasia" to replace "dysplasia".
Endoscopy is the most efficient tool for early detection of gastric neoplasia. Modern endoscopic devices such as high-definition endoscopy, magnifying endoscopy, chromoendoscopy and narrow-band imaging (NBI), can provide high resolution endoscopic pictures of color changes and minute structure alterations of gastric mucosa. However, although these endoscopic modalities can improve the detection rate of early gastric carcinoma on a certain extent, they can't enable microscopic observation of epithelial cells, gland architecture and vasculature. Therefore, multiple biopsies are still needed to diagnose endoscopic suspicious lesions, and the biopsy scars may interefere with the following short-term endoscopic therapy of these lesions if diagnosed as gastric intraepithelial neoplasia (GIN) by histopathology. Moreover, the histological processing of biopsy specimens usually takes of2-3days, which not only postpones therapeutic opportunity for patients with high-grade intraepithelial neoplasia (HGIN) and early gastric cancer, but also severes the patients'psychological and financial burdens.
Confocal laser endomicroscopy (CLE) is a novel endoscopic technology which can provide real-time cellular and subcellular visualization of gastric mucosa with the aid of intravenous surface application of fluorescent dyes, while at the same time displaying white-light endoscopic images. Previous studies found that CLE is a powerful new tool for early endoscopic diagnosis of intestinal metaplasia and gastric cancer. However, no investigation has yet applied CLE in the diagnosis of GIN.
Early diagnosis is essential for improving the prognosis of gastric cancer. Currently, although endoscopic biopsy is the gold standard for diagnosing gastric cancer, it still suffers from several drawbacks such as lesions'morphological alteration, bleeding and perforation. What's more, the delayed histopathological diagnosis will lead to later therapy and increased anxiety of patients. Thus a new endoscopic device is needed which can enable immediate histopathological diagnosis of suspicious lesions during white-light endoscopic examination. Several studies have been investigating the role of CLE in diagnosing gastric cancer in vivo, which showed that according to predefined endomicroscopic diagnostic criteria, gastric cancer can be accurately differentiated from non-cancerous lesions using CLE. However, all those studies applied intravenous fluorescein sodium as the contrast agent to obtain nonspecific endomicroscopic images, which cannot provide functional or molecular imaging of the gastric mucosa. Most recently, several researches revealed that using fluorescently labeled specific antibodies, CLE can achieve intravital molecular imaging of GI cancer.
Therefore, the aims of this study were as follows:(1) To create the endomicroscopic classification of gastric lesions, and to evaluate the diagnostic value of CLE for the identification and grading of GIN prospectively;(2) To evaluate the feasibility of CLE for molecular imaging of gastric cancer in vivo.
Methods
Part1:The establishment and evaluation of the endomicroscopic diagnostic criteria for GIN
This part was divided into two steps. In the first step, CLE was used to examine histologically confirmed normal gastric mucosa, nonneoplastic and neoplastic gastric lesions. CLE images were compared with horizontal sectioned histopathological pictures obtained from the same gastric lesions so as to establish the endomicroscopic classification for nonneoplastic and GIN lesions and the endomicroscpic scoring system for grading GIN. In the second step, consecutive patients were prospectively recruited according to predefined inclusion and exclusion criteria from July2009to January2010in Qilu Hospital. In vivo real-time CLE diagnosis was made for endoscopic suspicious lesions by the performing endoscopist who was blinded to the patients'previous histological diagnosis. CLE images of each lesion were stored in a specific folder, and were reevaluated after the endoscopy by another CLE investigator blinded to the patients'clinical history and endoscopic information. In addition, a post hoc assessment of inter-observer and intra-observer agreements for the CLE findings was performed according to the following protocol. A data set containing50confocal images of medium depth from50enrolled subjects were randomly selected and displayed to3independent endoscopists and one GI pathologist in a blinded fashion. To evaluate the intra-observer agreement, one investigator (R.J) reassessed these50pictures after a7days interval. Moreover, in an attempt to assess the feasibility of CLE for the differentiation between LGIN and HGIN, one confocal image of medium depth from each CLE diagnosed GIN lesion was further evaluated by an investigator according to the predefined scoring system in a blinded fashion.
Part2:Confocal laser endomicroscopy for in vivo molecular imaging of gastric cancer
This part was divided into3steps, including tumor cell line characterization, experiments of tumor-bearing mice, and human studies.
(1) The gastric cancer cell lines BGC823and SGC7901were incubated with AF488-MG7, isotype control antibody, or PBS respectively. Flowcytometry was firstly carried out, followed by CLE (FIVE1, Optiscan) and fluorescent microscopy. Finally, celluar immunohistochemistry was performed in both gastric cancer cell lines.
(2) Xenograft tumors were induced using the gastric cancer cell lines BGC823and SGC7901in BALB/c nu/nu mice. In order to evaluate the optimate imaging time and antibody concentration, full-body fluorescent imaging was firstly carried out using a cooled CCD camera (Kodak2000MM, Kodak Company, USA) after intracardiac administration of AF680-labelled MG7or isotype control antibody. Intravital CLE examination of xenograft tumors was then performed according to the results of full-body fluorescent imaging.
(3) Surgical specimens (5-10mm) or endoscopic biopsies of gastric cancerous tissue and non-cancerous gastric tissue were incubated with AF488-labelled MG7antibody, and imaged using CLE (FIVE, Optiscan) immediately. The CLE investigator was blinded to the patients'previous histopathological diagnosis of gastric lesions. MG7-specific fluorescence signal in CLE images was ranked as negative (0), weakly positive (+), moderately positive (++) or strong expression (+++). Then3experienced CLE investigators who were not involved in data collection were invited to give the semi-quantitative grading of MG7-Ag expression of all human specimens observed by CLE probe independently. H&E staing and immunohistochemistry were performed for all the specimens after CLE examinations. Spearman's correlation coefficient was also applied to assess the semi-quantitative results between CLE and IHC findings.
Results
Part1:In total,15093confocal images were obtained from91macroscopic lesions in the75patients. Using the prior described CLE diagnostic criteria for gastric lesions, the sensitivity, specificity, PPV, NPV, PLR and NLR of final CLE diagnosis of GIN were77.8%(95%CI,63.7%-87.5%),81.8%(95%CI,68.0%-90.5%),81.4%(95%CI,67.4%-90.3%),78.3%(95%CI,64.4%-87.7%),4.28(95%CI,2.24-8.16) and0.27(95%CI,0.16-0.48) respectively. There was no statistical difference between preliminary and final CLE diagnostic accuracy for GIN (P=0.549). The inter-observer agreement was substantial for the diagnosis of GIN among3endoscopists (mean kappa=0.70). We also investigated the inter-observer agreement between endoscopist and GI pathologist, and the mean kappa value was0.71. Intra-observer agreement was also graded as substantial (kappa=0.78). The intraepithelial neoplasia score≥5had a sensitivity of66.7%and a specificity of88.5%in discriminating HGIN from LGIN.
Part2:(1) The MG7-Ag expression was confirmed in the two gastric cancer cell lines BGC823and SGC7901by using Flow Cytometry. CLE (FIVE1, Optiscan) imaging showed membranous and cytoplasmic specific fluorescent signal. The immediate bench top fluorescence microscopic observation was similar to CLE images. IHC results also demonstrated specific staining of both tumor cell lines in the cell membrane and cytoplasm.(2) In the preliminary study of full-body imaging of tumor-bearing mouse, specific fluorescent signal was observed at24h after intracardiac injection of AF680-labelled MG7antibody (1μg/g body weight). In addition, the tumor fluorescence intensity reached its maximum at48h after antibody administration. Forty-eight hours after AF488-labelled MG7antibody injection, specific fluorescent signal could be visualized in both BGC823and SGC7901tumors; Ex vivo IHC results and fluorescent microscopic imaging of cryosections resembled in vivo endomicroscopic findings.(3) A total of46samples, including18surgical specimens and28biopsy specimens from23patients with gastric cancer, were analyzed prospectively. Specific signals (+to+++) were found in22/23samples diagnosed as gastric cancer, whereas non-cancerous samples revealed no or only weak (0or+, n=18, n=5) fluorescent signal. The correlation between different grades of IHC results of human samples and semi-quantitative assessment of in vivo MG7-Ag expression using CLE was good (Spearman's r=0.87, P＜0.001). The interobserver agreement of the semi-quantitative assessment scheme of CLE images was also excellent (ICC=0.883,95%CI0.819-0.929).
Conclusions
1. GIN, non-neoplastic gastric lesions, and normal gastric mucosa can be differenciated according to characteristic changes of gland architecture, cell morphology, and vassel architecture in CLE images.
2. HGIN can be differenciated from LGIN using CLE according to the predefined endomicroscopic scoring system.
3. The interobserver and intraobserver agreement are all substantial for the diagnosis of GIN using CLE, indicating the diagnostic criteria in this study is reliable.
4. Using fluorescently labeled MG7antibody, CLE can achive targeted molecular imaging in tumor-bearing mouse in vivo and in the fresh tissue of patients with gastric cancer; there is a good correlation between CLE fluorescent imaging and ex vivo immunohistochemistry results. Significance
Similar to the case with histopathologic features, distinct glandular derangements, cellular abnormalities, and microvascular alterations are identifiable for the diagnosis of GIN using CLE. The established endomicroscopic classification for gastric lesions can differentiate GIN lesions from non-neoplastic ones with a substantial inter-and intraobserver agreement, which indicates a good reliability of it. Moreover, HGIN can be differentiated from LGIN with a high specificity using CLE, thus endoscopists can make spot-decision regarding whether to perform endoscopic resection or otherwise. With the aid of fluorescently labeled specific antibody targeting gastric cancer, CLE is able to diagnose gastric cancer in the molecular level in vivo. This technique may have provided us with enhanced diagnosis of gastric cancer and accurate selection of MG7-Ag-positive patients. Although in vivo human studies are needed to further investigate its clinical role, data of the present study verified the feasibility of CLE mediated immunodiagnosis of gastric cancer from bench to bedside.
目前胃癌仍是世界上病死率排名第二位的恶性肿瘤。1988年,Correa提出的慢性萎缩性胃炎-胃黏膜肠上皮化生-胃黏膜不典型增生-肠型胃癌的多阶段胃癌发展模型已被较多临床随访研究、胃癌手术切除标本及基础实验等所验证。故萎缩性胃炎、胃黏膜肠上皮化生及不典型增生(Dysplasia)均为公认的胃黏膜癌前病变。尽管如此,胃黏膜不典型增生作为肠型胃癌形成的倒数第二步演变步骤,是胃腺癌最为直接的癌前病变,其早期诊断对防治胃癌具有十分重要的临床意义。2010年,世界卫生组织正式提出用“上皮内瘤变(Intraepithelial Neoplasia)"取代“不典型增生”。
内镜检查是早期发现胃黏膜瘤变的最有效手段。尤其是近年来应用于临床的先进的内镜技术如高清晰内镜、放大内镜、色素内镜及窄带成像(Narrow-band Imaging, NBI)等,均可提供胃黏膜表面颜色及细微结构变化的高清晰图像。然而,早期胃黏膜瘤变的内镜下宏观改变缺乏特异性,故虽然上述内镜技术可以-定程度提高早期胃癌的检出率,但仍无法观察到胃黏膜上皮细胞、腺体和微血管等显微结构的改变。因此,为确诊病变,内镜医师仍需留取多块活检标本,由此造成的活检瘢痕对胃黏膜上皮内瘤变(Gastric Intraepithelial Neoplasia, GIN)患者短期内实行内镜治疗亦有诸多不良影响。此外,活检标本的组织病理学检查需耗费2-3天时间,对于确诊为高级别上皮内瘤变(High-grade Intraepithelial Neoplasia, HGIN)和早期胃癌的患者来说,不但有可能延误治疗时机,而且增加了患者的精神与经济负担。
共聚焦激光显微内镜(Confocal Laser Endomicroscopy, CLE)是一种新型的内镜技术,可以在白光内镜观察的同时,通过静脉注射或黏膜表面喷洒荧光造影剂,实时观察消化道黏膜上皮细胞、腺体及血管等显微结构,获取类似病理组织学的显微内镜图像。CLE的问世为胃黏膜癌前病变及早期胃癌的诊断提供了新的有力工具。然而,目前尚无研究将其应用于胃黏膜上皮内瘤变的诊断。
早期诊断是改善胃癌患者预后的关键。内镜活检虽为目前公认的诊断胃癌的金标准,但仍存在致使病变形态改变、引发出血、穿孔等并发症的潜在缺陷。此外,常规内镜活检的病理组织学确诊病变后再行内镜下治疗还会延后治疗时间,增添患者的心理和医疗负担。因此,目前亟需一种能够实现实时显微组织观察的内镜技术,使医师在普通内镜检查的同时观察到病变的显微结构改变并立即做出诊断,采取相应的治疗措施,从而提高胃癌的检出率和诊断、治疗水平。近年来,已有多项研究致力于将CLE应用于在体诊断胃癌；研究结果显示,根据已制定的CLE形态学诊断标准,共聚焦激光显微内镜能够较为准确的鉴别胃黏膜癌变病变与非癌变病变。但上述研究均采用对患者静脉注射荧光素钠作为对比剂,获取胃黏膜显微结构的非特异性荧光成像,无法达到对胃肠道黏膜疾病功能性和分子水平改变进行观察的目的。近期几项研究表明,利用荧光物质标记的特异性抗体,CLE能够实现活体内分子水平的观察。
因此,本课题拟围绕胃黏膜上皮内瘤变和胃癌的显微内镜诊断方面展开研究,主要研究目的为：(1)探讨利用CLE对胃黏膜非瘤变和瘤变病变进行鉴别诊断的可行性,前瞻性评估CLE对胃黏膜上皮内瘤变进行实时诊断和分级诊断的效能；(2)系统研究CLE对胃癌活体分子成像的可行性。
方法
第一部分：共聚焦激光显微内镜在体诊断胃黏膜上皮内瘤变的可行性研究
研究过程分为两个阶段,第一阶段采用共聚焦激光显微内镜对已知正常胃黏膜、非瘤变和胃黏膜上皮内瘤变的病变进行检查,将所扫描部位的共聚焦激光显微内镜图像与相应部位横切面的组织病理学图像进行对比,制定共聚焦激光显微内镜对胃黏膜病变的鉴别诊断标准和GIN分级诊断的标准体系。第二阶段,前瞻性的纳入2009年7月至2010年1月间于齐鲁医院胃镜室预约行CLE检查且符合相应纳入标准的患者为研究对象。由CLE操作者对胃黏膜病变进行实时CLE诊断；由另外一名未参与实时诊断的有经验的内镜医师在检查结束后对所留取的图片进行盲法诊断。以病理组织学诊断结果为金标准,验证上述诊断标准。为了评估观察者间和观察者内的一致性,我们还邀请了3名内镜医师和1名病理医师独立地对前瞻性研究中的50处病变的共聚焦图像按本研究制定的诊断标准进行分类诊断。其中一名内镜医师间隔一周后再次对这50处病变的CLE图像进行诊断。
第二部分：共聚焦激光显微内镜对胃癌活体分子成像的研究
研究分为3个步骤,分别为细胞学研究、胃癌荷瘤鼠动物模型体内分子成像和人体新鲜组织体外研究。
(1)将BGC823和SGC7901这2种人源性胃癌细胞系分别与AF488-MG7抗体,同型对照抗体,或PBS体外共孵育,首先进行流式细胞仪分析,然后利用CLE (FIVEl, Optiscan)对未固定的活细胞进行显微荧光成像,荧光显微镜观察；最后进行MG7抗原细胞学免疫组织化学染色。
(2)建立上述两种胃癌细胞系的荷瘤鼠动物模型,心内注射AF680-MG7抗体或AF680-同型对照抗体,利用小动物活体成像系统(Kodak2000MM, Kodak,USA)对其进行整体动态观察,总结肿瘤特异性荧光成像的最佳抗体剂量及时间。然后根据上述结果进行CLE荷瘤鼠体内显微水平的分子成像。对CLE观察部位的组织进行MG7抗原免疫组织化学染色,以相同荷瘤鼠的肝脏组织为阴性对照。
(3)选取新鲜胃癌组织及相同患者的非癌组织的手术标本(5-10mm)或内镜活检标本为研究对象,与AF488-MG7抗体溶液共孵育,然后立即用CLE (FIVEl, Optiscan)进行观察。全部CLE操作均由同一研究者完成,该研究者在进行CLE观察时不知晓标本的病理组织学诊断和位置来源。将CLE图像利用半定量方法分为阴性(0),弱阳性(+),阳性(++)和强阳性(+++)4个等级,由另外3位有经验的CLE研究者分别盲法独立对CLE图片进行半定量分析,从而评估该CLE分子成像半定量评估体系的可靠性。CLE扫描结束后将标本行HE染色和MG7抗原免疫组化实验。以病理组织学诊断作为本实验的金标准。对所得实验结果利用Spearman相关系数评估CLE半定量结果与相应的免疫组化半定量结果的相关性。
结果
第一部分：
前瞻性研究共纳入75例患者,包括91处可见病变,留取CLE图像15093张。利用本研究中制定的CLE胃黏膜病变鉴别诊断标准,CLE最终诊断GIN的灵敏度、特异度、阳性预测值、阴性预测值、阳性似然比和阴性似然比分别为77.8%(95%CI,63.7%-87.5%),81.8%(95%CI,68.0%-90.5%),81.4%(95%CI,67.4%-90.3%),78.3%(95%CI,64.4%-87.7%),4.28(95%CI,2.24-8.16)口0.27(95%CI,0.16-0.48)。 CLE实时诊断与最终诊断的准确度比较无统计学差异(P=0.549)。
观察者间一致性的平均Kappa值为0.70(内镜医师之间)和0.71(内镜医师与病理医师之间)。观察者内一致性亦较高(kappa=0.78)。上皮内瘤变评分≥5用于区分高级别和低级别上皮内瘤变的灵敏度和特异度分别为66.7%和88.0%。
第二部分：
(1)流式细胞术检测结果显示本研究中所使用的BGC823和SGC7901胃癌细胞系均存在MG7-Ag的表达；CLE (FIVE1, Optiscan)图像显示细胞膜和胞浆的特异性荧光信号；即刻台式荧光显微镜观察结果与CLE相似。细胞免疫组化结果也验证了上述两种细胞系的细胞膜和胞浆中存在MG7-Ag的表达。
(2)荷瘤鼠整体动态荧光成像观察结果显示,心内注射AF680-MG7抗体(1μg/g体重)避光饲养24小时后,小动物活体成像仪摄取的图像中即可见肿瘤部位出现特异性荧光信号。该肿瘤部位的特异性荧光信号在48小时达最强。CLE观察肿瘤表面均可见特异性荧光信号；体外免疫组化结果和台式荧光显微镜观察冰冻切片的图像与体内CLE图像相似。
(3)共前瞻性纳入了46例新鲜的人组织标本,包括18例手术标本和28例内镜活检标本(来自23例胃癌患者)。23例胃癌组织标本中有22例在CLE实验中观察到了特异性荧光信号(+～+++),而非癌组织标本则无或仅有弱荧光信号(0或+,n=18, n=5)。CLE半定量分析结果与不同等级的免疫组化结果的相关性较好(Spearman r=0.87, P<0.001)。CLE图像半定量分析方法的观察者间一致性较高(ICC=0.883,95%可信区间：0.819～0.929)。
结论
1.通过识别共聚焦显微图像中腺体结构、细胞形态和血管结构的特征性改变,可以区分正常胃黏膜、非瘤变病变及胃黏膜上皮内瘤变病变。
2.通过制定共聚焦显微图像瘤变改变的评分系统,可初步区分低级别和高级别上皮内瘤变。
3.CLE诊断胃黏膜上皮内瘤变的观察者间和观察者内一致性较高,具有较好的可靠性。
4.利用荧光标记的MG7抗体,CLE可以对胃癌荷瘤鼠进行活体内显微水平的靶向分子成像；CLE对胃癌患者的新鲜黏膜组织体外荧光分子成像的结果与传统的体外免疫组织化学染色结果相关性好。
研究意义
本研究证实CLE可清晰识别GIN特征性的细胞异常、结构紊乱和微血管变化,建立的CLE下胃黏膜病变的诊断标准可实时区分GIN病变与非瘤变病变。CLE对GIN的分类诊断标准的观察者间及观察者内一致性均较高,说明该诊断标准的可靠性较好。利用CLE图像中GIN的改变的严重程度的差异可以对GIN进行分级诊断,高特异度的实时鉴别诊断HGIN病变,从而使内镜医师对病变部位即刻进行内镜下治疗,避免治疗时机的延误、活检疤痕对内镜治疗的不良影响以及医疗资源的重复消耗。本研究首次利用荧光标记的MG7抗体,以CLE为观察手段,实现了对胃癌荷瘤鼠的活体内靶向分子成像和人体活组织的体外分子成像。CLE作为一种新的在体显微分子成像技术,有助于提高胃癌的诊断效能,准确筛选MG7-Ag阳性的患者。尽管该项技术的临床价值尚待进一步人体内研究的验证,我们目前的研究结果已初步确定了CLE辅助下的对胃癌患者进行活体免疫学诊断的可行性。
Backgrounds and aims
Gastric cancer remains the world's second leading cause of cancer-related death. In1988, the multistep carcinogenesis process of gastric carcinoma was proposed by Correa, and it was described as atrophic gastritis-intestinal metaplasia-dysplasia-intestinal type gastric cancer. This theory has been validated by many follow-up clinical trials todate. Therefore, atrophic gastritis, intestinal metaplasia, and dysplasia are all widely considered as premalinant lesions of the gastric mucosa. Nevertheless, as the penultimate step for the development of intestinal type gastric cancer, gastric dysplasia is the most threatening precancerous changes. Thus the early diagnosis of gastric dysplasia is significant for the prevention and treatment of gastric cancer. In2010, the World Health Organization suggested to use the nomenclature of "intraepithelial neoplasia" to replace "dysplasia".
Endoscopy is the most efficient tool for early detection of gastric neoplasia. Modern endoscopic devices such as high-definition endoscopy, magnifying endoscopy, chromoendoscopy and narrow-band imaging (NBI), can provide high resolution endoscopic pictures of color changes and minute structure alterations of gastric mucosa. However, although these endoscopic modalities can improve the detection rate of early gastric carcinoma on a certain extent, they can't enable microscopic observation of epithelial cells, gland architecture and vasculature. Therefore, multiple biopsies are still needed to diagnose endoscopic suspicious lesions, and the biopsy scars may interefere with the following short-term endoscopic therapy of these lesions if diagnosed as gastric intraepithelial neoplasia (GIN) by histopathology. Moreover, the histological processing of biopsy specimens usually takes of2-3days, which not only postpones therapeutic opportunity for patients with high-grade intraepithelial neoplasia (HGIN) and early gastric cancer, but also severes the patients'psychological and financial burdens.
Confocal laser endomicroscopy (CLE) is a novel endoscopic technology which can provide real-time cellular and subcellular visualization of gastric mucosa with the aid of intravenous surface application of fluorescent dyes, while at the same time displaying white-light endoscopic images. Previous studies found that CLE is a powerful new tool for early endoscopic diagnosis of intestinal metaplasia and gastric cancer. However, no investigation has yet applied CLE in the diagnosis of GIN.
Early diagnosis is essential for improving the prognosis of gastric cancer. Currently, although endoscopic biopsy is the gold standard for diagnosing gastric cancer, it still suffers from several drawbacks such as lesions'morphological alteration, bleeding and perforation. What's more, the delayed histopathological diagnosis will lead to later therapy and increased anxiety of patients. Thus a new endoscopic device is needed which can enable immediate histopathological diagnosis of suspicious lesions during white-light endoscopic examination. Several studies have been investigating the role of CLE in diagnosing gastric cancer in vivo, which showed that according to predefined endomicroscopic diagnostic criteria, gastric cancer can be accurately differentiated from non-cancerous lesions using CLE. However, all those studies applied intravenous fluorescein sodium as the contrast agent to obtain nonspecific endomicroscopic images, which cannot provide functional or molecular imaging of the gastric mucosa. Most recently, several researches revealed that using fluorescently labeled specific antibodies, CLE can achieve intravital molecular imaging of GI cancer.
Therefore, the aims of this study were as follows:(1) To create the endomicroscopic classification of gastric lesions, and to evaluate the diagnostic value of CLE for the identification and grading of GIN prospectively;(2) To evaluate the feasibility of CLE for molecular imaging of gastric cancer in vivo.
Methods
Part1:The establishment and evaluation of the endomicroscopic diagnostic criteria for GIN
This part was divided into two steps. In the first step, CLE was used to examine histologically confirmed normal gastric mucosa, nonneoplastic and neoplastic gastric lesions. CLE images were compared with horizontal sectioned histopathological pictures obtained from the same gastric lesions so as to establish the endomicroscopic classification for nonneoplastic and GIN lesions and the endomicroscpic scoring system for grading GIN. In the second step, consecutive patients were prospectively recruited according to predefined inclusion and exclusion criteria from July2009to January2010in Qilu Hospital. In vivo real-time CLE diagnosis was made for endoscopic suspicious lesions by the performing endoscopist who was blinded to the patients'previous histological diagnosis. CLE images of each lesion were stored in a specific folder, and were reevaluated after the endoscopy by another CLE investigator blinded to the patients'clinical history and endoscopic information. In addition, a post hoc assessment of inter-observer and intra-observer agreements for the CLE findings was performed according to the following protocol. A data set containing50confocal images of medium depth from50enrolled subjects were randomly selected and displayed to3independent endoscopists and one GI pathologist in a blinded fashion. To evaluate the intra-observer agreement, one investigator (R.J) reassessed these50pictures after a7days interval. Moreover, in an attempt to assess the feasibility of CLE for the differentiation between LGIN and HGIN, one confocal image of medium depth from each CLE diagnosed GIN lesion was further evaluated by an investigator according to the predefined scoring system in a blinded fashion.
Part2:Confocal laser endomicroscopy for in vivo molecular imaging of gastric cancer
This part was divided into3steps, including tumor cell line characterization, experiments of tumor-bearing mice, and human studies.
(1) The gastric cancer cell lines BGC823and SGC7901were incubated with AF488-MG7, isotype control antibody, or PBS respectively. Flowcytometry was firstly carried out, followed by CLE (FIVE1, Optiscan) and fluorescent microscopy. Finally, celluar immunohistochemistry was performed in both gastric cancer cell lines.
(2) Xenograft tumors were induced using the gastric cancer cell lines BGC823and SGC7901in BALB/c nu/nu mice. In order to evaluate the optimate imaging time and antibody concentration, full-body fluorescent imaging was firstly carried out using a cooled CCD camera (Kodak2000MM, Kodak Company, USA) after intracardiac administration of AF680-labelled MG7or isotype control antibody. Intravital CLE examination of xenograft tumors was then performed according to the results of full-body fluorescent imaging.
(3) Surgical specimens (5-10mm) or endoscopic biopsies of gastric cancerous tissue and non-cancerous gastric tissue were incubated with AF488-labelled MG7antibody, and imaged using CLE (FIVE, Optiscan) immediately. The CLE investigator was blinded to the patients'previous histopathological diagnosis of gastric lesions. MG7-specific fluorescence signal in CLE images was ranked as negative (0), weakly positive (+), moderately positive (++) or strong expression (+++). Then3experienced CLE investigators who were not involved in data collection were invited to give the semi-quantitative grading of MG7-Ag expression of all human specimens observed by CLE probe independently. H&E staing and immunohistochemistry were performed for all the specimens after CLE examinations. Spearman's correlation coefficient was also applied to assess the semi-quantitative results between CLE and IHC findings.
Results
Part1:In total,15093confocal images were obtained from91macroscopic lesions in the75patients. Using the prior described CLE diagnostic criteria for gastric lesions, the sensitivity, specificity, PPV, NPV, PLR and NLR of final CLE diagnosis of GIN were77.8%(95%CI,63.7%-87.5%),81.8%(95%CI,68.0%-90.5%),81.4%(95%CI,67.4%-90.3%),78.3%(95%CI,64.4%-87.7%),4.28(95%CI,2.24-8.16) and0.27(95%CI,0.16-0.48) respectively. There was no statistical difference between preliminary and final CLE diagnostic accuracy for GIN (P=0.549). The inter-observer agreement was substantial for the diagnosis of GIN among3endoscopists (mean kappa=0.70). We also investigated the inter-observer agreement between endoscopist and GI pathologist, and the mean kappa value was0.71. Intra-observer agreement was also graded as substantial (kappa=0.78). The intraepithelial neoplasia score≥5had a sensitivity of66.7%and a specificity of88.5%in discriminating HGIN from LGIN.
Part2:(1) The MG7-Ag expression was confirmed in the two gastric cancer cell lines BGC823and SGC7901by using Flow Cytometry. CLE (FIVE1, Optiscan) imaging showed membranous and cytoplasmic specific fluorescent signal. The immediate bench top fluorescence microscopic observation was similar to CLE images. IHC results also demonstrated specific staining of both tumor cell lines in the cell membrane and cytoplasm.(2) In the preliminary study of full-body imaging of tumor-bearing mouse, specific fluorescent signal was observed at24h after intracardiac injection of AF680-labelled MG7antibody (1μg/g body weight). In addition, the tumor fluorescence intensity reached its maximum at48h after antibody administration. Forty-eight hours after AF488-labelled MG7antibody injection, specific fluorescent signal could be visualized in both BGC823and SGC7901tumors; Ex vivo IHC results and fluorescent microscopic imaging of cryosections resembled in vivo endomicroscopic findings.(3) A total of46samples, including18surgical specimens and28biopsy specimens from23patients with gastric cancer, were analyzed prospectively. Specific signals (+to+++) were found in22/23samples diagnosed as gastric cancer, whereas non-cancerous samples revealed no or only weak (0or+, n=18, n=5) fluorescent signal. The correlation between different grades of IHC results of human samples and semi-quantitative assessment of in vivo MG7-Ag expression using CLE was good (Spearman's r=0.87, P＜0.001). The interobserver agreement of the semi-quantitative assessment scheme of CLE images was also excellent (ICC=0.883,95%CI0.819-0.929).
Conclusions
1. GIN, non-neoplastic gastric lesions, and normal gastric mucosa can be differenciated according to characteristic changes of gland architecture, cell morphology, and vassel architecture in CLE images.
2. HGIN can be differenciated from LGIN using CLE according to the predefined endomicroscopic scoring system.
3. The interobserver and intraobserver agreement are all substantial for the diagnosis of GIN using CLE, indicating the diagnostic criteria in this study is reliable.
4. Using fluorescently labeled MG7antibody, CLE can achive targeted molecular imaging in tumor-bearing mouse in vivo and in the fresh tissue of patients with gastric cancer; there is a good correlation between CLE fluorescent imaging and ex vivo immunohistochemistry results. Significance
Similar to the case with histopathologic features, distinct glandular derangements, cellular abnormalities, and microvascular alterations are identifiable for the diagnosis of GIN using CLE. The established endomicroscopic classification for gastric lesions can differentiate GIN lesions from non-neoplastic ones with a substantial inter-and intraobserver agreement, which indicates a good reliability of it. Moreover, HGIN can be differentiated from LGIN with a high specificity using CLE, thus endoscopists can make spot-decision regarding whether to perform endoscopic resection or otherwise. With the aid of fluorescently labeled specific antibody targeting gastric cancer, CLE is able to diagnose gastric cancer in the molecular level in vivo. This technique may have provided us with enhanced diagnosis of gastric cancer and accurate selection of MG7-Ag-positive patients. Although in vivo human studies are needed to further investigate its clinical role, data of the present study verified the feasibility of CLE mediated immunodiagnosis of gastric cancer from bench to bedside.
引文
1. Yang L. Incidence and mortality of gastric cancer in China. World J Gastroenterol 2006; 12:17-20.
2. Correa P. Human gastric carcinogenesis:a multistep and multifactorial process--First American Cancer Society Award Lecture on Cancer Epidemiology and Prevention. Cancer Res 1992;52:6735-40.
3. Schlemper RJ, Riddell RH, Kato Y, et al. The Vienna classification of gastrointestinal epithelial neoplasia. Gut 2000;47:251-5.
4. Yamada H, Ikegami M, Shimoda T, et al. Long-term follow-up study of gastric adenoma/dysplasia. Endoscopy 2004;36:390-6.
5. Dinis-Ribeiro M, da Costa-Pereira A, Lopes C, et al. Magnification chromoendoscopy for the diagnosis of gastric intestinal metaplasia and dysplasia. Gastrointest Endosc 2003;57:498-504.
6. Areia M, Amaro P, Dinis-Ribeiro M, et al. External validation of a classification for methylene blue magnification chromoendoscopy in premalignant gastric lesions. Gastrointest Endosc 2008;67:1011-8.
7. Zhang JN, Li YQ, Zhao YA, et al. Classification of gastric pit patterns by confocal endomicroscopy. Gastrointest Endosc 2008;67:843-53.
8. Guo YT, Li YQ, Yu T, et al. Diagnosis of gastric intestinal metaplasia with confocal laser endomicroscopy in vivo:a prospective study. Endoscopy 2008;40:547-53.
9. Kiesslich R, Gossner L, Goetz M, et al. In vivo histology of Barrett's esophagus and associated neoplasia by confocal laser endomicroscopy. Clin Gastroenterol Hepatol 2006;4:979-87.
10. Dunbar KB, Okolo P,3rd, Montgomery E, et al. Confocal laser endomicroscopy in Barrett's esophagus and endoscopically inapparent Barrett's neoplasia:a prospective, randomized, double-blind, controlled, crossover trial. Gastrointest Endosc 2009;70:645-54.
11. Ming SC, Bajtai A, Correa P, et al. Gastric dysplasia. Significance and pathologic criteria. Cancer 1984;54:1794-801.
12. Goetz M, Ziebart A, Foersch S, et al. In vivo molecular imaging of colorectal cancer with confocal endomicroscopy by targeting epidermal growth factor receptor. Gastroenterology 2010; 138:435-46.
13. Foersch S, Kiesslich R, Waldner MJ, et al. Molecular imaging of VEGF in gastrointestinal cancer in vivo using confocal laser endomicroscopy. Gut 2010; 59:1046-55.
14. Hsiung PL, Hardy J, Friedland S, et al. Detection of colonic dysplasia in vivo using a targeted heptapeptide and confocal microendoscopy. Nat Med 2008; 14: 454-8.
1. Winawer S, Fletcher R, Rex D, et al. Colorectal cancer screening and surveillance: clinical guidelines and rationale-Update based on new evidence. Gastroenterology 2003;124:544-560
2. Wehrmann K, Fruhmorgen P. [Removing adenomas reduces colon carcinoma risk up to 90%. Effective cancer prevention with the endoscope]. MMW Fortschritte der Medizin 2000;142:26-29
3. Togashi K, Konishi F, Ishizuka T, et al. Efficacy of magnifying endoscopy in the differential diagnosis of neoplastic and non-neoplastic polyps of the large bowel. Diseases of the colon and rectum 1999;42:1602-1608
4. Kiesslich R, Goetz M, Lammersdorf K, et al. Chromoscopy-guided endomicroscopy increases the diagnostic yield of intraepithelial neoplasia in ulcerative colitis. Gastroenterology 2007;132:874-882
5. Kiesslich R, Gossner L, Goetz M, et al. In vivo histology of Barrett's esophagus and associated neoplasia by confocal laser endomicroscopy. Clin Gastroenterol Hepatol 2006;4:979-987
6. Sanduleanu S, Driessen A, Hameeteman W, et al. Inflammatory cloacogenic polyp:diagnostic features by confocal endomicroscopy. Gastrointestinal endoscopy 2009;69:595-598
7. Sanduleanu S, Driessen A, Gomez-Garcia E, et al. In Vivo Diagnosis and Classification of Colorectal Neoplasia by Chromoendoscopy-Guided Confocal Laser Endomicroscopy. Clin Gastroenterol Hepatol 2009
8. Kiesslich R, Burg J, Vieth M, et al. Confocal laser endoscopy for diagnosing intraepithelial neoplasias and colorectal cancer in vivo. Gastroenterology 2004;127:706-713
9. Kiesslich R, Goetz M, Angus EM, et al. Identification of epithelial gaps in human small and large intestine by confocal endomicroscopy. Gastroenterology 2007;133:1769-1778
10. Guo YT, Li YQ, Yu T, et al. Diagnosis of gastric intestinal metaplasia with confocal laser endomicroscopy in vivo:a prospective study. Endoscopy 2008;40:547-553
11. Roy HK, Gomes A, Turzhitsky V, et al. Spectroscopic microvascular blood detection from the endoscopically normal colonic mucosa:biomarker for neoplasia risk. Gastroenterology 2008;135:1069-1078
12. Hamilton SR, Aaltonen, L.A. Pathology and Genetics. Tumours of the Digestive System. WHO Classification of Tumours 2000;2:2
13. Levin B, Lieberman DA, McFarland B, et al. Screening and surveillance for the early detection of colorectal cancer and adenomatous polyps,2008:a joint guideline from the American Cancer Society, the US Multi-Society Task Force on Colorectal Cancer, and the American College of Radiology. Gastroenterology 2008;134:1570-1595
14. Kiesslich R, von Bergh M, Hahn M, Hermann G, Jung M. Chromoendoscopy with indigocarmine improves the detection of adenomatous and nonadenomatous lesions in the colon. Endoscopy 2001;33:1001-1006
15. Eisen GM, Kim CY, Fleischer DE, et al. High-resolution chromoendoscopy for classifying colonic polyps:a multicenter study. Gastrointestinal endoscopy 2002;55:687-694
16. van den Broek FJ, Reitsma JB, Curvers WL, Fockens P, Dekker E. Systematic review of narrow-band imaging for the detection and differentiation of neoplastie and nonneoplastic lesions in the colon (with videos). Gastrointestinal endoscopy 2009;69:124-135
17. Hurlstone DP, Thomson M, Brown S, et al. Confocal endomicroscopy in ulcerative colitis:differentiating dysplasia-associated lesional mass and adenoma-like mass. Clin Gastroenterol Hepatol 2007;5:1235-1241
18. Wang HW, Willis J, Canto MI, Sivak MV, Jr., Izatt JA. Quantitative laser scanning confocal autofluorescence microscopy of normal, premalignant, and malignant colonic tissues. IEEE transactions on bio-medical engineering 1999;46:1246-1252
19. Buchner AM, Shahid MW, Heckman MG, et al. Comparison of Probe-Based Confocal Laser Endomicroscopy with Virtual Chromoendoscopy for Classification of Colon Polyps. Gastroenterology 2009
20. Goetz M, Toermer T, Vieth M, et al. Simultaneous confocal laser endomicroscopy and chromoendoscopy with topical cresyl violet. Gastrointestinal endoscopy 2009;70:959-968
21. Snover DC, Jass JR, Fenoglio-Preiser C, Batts KP. Serrated polyps of the large intestine:a morphologic and molecular review of an evolving concept. American journal of clinical pathology 2005;124:380-391
22. Goetz M, Ziebart A, Foersch S, et al. In vivo molecular imaging of colorectal cancer with confocal endomicroscopy by targeting epidermal growth factor receptor. Gastroenterology 2010; 138:435-446
1. Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics,2002. CA Cancer J Clin 2005; 55:74-108.
2. Sakai Y, Eto R, Kasanuki J, et al. Chromoendoscopy with indigo carmine dye added to acetic acid in the diagnosis of gastric neoplasia:a prospective comparative study. Gastrointest Endosc 2008; 68:635-41.
3. Yao K, Anagnostopoulos GK, Ragunath K. Magnifying endoscopy for diagnosing and delineating early gastric cancer. Endoscopy 2009; 41:462-7.
4. Ohashi A, Niwa Y, Ohmiya N, et al. Quantitative analysis of the microvascular architecture observed on magnification endoscopy in cancerous and benign gastric lesions. Endoscopy 2005; 37:1215-9.
5. Otsuka Y, Niwa Y, Ohmiya N, et al. Usefulness of magnifying endoscopy in the diagnosis of early gastric cancer. Endoscopy 2004; 36:165-9.
6. Kaise M, Kato M, Urashima M, et al. Magnifying endoscopy combined with narrow-band imaging for differential diagnosis of superficial depressed gastric lesions. Endoscopy 2009; 41:310-5.
7. Kakeji Y, Yamaguchi S, Yoshida D, et al. Development and assessment of morphologic criteria for diagnosing gastric cancer using confocal endomicroscopy: an ex vivo and in vivo study. Endoscopy 2006; 38:886-90.
8. Kitabatake S, Niwa Y, Miyahara R, et al. Confocal endomicroscopy for the diagnosis of gastric cancer in vivo. Endoscopy 2006; 38):1110-4.
9. Li WB, Zuo XL, Li CQ, et al. Diagnostic value of confocal laser endomicroscopy for gastric superficial cancerous lesions. Gut 2011; 60:299-306.
10. Goetz M, Ziebart A, Foersch S, et al. In vivo molecular imaging of colorectal cancer with confocal endomicroscopy by targeting epidermal growth factor receptor. Gastroenterology 2010; 138:435-46.
11. Foersch S, Kiesslich R, Waldner MJ, et al. Molecular imaging of VEGF in gastrointestinal cancer in vivo using confocal laser endomicroscopy. Gut 2010; 59: 1046-55.
12. Hsiung PL, Hardy J, Friedland S, et al. Detection of colonic dysplasia in vivo using a targeted heptapeptide and confocal microendoscopy. Nat Med 2008; 14: 454-8.
13. Keller R, Winde G, Eisenhawer C, et al. Immunoscopy--a technique combining endoscopy and immunofluorescence for diagnosis of colorectal carcinoma. Gastrointest Endosc 1998; 47:154-61.
14. Keller R, Winde G, Terpe HJ, Foerster EC, Domschke W. Fluorescence endoscopy using a fluorescein-labeled monoclonal antibody against carcinoembryonic antigen in patients with colorectal carcinoma and adenoma. Endoscopy 2002; 34:801-7.
15. Folli S, Wagnieres G, Pelegrin A, et al. Immunophotodiagnosis of colon carcinomas in patients injected with fluoresceinated chimeric antibodies against carcinoembryonic antigen. Proc Natl Acad Sci U S A 1992; 89:7973-7.
16. Takenoshita S, Hashizume T, Asao T, et al. Immunoscintigraphy using 99mTc-labeled anti-CEA monoclonal antibody for patients with colorectal cancer. Anticancer Res 1995; 15:471-5.
17. Marten K, Bremer C, Khazaie K, et al. Detection of dysplastic intestinal adenomas using enzyme-sensing molecular beacons in mice. Gastroenterology 2002; 122: 406-14.
18. Zhang H, Morgan D, Cecil G, et al. Biochromoendoscopy:molecular imaging with capsule endoscopy for detection of adenomas of the GI tract. Gastrointest Endosc 2008; 68:520-7.
19. Fan DM, Zhang XY, Chen XT, et al. Establishment of four monoclonal antibodies to a poorly differentiated gastric cancer cell line MKN-46-9 and immunohistochemical study on their corresponding antigens. Chin J Med PLA 1988; 13:12-5.
20. Jin B, Wang X, Jin Y, et al. Detection of serum gastric cancer-associated MG7-Ag from gastric cancer patients using a sensitive and convenient ELISA method. Cancer Invest 2009; 27:227-33.
21.Guo DL, Dong M, Wang L, Sun LP, Yuan Y. Expression of gastric cancer-associated MG7 antigen in gastric cancer, precancerous lesions and H. pylori -associated gastric diseases. World J Gastroenterol 2002; 8:1009-13.
22. Zhang X, Hong L, Chan WY, et al. Expression of MG7-Ag in patients with gastric cancer correlates with weaker T cell immune response and more proinflammatory cytokine secretion. Biochem Cell Biol 2006; 84:135-41.
23. Liu J, Hu JL, Zhang XY, et al. The value of MG7 antigen in predicting cancerous change in dysplastic gastric mucosa. Int J Clin Pract 2002; 56:169-72.
24. Kiesslich R, Goetz M, Vieth M, Galle PR, Neurath MR Confocal laser endomicroscopy. Gastrointestinal endoscopy clinics of North America 2005; 15: 715-31.
25. Chang CC, Chen SH, Lin CP, et al. Premedication with pronase or N-acetylcysteine improves visibility during gastroendoscopy:an endoscopist-blinded, prospective, randomized study. World J Gastroenterol 2007; 13:444-7.
26. Fleiss JL, Cohen J. The equivalence of weighted kappa and the intraclass correlation coefficient as measures of reliability. Educ Psychol Meas 1973; 33: 613-19.
27. Conte WL, Kamishina H, Reep RL. Multiple neuroanatomical tract-tracing using fluorescent Alexa Fluor conjugates of cholera toxin subunit B in rats. Nat Protoc 2009; 4:1157-66.
28. Goetz M, Kiesslich R. Advances of endomicroscopy for gastrointestinal physiology and diseases. Am J Physiol Gastrointest Liver Physiol 2010; 298: G797-806.
29. Tong Q, Liu K, Lu XM, Shu XG, Wang GB. Construction and characterization of a novel fusion protein MG7-scFv/SEB against gastric cancer. J Biomed Biotechnol 2010; 2010:121094.
30. Hoffman RM. Orthotopic metastatic mouse models for anticancer drug discovery and evaluation:a bridge to the clinic. Invest New Drugs 1999; 17:343-59.
2. Correa P. Human gastric carcinogenesis:a multistep and multifactorial process--First American Cancer Society Award Lecture on Cancer Epidemiology and Prevention. Cancer Res 1992;52:6735-40.
3. Schlemper RJ, Riddell RH, Kato Y, et al. The Vienna classification of gastrointestinal epithelial neoplasia. Gut 2000;47:251-5.
4. Yamada H, Ikegami M, Shimoda T, et al. Long-term follow-up study of gastric adenoma/dysplasia. Endoscopy 2004;36:390-6.
5. Dinis-Ribeiro M, da Costa-Pereira A, Lopes C, et al. Magnification chromoendoscopy for the diagnosis of gastric intestinal metaplasia and dysplasia. Gastrointest Endosc 2003;57:498-504.
6. Areia M, Amaro P, Dinis-Ribeiro M, et al. External validation of a classification for methylene blue magnification chromoendoscopy in premalignant gastric lesions. Gastrointest Endosc 2008;67:1011-8.
7. Zhang JN, Li YQ, Zhao YA, et al. Classification of gastric pit patterns by confocal endomicroscopy. Gastrointest Endosc 2008;67:843-53.
8. Guo YT, Li YQ, Yu T, et al. Diagnosis of gastric intestinal metaplasia with confocal laser endomicroscopy in vivo:a prospective study. Endoscopy 2008;40:547-53.
9. Kiesslich R, Gossner L, Goetz M, et al. In vivo histology of Barrett's esophagus and associated neoplasia by confocal laser endomicroscopy. Clin Gastroenterol Hepatol 2006;4:979-87.
10. Dunbar KB, Okolo P,3rd, Montgomery E, et al. Confocal laser endomicroscopy in Barrett's esophagus and endoscopically inapparent Barrett's neoplasia:a prospective, randomized, double-blind, controlled, crossover trial. Gastrointest Endosc 2009;70:645-54.
11. Ming SC, Bajtai A, Correa P, et al. Gastric dysplasia. Significance and pathologic criteria. Cancer 1984;54:1794-801.
12. Goetz M, Ziebart A, Foersch S, et al. In vivo molecular imaging of colorectal cancer with confocal endomicroscopy by targeting epidermal growth factor receptor. Gastroenterology 2010; 138:435-46.
13. Foersch S, Kiesslich R, Waldner MJ, et al. Molecular imaging of VEGF in gastrointestinal cancer in vivo using confocal laser endomicroscopy. Gut 2010; 59:1046-55.
14. Hsiung PL, Hardy J, Friedland S, et al. Detection of colonic dysplasia in vivo using a targeted heptapeptide and confocal microendoscopy. Nat Med 2008; 14: 454-8.
1. Winawer S, Fletcher R, Rex D, et al. Colorectal cancer screening and surveillance: clinical guidelines and rationale-Update based on new evidence. Gastroenterology 2003;124:544-560
2. Wehrmann K, Fruhmorgen P. [Removing adenomas reduces colon carcinoma risk up to 90%. Effective cancer prevention with the endoscope]. MMW Fortschritte der Medizin 2000;142:26-29
3. Togashi K, Konishi F, Ishizuka T, et al. Efficacy of magnifying endoscopy in the differential diagnosis of neoplastic and non-neoplastic polyps of the large bowel. Diseases of the colon and rectum 1999;42:1602-1608
4. Kiesslich R, Goetz M, Lammersdorf K, et al. Chromoscopy-guided endomicroscopy increases the diagnostic yield of intraepithelial neoplasia in ulcerative colitis. Gastroenterology 2007;132:874-882
5. Kiesslich R, Gossner L, Goetz M, et al. In vivo histology of Barrett's esophagus and associated neoplasia by confocal laser endomicroscopy. Clin Gastroenterol Hepatol 2006;4:979-987
6. Sanduleanu S, Driessen A, Hameeteman W, et al. Inflammatory cloacogenic polyp:diagnostic features by confocal endomicroscopy. Gastrointestinal endoscopy 2009;69:595-598
7. Sanduleanu S, Driessen A, Gomez-Garcia E, et al. In Vivo Diagnosis and Classification of Colorectal Neoplasia by Chromoendoscopy-Guided Confocal Laser Endomicroscopy. Clin Gastroenterol Hepatol 2009
8. Kiesslich R, Burg J, Vieth M, et al. Confocal laser endoscopy for diagnosing intraepithelial neoplasias and colorectal cancer in vivo. Gastroenterology 2004;127:706-713
9. Kiesslich R, Goetz M, Angus EM, et al. Identification of epithelial gaps in human small and large intestine by confocal endomicroscopy. Gastroenterology 2007;133:1769-1778
10. Guo YT, Li YQ, Yu T, et al. Diagnosis of gastric intestinal metaplasia with confocal laser endomicroscopy in vivo:a prospective study. Endoscopy 2008;40:547-553
11. Roy HK, Gomes A, Turzhitsky V, et al. Spectroscopic microvascular blood detection from the endoscopically normal colonic mucosa:biomarker for neoplasia risk. Gastroenterology 2008;135:1069-1078
12. Hamilton SR, Aaltonen, L.A. Pathology and Genetics. Tumours of the Digestive System. WHO Classification of Tumours 2000;2:2
13. Levin B, Lieberman DA, McFarland B, et al. Screening and surveillance for the early detection of colorectal cancer and adenomatous polyps,2008:a joint guideline from the American Cancer Society, the US Multi-Society Task Force on Colorectal Cancer, and the American College of Radiology. Gastroenterology 2008;134:1570-1595
14. Kiesslich R, von Bergh M, Hahn M, Hermann G, Jung M. Chromoendoscopy with indigocarmine improves the detection of adenomatous and nonadenomatous lesions in the colon. Endoscopy 2001;33:1001-1006
15. Eisen GM, Kim CY, Fleischer DE, et al. High-resolution chromoendoscopy for classifying colonic polyps:a multicenter study. Gastrointestinal endoscopy 2002;55:687-694
16. van den Broek FJ, Reitsma JB, Curvers WL, Fockens P, Dekker E. Systematic review of narrow-band imaging for the detection and differentiation of neoplastie and nonneoplastic lesions in the colon (with videos). Gastrointestinal endoscopy 2009;69:124-135
17. Hurlstone DP, Thomson M, Brown S, et al. Confocal endomicroscopy in ulcerative colitis:differentiating dysplasia-associated lesional mass and adenoma-like mass. Clin Gastroenterol Hepatol 2007;5:1235-1241
18. Wang HW, Willis J, Canto MI, Sivak MV, Jr., Izatt JA. Quantitative laser scanning confocal autofluorescence microscopy of normal, premalignant, and malignant colonic tissues. IEEE transactions on bio-medical engineering 1999;46:1246-1252
19. Buchner AM, Shahid MW, Heckman MG, et al. Comparison of Probe-Based Confocal Laser Endomicroscopy with Virtual Chromoendoscopy for Classification of Colon Polyps. Gastroenterology 2009
20. Goetz M, Toermer T, Vieth M, et al. Simultaneous confocal laser endomicroscopy and chromoendoscopy with topical cresyl violet. Gastrointestinal endoscopy 2009;70:959-968
21. Snover DC, Jass JR, Fenoglio-Preiser C, Batts KP. Serrated polyps of the large intestine:a morphologic and molecular review of an evolving concept. American journal of clinical pathology 2005;124:380-391
22. Goetz M, Ziebart A, Foersch S, et al. In vivo molecular imaging of colorectal cancer with confocal endomicroscopy by targeting epidermal growth factor receptor. Gastroenterology 2010; 138:435-446
1. Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics,2002. CA Cancer J Clin 2005; 55:74-108.
2. Sakai Y, Eto R, Kasanuki J, et al. Chromoendoscopy with indigo carmine dye added to acetic acid in the diagnosis of gastric neoplasia:a prospective comparative study. Gastrointest Endosc 2008; 68:635-41.
3. Yao K, Anagnostopoulos GK, Ragunath K. Magnifying endoscopy for diagnosing and delineating early gastric cancer. Endoscopy 2009; 41:462-7.
4. Ohashi A, Niwa Y, Ohmiya N, et al. Quantitative analysis of the microvascular architecture observed on magnification endoscopy in cancerous and benign gastric lesions. Endoscopy 2005; 37:1215-9.
5. Otsuka Y, Niwa Y, Ohmiya N, et al. Usefulness of magnifying endoscopy in the diagnosis of early gastric cancer. Endoscopy 2004; 36:165-9.
6. Kaise M, Kato M, Urashima M, et al. Magnifying endoscopy combined with narrow-band imaging for differential diagnosis of superficial depressed gastric lesions. Endoscopy 2009; 41:310-5.
7. Kakeji Y, Yamaguchi S, Yoshida D, et al. Development and assessment of morphologic criteria for diagnosing gastric cancer using confocal endomicroscopy: an ex vivo and in vivo study. Endoscopy 2006; 38:886-90.
8. Kitabatake S, Niwa Y, Miyahara R, et al. Confocal endomicroscopy for the diagnosis of gastric cancer in vivo. Endoscopy 2006; 38):1110-4.
9. Li WB, Zuo XL, Li CQ, et al. Diagnostic value of confocal laser endomicroscopy for gastric superficial cancerous lesions. Gut 2011; 60:299-306.
10. Goetz M, Ziebart A, Foersch S, et al. In vivo molecular imaging of colorectal cancer with confocal endomicroscopy by targeting epidermal growth factor receptor. Gastroenterology 2010; 138:435-46.
11. Foersch S, Kiesslich R, Waldner MJ, et al. Molecular imaging of VEGF in gastrointestinal cancer in vivo using confocal laser endomicroscopy. Gut 2010; 59: 1046-55.
12. Hsiung PL, Hardy J, Friedland S, et al. Detection of colonic dysplasia in vivo using a targeted heptapeptide and confocal microendoscopy. Nat Med 2008; 14: 454-8.
13. Keller R, Winde G, Eisenhawer C, et al. Immunoscopy--a technique combining endoscopy and immunofluorescence for diagnosis of colorectal carcinoma. Gastrointest Endosc 1998; 47:154-61.
14. Keller R, Winde G, Terpe HJ, Foerster EC, Domschke W. Fluorescence endoscopy using a fluorescein-labeled monoclonal antibody against carcinoembryonic antigen in patients with colorectal carcinoma and adenoma. Endoscopy 2002; 34:801-7.
15. Folli S, Wagnieres G, Pelegrin A, et al. Immunophotodiagnosis of colon carcinomas in patients injected with fluoresceinated chimeric antibodies against carcinoembryonic antigen. Proc Natl Acad Sci U S A 1992; 89:7973-7.
16. Takenoshita S, Hashizume T, Asao T, et al. Immunoscintigraphy using 99mTc-labeled anti-CEA monoclonal antibody for patients with colorectal cancer. Anticancer Res 1995; 15:471-5.
17. Marten K, Bremer C, Khazaie K, et al. Detection of dysplastic intestinal adenomas using enzyme-sensing molecular beacons in mice. Gastroenterology 2002; 122: 406-14.
18. Zhang H, Morgan D, Cecil G, et al. Biochromoendoscopy:molecular imaging with capsule endoscopy for detection of adenomas of the GI tract. Gastrointest Endosc 2008; 68:520-7.
19. Fan DM, Zhang XY, Chen XT, et al. Establishment of four monoclonal antibodies to a poorly differentiated gastric cancer cell line MKN-46-9 and immunohistochemical study on their corresponding antigens. Chin J Med PLA 1988; 13:12-5.
20. Jin B, Wang X, Jin Y, et al. Detection of serum gastric cancer-associated MG7-Ag from gastric cancer patients using a sensitive and convenient ELISA method. Cancer Invest 2009; 27:227-33.
21.Guo DL, Dong M, Wang L, Sun LP, Yuan Y. Expression of gastric cancer-associated MG7 antigen in gastric cancer, precancerous lesions and H. pylori -associated gastric diseases. World J Gastroenterol 2002; 8:1009-13.
22. Zhang X, Hong L, Chan WY, et al. Expression of MG7-Ag in patients with gastric cancer correlates with weaker T cell immune response and more proinflammatory cytokine secretion. Biochem Cell Biol 2006; 84:135-41.
23. Liu J, Hu JL, Zhang XY, et al. The value of MG7 antigen in predicting cancerous change in dysplastic gastric mucosa. Int J Clin Pract 2002; 56:169-72.
24. Kiesslich R, Goetz M, Vieth M, Galle PR, Neurath MR Confocal laser endomicroscopy. Gastrointestinal endoscopy clinics of North America 2005; 15: 715-31.
25. Chang CC, Chen SH, Lin CP, et al. Premedication with pronase or N-acetylcysteine improves visibility during gastroendoscopy:an endoscopist-blinded, prospective, randomized study. World J Gastroenterol 2007; 13:444-7.
26. Fleiss JL, Cohen J. The equivalence of weighted kappa and the intraclass correlation coefficient as measures of reliability. Educ Psychol Meas 1973; 33: 613-19.
27. Conte WL, Kamishina H, Reep RL. Multiple neuroanatomical tract-tracing using fluorescent Alexa Fluor conjugates of cholera toxin subunit B in rats. Nat Protoc 2009; 4:1157-66.
28. Goetz M, Kiesslich R. Advances of endomicroscopy for gastrointestinal physiology and diseases. Am J Physiol Gastrointest Liver Physiol 2010; 298: G797-806.
29. Tong Q, Liu K, Lu XM, Shu XG, Wang GB. Construction and characterization of a novel fusion protein MG7-scFv/SEB against gastric cancer. J Biomed Biotechnol 2010; 2010:121094.
30. Hoffman RM. Orthotopic metastatic mouse models for anticancer drug discovery and evaluation:a bridge to the clinic. Invest New Drugs 1999; 17:343-59.