【作者】 郭玉婷；

【导师】 李延青；

【作者基本信息】 山东大学 ， 内科学， 2008， 博士

【摘要】 研究背景和目的胃癌是世界范围内死亡率高居第二位的肿瘤性疾病。胃粘膜的肠上皮化生(gastric intestinal metaplasia,GIM)是肠型胃癌的危险因素,被认为是肠型胃癌的癌前期病变。若能及早通过内镜下识别和治疗使其病变逆转,不失为防治胃癌的有效途径。由于GIM的多灶性分布,在普通内镜及其他内镜下表现缺乏特异性,胃镜下诊断与组织学诊断的一致性较差,目前尚无明确定义的内镜下诊断标准,仍需依靠活组织病理学诊断才能最终确诊。共聚焦激光显微内镜(confocal laser endomicroscopy,CLE)是最新开发出来的内镜,是在传统的可视内镜的顶端安装了共聚焦激光显微镜,是共聚焦激光显微镜和传统电子内镜的有机结合体,除作标准电子内镜检查外,还能进行共聚焦显微镜检查,模拟体内表面下结构的组织学图像,在内镜检查的同时获得消化道上皮及上皮下高度放大的横切面的图像,对粘膜和粘膜下层做即时的高分辨率的组织学诊断,达到光学活检的目的。与其他光学技术相比,CLE的优势在于它不仅使上皮表面成像,而且可使上皮下成像。这种放大1000倍的图像可清晰辨认组织结构、细胞及亚细胞结构,做即时的高分辨率的组织学诊断。本研究拟在应用CLE识别正常胃粘膜及GIM时的各种细胞及亚细胞结构,制定GIM共聚焦图像下的诊断、分型和分级标准,并以病理诊断作为金标准评价其诊断效率,最后应用CLE诊断GIM的范围和分布,全面评估CLE对GIM的诊断价值。材料和方法1共聚焦激光显微内镜对肠上皮化生细胞及亚细胞结构的识别采用日本Pentax EC3870KCLE,荧光素钠和吖啶黄素为对比剂。选取28位已知GIM的患者,分别对患者食道、胃及十二指肠粘膜进行共聚焦激光扫描。在正常食道可见食管鳞状上皮细胞,应用吖啶黄素后可见清晰的细胞核。为确认鳞状上皮细胞及细胞核,同时进行了食管鳞状上皮细胞体外培养,吖啶黄素染色,再用CLE观察确定。正常胃粘膜可见正常的胃粘膜柱状上皮,肠化部位可见除可见杯状细胞外,可见细长而色泽明亮的柱状上皮细胞,并在柱状上皮细胞表面可见一条清晰而黑色的线。十二指肠粘膜可见细长而明亮的柱状上皮细胞及杯状细胞。观察后分别进行活检,进行常规病理组织学检查、HID/AB和AB/PAS组织化学染色、CD10免疫组化检查、透射电镜和扫描电镜检查。2共聚焦激光显微内镜下胃粘膜肠上皮化生诊断标准的制定及评价第一阶段选取已知GIM的病人用CLE检查,确定在CLE下GIM的诊断标准。第二阶段再选取慢性胃炎或怀疑有GIM的患者进行CLE检查,首先普通内镜观察,判断是否存在GIM,然后用CLE扫描观察,根据CLE标准进行诊断,病理诊断作为金标准评估其诊断效率。3共聚焦激光显微内镜对胃粘膜肠上皮化生分级和分型的诊断第一阶段选取已知GIM的病人用CLE检查,确定在CLE下GIM的分型和分级诊断标准。第二阶段再选取怀疑有GIM患者进行CLE检查,根据CLE诊断标准诊断其分型和分级,组织病理诊断为金标准,评估其诊断效率,并对几种胃癌相关基因进行了检测。4共聚焦激光显微内镜对胃粘膜肠上皮化生范围和程度的诊断选取慢性胃炎和可疑胃癌伴有GIM患者,应用CLE扫描观察每位患者胃粘膜的11个部位,即时诊断是否有GIM,并对阳性部位活检。根据GIM的阳性部位数,分为局灶性、多灶性和广泛性GIM;根据GIM的分布分为局灶型、胃窦型、小弯型和弥漫型。评价不同范围和分布方式的GIM与胃癌及其他癌前病变的关系。结果1分别对28位已知GIM的患者的124个部位进行共聚焦扫描,获得5750幅图像。共聚焦显微内镜可以容易识别在体的食管正常鳞状上皮细胞及其细胞核,与体外培养鳞状上皮细胞吖啶黄素染色后观察一致,与病理检查一致。胃内正常的胃粘膜柱状上皮在共聚焦激光显微内镜图像上为典型的铺路石样改变。杯状细胞表现为大而黑色的细胞,散布于柱状排列的上皮细胞之间,有特异的形态学特征,易于识别。肠化部位及十二指肠出现的细长色泽明亮的细胞为吸收上皮细胞,HID-AB和AB-PAS粘液染色中不着色,与杯状细胞和正常胃柱状吸收上皮细胞明显不同,细胞表面清晰而黑色线CD10免疫组化染色为阳性,在扫描电镜及透射电镜可证实为吸收上皮细胞及刷状缘。2 GIM在CLE下的诊断标准是,如果在共聚焦图像下出现以下三个特征之一即可诊断为该部位存在GIM:1)存在杯状细胞,表现为大而黑色的细胞;2)存在柱状吸收细胞和刷状缘,特征是比胃粘膜柱状上皮细胞细长而色泽明亮的细胞,刷状缘表现为上皮细胞的表面一条清晰而黑色的线;3)胃小凹呈绒毛状改变。在第二阶段的研究中,共有53位病人接受CLE检查,扫描了267个部位。其中36位病人的160个部位被组织病理诊断为GIM,普通内镜及CLE对GIM的诊断敏感性分别是36.88%和98.13%,特异性分别是91.59%和95.33%,阳性预测值分别是86.76%和96.91%,隐性预测值分别是49.25%和97.14%,普通内镜及CLE与病理诊断的一致性检验中,K值分别是0.25和0.94。3 GIM在CLE下,根据杯状细胞的形态、是否存在吸收上皮细胞或刷状缘、胃小凹和固有层血管形态进一步被分为完全型和不完全型。根据肠化面积的多少分为轻中重度GIM。共有53位病人接受CLE检查,扫描了267个部位。在CLE诊断的98处完全型GIM中,有83处得到组织学的证实,CLE对完全型GIM诊断的敏感性、特异性、阳性预测值和阴性预测值分别是68.03%、89.66%、84.69%和76.92%;在在CLE诊断的64处不完全型GIM中,有26处得到组织学的证实,CLE对不完全型GIM诊断的敏感性、特异性、阳性预测值和阴性预测值分别是68.42%、83.41%、40.63%和94.09%。CLE与病理分型诊断的κ值是0.67。在可以进行GIM分级诊断的146个部位中,88个部位诊断为轻度GIM,其中的74个部位得到病理的证实;33个部位被CLE诊断为中度,其中的25个与病理诊断一致;25个被CLE诊断为重度的部位中,20个也得到病理诊断的证实。共聚焦激光显微内镜对轻度GIM诊断的敏感性和特异性分别是90.2%和78.1%,对中度GIM诊断的敏感性和特异性分别是69.4%和92.7%,对重度GIM诊断的敏感性和特异性分别是71.4%和95.8%。CLE与病理进行GIM轻、中、重度分级诊断的K值分别是0.69、0.64和0.70,具有较好的一致性。胃癌相关基因CDX2、Ki67及APC在不同亚型及不同严重程度GIM中有不同的表达。4共70位确诊为慢性胃炎和胃癌伴有GIM患者入选本研究,47.1%的患者GIM范围为局灶性,41.4%为多灶性,11.4%为广泛性。GIM的范围与胃癌及癌前病变有密切关系,多发性和广泛性GIM伴有更多的胃粘膜萎缩,广泛性GIM更多提示了不典型增生和胃癌。37.1%患者GIM为局灶型分布,21.4%为胃窦型,31.4%为小弯型,10.0%为弥漫型分布。GIM的分布与胃癌及癌前病变有密切关系,小弯型和弥漫型分布伴有更多的不典型增生和胃癌。结论CLE是一种新开发的诊断工具,可以在内镜检查的同时提供即时可靠的组织学诊断。它可以准确识别正常及GIM时的多种细胞及亚细胞结构,可以非常准确地诊断GIM,并可进行分型、分级诊断,也可以判断GIM的范围和分布方式。意义CLE是一种新开发的诊断工具,可以在内镜检查的同时提供即时可靠的组织学诊断,CLE可以非常准确地诊断GIM及分型、分级,判断其范围和分布。GIM是肠型胃癌的危险因素,但多数GIM是癌前状态,而非癌前病变。本研究评价了CLE在筛选和检测GIM这一癌前病变中的作用,可以为患者提供这一病变发展成为胃癌的危险性和随防计划。CLE是光学活检领域内革命性的创新技术,这种全新的诊断工具,观察到的图像毫无疑问使内镜检查进入了一个新的时代,标志着内镜检查从表层走向深层,从形态学迈向组织学的质变。可以预测,CLE将在胃肠内镜方面发挥非常重要的诊断作用。更多还原

【Abstract】 Background and aimsGastric cancer is the second leading cause of cancer related mortality worldwide. Gastric intestinal metaplasia(GIM)is a risk factor that leads to the development of intestinal-type gastric cancer and is generally regarded as a precancerous condition.IfGIM was identified under endoscope earlier,effective strategies could be developed to detect the early,curable phase of gastric cancer and prevent its progression.GIM is multifocal and is mostly indistinguishable by the conventional endoscopy or even by other new endoscopic techniques.Conventional endoscopic identification of intestinal metaplasia has a high rate of interobserver variability and correlates poorly with the histological finding.None of them can distinguish the structure of individual cells,or allow the endoscopic criteria of GIM to be defined. Histologic analysis of biopsy material remains the gold standard for the final diagnosis of GIM.Recently,confocal laser endomicroscopy(CLE)has been developed which is integration of a confocal laser microscope in the distal tip of a conventional videoendoscope.The components enable confocal microscopy in addition to standard videoendoscopy.The new device can provide real-time,high magnification, cross-sectional images of the gastrointestinal epithelium during routine endoscopy without the need for biopsy and thus histopathology has been termed optical biopsy. Compared with other new optics techniques,the greatest advantage of the CLE is that it can enables surface and subsurface imaging of living cells in the mucosa during ongoing colonoscopy.The confocal images that approximately 1000-fold magnification readily permits single cells in the gastrointestinal tract to be resolved.The aim of this study were to determine if CLE could identify cells and subcellular structures of normal and intestinal metaplasia mucosa,definite the confocal criteria of GIM,definite the criteria of its grading and subtype,and evaluate the efficacy of CLE for the extent and topographic patterns of GIM assessment in vivo.Methods1 Identification of cells and subcellular structures in gastric intestinal metaplasia by confocal laser endomicroscopyPatients with known GIM underwent CLE(Pentax EC-3870K;Pentax,Tokyo, Japan).Fluorescein sodium and acriflavine hydrochloride was used as contrast agent. Esophagus,stomach and duodenum were examined with the CLE system.In normal esophagus,squamous epithelial cell showed rhombus single cells at high resolution with clear visible borders.The nucleus can be stained clearly with acriflavine hydrochloride.Furthermore,squamous epithelial cells were cultured and observed with CLE in vitro for identification.Gastric type columnar epithelial cells and mucin-containing goblet cells can easily recognized in stomach under CLE images.In some areas,a more slender,and brighter than columnar epithelial cells of normal gastric mucosa can be seen with a clear dark line at the surface of the epithelium. These cells and structures also are seen in duodenum.The histologic specimens from each site were compared with the targeted confocal images.All of the biopsy specimens were sectioned vertically and transversely to facilitate the comparison between histology and confocal images.All the biopsy specimens were stained with hematoxylin and eosin.Biopsy specimens diagnosed as GIM and duodenum were further stained by AB-PAS mucin staining,HID-AB mucin staining and CD10 immunohistochemistry.These slender and brighter cells and the clear dark line at the surface of the epithelium were identified with scanning electron microscope(SEM) and transmission electron microscope(TEM). 2 Definition and evaluation of gastric intestinal metaplasia with confocal laser endomicroscopy in VivoIn first phase,28 patients with known GIM underwent CLE,and CLE criteria for diagnosis of GIM were developed.In addition,53 consecutive patients with known or suspected GIM were prospectively evaluated in second phase.Standard and abnormal appearance areas were examined with the CLE system.Fluorescein was used as contrast agent.Afterwards,a targeted biopsy was done at the same sites.The results of histoathological biopsy specimens taken from the corresponding sites of gastric mucosa under CLE were regarded as gold standard.3 Diagnostic value of confocal laser endomicroscopy for the prediction of the subtype and grading of gastric intestinal metaplasiaIn first phase,28 patients with known GIM underwent CLE,and CLE criteria for classifying and grading GIM were developed.In addition,53 consecutive patients with known or suspected GIM were prospectively evaluated in second phase. Standard and abnormal appearance areas were examined with the CLE system. Fluorescein was used as contrast agent.Afterwards,a targeted biopsy was done at the same sites.The results of histoathological biopsy specimens taken from the corresponding sites of gastric mucosa under CLE were regarded as gold standard.The expression of three gastric carcinoma related gene were detected in different subtype and severity GIM.4 Diagnostic value of confocal laser endomicroscopy for the prediction of the extent and topographic patterns of gastric intestinal metaplasiaSeventy patients with known GIM underwent CLE.Fluorescein was used as contrast agent.Endoscopic gastric biopsy specimens were obtained using a jumbo forceps from the 11 gastric sites.Five specimens were from the antrum,six from the corpus and one from the incisura angularis.The presence of GIM was made immediately by the endoscopist at the time of the procedure.The extent of GIM was categoried focal,multifocal and extensive GIM.Four topographical patterns of intestinalization emerged:"Focal,","Antrum-predominant,Magenstraβe" and "Diffuse,".The histological evaluation remains the gold standard for the final diagnosis of intestinal metaplasia.Results1 All of 28 patients under went CLE with known GIM.A total of 5750 CLE images were obtained from 124 areas under CLE images.The confocal images showed the normal squamous epithelial cells their nucleus on the surface of the esophagus at high resolution in vivo.These patterns can be directly compared with cultured cells in vitro and H&E-stained sections of biopsy specimens cut parallel to the tissue surface.At the surface of the stomach,epithelium a typical cobblestone structure.The mucin-containing goblet cells showed very dark and big within the columnar-lined epithelium.Goblet cells had specific appearance and easily recognized.The more slender and brighter than columnar epithelial cells of normal gastric mucosa were identified as absorptive columnar epithelial cells.These cells were colorless by AB/PAS and HID/AB mucin staining,difference from goblet cells (blue)and gastric epithilum(purple).The clear dark line at the surface of the epithelium were identified as brush border by CD 10 immunohistochemistry,SEM and TEM.2 GIM was identified if any of the following three features were present in an image field:goblet cells,columnar absorptive cells and brush border,and villiform foveolar epithelium.In a prospective study,a total 267 sites from 53 patients were obtained.160 from 36 patients were diagnosed histopathologically as GIM.The sensitivities of conventional endoscopy and CLE for GIM were 36.88%vs.98.13%, the specificities were 91.59%vs.95.33%,the positive predictive value were 86.76% vs.96.91%,and the negative predictive value were 49.25%vs.97.14%,respectively. The kappa value for the correlation with histological findings was 0.25 for conventional endoscopy vs.0.94 for CLE.3 In the CLE images,GIM was classified as complete or incomplete,according to the shape of goblet cells,the presence of absorptive cells or brush border,and the architecture of vessels and crypts.In a prospective study,a total 267 sites from 53 patients were obtained.Among the 98 sites with complete GIM according to CLE, this was confirmed in 83 by histopathology.The sensitivity,specificity,positive predictive value and negative predictive value of CLE for the diagnosis of complete GIM were 68.03%,89.66%,84.69%and 76.92%,respectively.Among the 64 sites with incomplete GIM according to CLE,this was confirmed histologically in 26.The sensitivity,specificity,positive predictive value and negative predictive value of CLE for the diagnosis of incomplete GIM were 68.42%,83.41%,40.63%and 94.09%, respectively.The kappa score for the agreement between CLE and histopathology was 0.67.Among 146 GIM positive areas,88 were identified as mild GIM by CLE,in which 74 confirmed by histopathology.Thirty-thee areas were diagnosed as moderate GIM by CLE,25 of them were confirmed by histopathology.Twenty-eight areas were diagnosed as marked GIM by CLE,20 of them were confirmed by histopathology. The sensitivity and specificity of CLE were 90.2%and 78.1%for the diagnosis of mild GIM,69.4%and 92.2%for morderate GIM,71.4%and 95.8%for marked GIM,respectively.The kappa coefficient of CLE criteria and the histopathological grading for mild,moderate and marked IM were 0.69,0.64 and 0.70,respectively. There were differences for the expression of CDX2,Ki67 and APC among different subtype and severity GIM.4 A total of 70 gastric carcinoma and chronic atrophy gastritis patients with GIM were recruited.47.1%of them were focal GIM,41.4%were multifocal GIM and 11.4%were extensive GIM.The extent of GIM is associated with the gastric cancer and associated with lesion precancerous.Multifocal GIM and ententive GIM was significantly associated with the presence of cancer and gastric atrophy.Extensive GIM was significantly associated with the presence of cancer and dysplasia.Of the entire study population of 70 subjects,37.1%of them were focal pattern,21.4% were antrum-predominant pattern,31.4%of them were Magenstraβe pattern,10.0% of them were diffuse pattern.The topographic patterns of GIM is associated with gastric cancer and lesion precancerous.MagenstraBe and diffuse topographic patterns was significantly associated with the presence of cancer and dysplasia.Conclusion:Confocal laser endomicroscopy is a newly developed diagnostic tool and may offer an instant and reliable diagnostic tool for in vivo histology.CLE can identify cells and subcellular structures in normal gastric mucosa and GIM at high resolution in vivo,enable classification,grading,extent and topographic patterns of GIM with high accuracy during ongoing endoscopy.SignificanceConfocal laser endomicroscopy is a newly developed diagnostic tool and may offer an instant and reliable diagnostic tool for in vivo histology.CLE can diagnose GIM with high accuracy during ongoing endoscopy,as well as its grading and subtype. The extent and topographic patterns of GIM also be evaluated.GIM is a risk factor that leads to the development of intestinal-type gastric cancer and is generally regarded as a precancerous condition.Most GIMs are only "precancerous conditions" rather than "precancerous lesions".This present study will help determine the role of CLE in screening and surveillance of premalignant conditions.It could serve as a reasonably good predictor of cancer risk and provide appropriate follow-up in an individual patient.Endoscopic confocal imaging systems are revolutionary instruments in the emerging realm of optical biopsy techniques.The new detailed images seen with CLE unequivocally are the beginning of a new era.It is tempting to speculate that CLE will play an important diagnostic role in the future during gastrointestinal endoscopy.更多还原