共聚焦显微内镜在下消化道病变分子成像和功能成像中的应用研究
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摘要
研究背景和目的
结直肠癌(colorectal cancer, CRC)是目前消化道最常见的恶性肿瘤之一。迄今为止,公认的结直肠癌发病机制为“腺瘤—癌”顺序模式。对结直肠癌及其癌前病变的早期发现、早期诊断和鉴别诊断能够显著提高患者的生存率。炎症性肠病(inflammatory bowel disease, IBD)是一组病因尚未完全清楚肠道免疫系统调节紊乱的非特异性慢性肠道炎症性疾病。虽然其发展成为结直肠癌的概率比较低,但炎症性肠病癌变长期以来却被认为是其最严重的并发症。对疾病的早期评估和干预是成功的预防策略之一,能够显著改善患者的预后。
迄今为止,内镜检查和活检诊断仍是许多下消化道疾病包括结直肠瘤变和炎症性肠病的诊断金标准。共聚焦激光显微内镜(confocal laser endomicroscopy,CLE)的研发为下消化道疾病的早期诊断提供了强有力的技术支持。共聚焦显微内镜是一种新的显微内镜成像技术,可使图像放大1000倍,实现了在体内实时显示胃肠道组织、细胞和亚细胞结构,能够准确鉴别正常、增生或瘤变的黏膜,被誉为“光学活检”,使在体研究胃肠道生理、病理分子机制成为可能。近年来,共聚焦显微内镜分子成像及功能成像方面的研究正逐渐成为研究热点。
分子成像能够检测肿瘤细胞的特征性分子表达,显微内镜分子成像实现了分子成像从体外至体内的重大突破,它结合了分子探针和显微内镜技术,实现在体实时检测肿瘤分子的表达水平。表皮生长因子受体(epidermal growth factor receptor, EGFR)是目前研究较为深入的肿瘤血管新生相关分子标记物。目前已经有利用共聚焦内镜在体对人结直肠癌动物菏瘤鼠肿瘤组织EGFR分子成像的相关报道,首次实现了结直肠癌的EGFR体内分子成像,该研究还证实了共聚焦激光显微内镜分子成像技术可体内实时鉴别不同类别种植瘤的EGFR表达水平差异。此外,作者还通过体外对人新鲜结直肠癌组织标本表面喷洒荧光标记的EGFR抗体探针的方式,利用FIVE1小型共聚焦探针行EGFR分子成像,结果表明共聚焦系统可检测肿瘤组织和非肿瘤组织的EGFR表达差异。该实验为EGFR分子成像由动物实验向人体应用过渡提供了理论依据。
依据形态学和功能学诊断标准,显微内镜功能成像能够对细胞、组织的病理生理动态过程进行在体实时成像,如细胞脱落、细胞迁移、坏死和凋亡及组织的血流灌注及其改变。上皮细胞的不断更新(细胞脱落,gaps)是一个可能导致胃肠道上皮屏障破坏的潜在因素。研究表明在炎症环境下,上皮增生更新加快,上皮屏障功能破坏的危险性则变得复杂化。Kiesslich等人的研究显示,动物腹腔注射TNF-α诱导小肠炎症时有20%的gaps存在通透性改变。一项研究利用CLE观察IBD患者及对照组静脉注射荧光素钠后gaps的密度,发现IBD患者回肠末端黏膜上皮细胞gaps的密度明显高于正常对照组。还有研究显示,IBD患者回肠末端gaps密度升高能够预测疾病的复发。既往的研究发现,益生菌能够通过多种机制通过促进紧密连接蛋白成熟等多种方式保护紧密连接蛋白,继而降低肠道上皮通透性、维持肠道上皮的屏障功能；此外,研究证实,多种益生菌及其蛋白成分能有效的减少炎性因子诱导或坏死性小肠结肠炎的上皮细胞凋亡。因此,我们推测益生菌可能能够修复炎症性肠病回肠末端黏膜上皮细胞gaps。然而,迄今为止,尚无在体观察益生菌对肠道上皮gaps的影响及其机制的研究报道。
本研究拟围绕共聚焦激光显微内镜在下消化道病变分子成像和功能成像中的应用展开研究,研究目的包括：1.评估共聚焦显微内镜在体对结直肠癌和腺瘤患者行EGFR分子成像的价值,并比较在体共聚焦显微内镜分子成像技术和体外活检组织免疫组化技术在评价EGFR表达水平上的价值；2.利用共聚焦显微内镜对炎症性肠病患者回肠末端上皮细胞脱落(gaps)行功能成像并检测其密度,探索益生菌对上皮gaps的修复作用及其可能的机制。
研究方法：
第一部分：共聚焦显微内镜对结直肠瘤变在体分子成像的研究
自2011年1月至2011年6月,于我院就诊的40例已知结直肠瘤变的连续门诊或住院患者入组研究,所有的患者均依据术前的结肠镜检查安排了外科手术或内镜下切除手术。研究分为两个阶段：
第一阶段,先期研究,为了探索合适的分子探针浓度和孵育时间,共聚焦显微结肠镜(Pentax EC-3870K)白光模式下,用喷洒管对3名已知结直肠癌的患者肿瘤组织表面喷洒5ml Alexa Fluor488绿色荧光标记的EGFR抗体探针(探针浓度分别为1：50,1：100和1：500),10min及15min后开启共聚焦显微内镜模式行分子成像,随后于分子成像部位行靶向活检。结果显示,分子探针浓度在1：50、孵育时间在10min时即可达到较好的分子成像质量。
第二阶段,前瞻性研究,37例已知结直肠瘤变患者入组研究,充分冲洗组织表面后,在体对患者肿瘤组织或腺瘤组织表面喷洒1：50浓度的EGFR抗体探针,10min后利用共聚焦显微结肠镜行在体EGFR分子成像。此外,对10例结直肠瘤变患者,我们除对瘤变组织行分子成像外,同时对邻近的正常黏膜组织进行了EGFR在体分子成像。所有在体分子成像的部位均行靶向活检行体外EGFR免疫组织化学检查和HE病理检查。共聚焦在体分子成像的图像均予以事后分析,每位患者选取荧光强度最强的1张图片,选取该图片3个60*601μm荧光强度最强的感兴趣区域(region of interest, ROI),用ImageJ软件进行灰度值分析。名有经验的消化病理医师按维也纳分型对病理结果做出盲法诊断。此外,4名参与研究的患者于内镜结束后的4-6周后采取外周血检测血人抗小鼠抗体变化。
第二部分：共聚焦显微内镜对炎症性肠病回肠gaps功能成像的研究
研究分为两个步骤,分别为临床研究和动物研究。
第一部分,临床研究,纳入已知或怀疑炎症性肠病来我院就诊的门诊患者,对照组为健康查体者及息肉切除术后复查患者。在充分的肠道准备后在静脉全麻下对所有入组患者行共聚焦显微结肠镜(Pentax EC-3870K)检查,所有患者在进镜至回肠末端,静脉注射6ml荧光素钠,开启共聚焦模式对至少5个白光模式下正常的黏膜部位上皮细胞gaps进行观察并采集图像。事后采用盲法对采集的图像进行分析,计数患者回肠末端gaps密度。
第二部分,动物研究,动物模型分为TNBS造模IBD大鼠组、益生菌VSL#3治疗IBD大鼠组和正常对照大鼠组三组,利用小型动物活体共聚焦成像系统FIVE1(Optiscan公司)在体观察三组动物模型回肠末端上皮gaps密度(每1000个上皮细胞中gaps的数目),对大鼠回肠末端黏膜活检组织行免疫组化检测紧密连接蛋白Occludin和ZO-1的表达量,利用酶联免疫吸附试验(ELISA)检测黏膜炎症因子IL-1β表达,研究益生菌治疗对IBD大鼠回肠末端gaps的修复作用。此外,我们还对大鼠腹腔注射不同剂量的IL-1β后观察大鼠回肠末端上皮gaps密度的变化,探讨炎症因子IL-1β对上皮细胞gaps的影响。
研究结果：
第一部分：共聚焦显微内镜对结直肠瘤变在体分子成像的研究
前瞻性研究共纳入37例结直肠瘤变患者(21例男性,16例女性),表面喷洒AF488标记EGFR抗体后利用共聚焦内镜行分子成像显示,18例结直肠癌(18/19,94.7%)和12例结直肠腺瘤(12/18,66.7%)可检测到EGFR特异性荧光信号。18例EGFR分子成像阳性的结直肠癌组织中包括8例高分化(8/8)、10例中分化(10/11)和0例低分化(0/0)。12例分子成像阳性的结直肠腺瘤组织中包括6例高级别上皮内瘤变(6/10)和6例低级别上皮内瘤变(6/8)。对共聚焦图像灰度值分析显示,结直肠癌EGFR分子成像平均灰度值为52.84±5.73,结直肠腺瘤为44.31±4.86(p=0.329)。而对正常结肠黏膜行EGFR分子成像显示,仅有3例呈弱阳性,其余7例均为阴性。10例正常黏膜分子成像的平均灰度值为36.23±7.90,对应的10例结直肠瘤变组织平均灰度值为53.78±7.06(p<0.001)。体外组织EGFR免疫组化和体内EGFR分子成像的一致性的Kappa值较高,为0.788。4-6周后采集的4例血清样本显示,所有样本HAMA滴度值均未有明显升高。
第二部分：共聚焦显微内镜对炎症性肠病回肠gaps功能成像的研究
共有43例患者入组临床研究,其中,19例溃疡性结肠炎患者,4例克罗恩病患者与20例正常对照患者。与正常对照组(1.50gaps/1000细胞)相比,IBD患者的回肠末端上皮细胞gaps密度显著增加(38.00gaps/1000细胞,p<0.001)。动物研究中,与正常对照组大鼠(14.17±4.17gaps/1000细胞)相比,IBD组大鼠回肠末端上皮gaps密度(47.83±7.57gaps/1000细胞)显著升高(p<0.001)。益生菌VSL#3治疗IBD组大鼠回肠末端gaps密度较IBD组大鼠显著降低(19.33±4.84gaps/1000细胞,p<0.001)。腹腔注射10或5μg/kg IL-1β后,大鼠回肠末端gaps密度均显著升高(205.00±23.48gaps/1000细胞和157.67±28.36gaps/1000细胞)。益生菌治疗IBD组大鼠回肠末端黏膜紧密连接蛋白Occludin和ZO-1表达升高,炎症因子IL-1β表达下降。
研究结论：
1.本研究首次证实了利用共聚焦显微内镜和荧光标记EGFR抗体分子探针可实现对结直肠瘤变患者的在EGFR分子成像。这种新型体内分子成像技术为结直肠癌的分子靶向治疗提供了理论依据。
2.共聚焦内镜可以对细胞脱落gaps行在体功能成像,共聚焦内镜观察下,IBD患者回肠末端gaps密度显著升高,在体对IBD动物模型行功能成像表明,益生菌可能通过降低炎症因子表达,提高紧密连接蛋白表达的方式修复IBD动物模型回肠末端黏膜gaps。
研究意义：
本研究第一次利用共聚焦显微内镜,通过表面喷洒荧光标记EGFR抗体探针的方式实现了对结直肠瘤变患者在体EGFR分子成像。共聚焦显微内镜能够检测出靶向EGFR分子的特异性细胞荧光信号。对成像部位行体外EGFR免疫组化检测与在体EGFR分子成像一致性较高。通过计算共聚焦分子成像灰度值的方式能够区分结直肠瘤变和正常结直肠黏膜。EGFR显微内镜分子成像的实现,将有助于早期发现结直肠癌,并对其行分型诊断和指导分子靶向治疗。此外,共聚焦显微内镜能够对IBD患者回肠末端gaps行功能成像,研究还首次证实了益生菌能够通过降低炎症因子和提高紧密连接蛋白的表达等多种机制修复上皮细胞gaps,这可能是益生菌治疗IBD的机制之一。
Background and aims:
Colorectal cancer (CRC) is one of the most common malignant tumors of gastrointestinal tract. The well-accepted pathogenetic framework for colorectal tumorigenesis is an adenoma-carcinoma sequence. Early detection, diagnosis and differential diagnosis of colorectal cancer and precancerous lesions could significantly increase the overall survival rate of patients. Inflammatory bowel disease (IBD) is a kind of nonspecific chronic bowel inflammatory disease with unclear pathogenesis and characterized by intestinal immune system disorder. Although the probability of IBD become colorectal cancer is low, colorectal neoplasia is recognized as the most severe complication of IBD. Early assessment and intervention for IBD is one of the successful prevention strategies, improving the prognosis of IBD patients.
To date, endoscopy and pathology are the gold standard of many lower gastrointestinal tract diease, including colorectal neoplasia and inflammatory bowel diease. With the advances in in optical instruments, e.g. confocal laser endomicroscopy (CLE), the early detection of lower gastrointestinal tract diease became possible. Confocal endomicroscopy is new endomicroscopic technique with a1000fold magnification, could observe the gastrointestinal tract in vivo at tissue, cellular and subcellular level, and differetiate normal, hyperplastic and neoplastic mucosa, which is named as "optical biopsy". With CLE, we can investigate the physiological and pathological mechanism gastrointestinal trat in vivo. Molecular imaging and functional imaging using CLE are rapidly advanced subjects of gastrointestinal endoscopy.
The specific molecular expression of tumor cells can be detected by molecular imaging. Molecular imaging using CLE realized the breakthrough of molecular imaging from ex vivo to in vivo, combining molecular probe and endomicroscopic technique, detecting the molecular expression of tumor cells in vivo. Epidermal growth factor receptor (EGFR) is one of the molecular markers of tumor angiogenesis. A study has reported in vivo molecular imaging of EGFR in human CRC mice model, firstly achieving in vivo molecular imaging of EGFR in CRC. The study demonstrated that molecular imaging using CLE could differentiate CRC with different EGFR expression. Moreover, molecular imaging was also carried out in fresh CRC tissue by topically applied with fluorescein labelled EGFR antibody. This study provided a theoratical basis for the transition of molecular imaging from animal experiment to clinical use.
Based on the morphological and functional diagnostic criteria, functional imaging using CLE could observe the pathophysiological process of tissue and cell in vivo, e.g. cell shedding, cell migration, cell necrosis and apoptosis and tissue perfusion. Cell shedding (gaps) is a potential factor that might cause gastrointestinal epithelial barrier dysfunction. Studies have shown that inflammation could drive the epithelial cells to increased shedding. A study by Kiesslich et al has demonstrated20%of gaps were permeable in animal intraperitoneally injected with TNF-α. Other studies have shown that the gaps density in the termial ileum of IBD patients was significantly higher than controls by CLE imaging, and the increased epithelial gaps could predict the relapse of disease. Probiotics could maintain intestinal epithelial barrier by increasing tight junction protein expression, decrease epithelial cell apoptosis induced by inflammatory factors and necrotizing enterocolitis, and decrease the intestinal permeability. However, to date, no study has investigate the influence of probiotics on intestinal epithelial gaps by in vivo observation and the possible mechanisms.
The aims of the current study were:
(1) to evaluate the use of CLE for in vivo molecular imaging of EGFR in patients with colorectal neoplasia and to perform a comparative analysis of in vitro detection of EGFR expression with IHC and in vivo EGFR molecular imaging using CLE;
(2) to investigate the gaps density in patients with IBD by CLE, and to evaluate the function of probiotics application to the epithelial gaps in IBD rat model using CLE, and discuss the possible mechanisms.
Methods:
Part One: In vivo molecular imaging of colorectal neoplasia by confocal laser endomicroscopy.
From January2011to June2011,40consecutive patients known to have colorectal neoplasia from previous examinations as outpatients or inpatients at our hospital were enrolled in our study. In all patients, surgical or endoscopic resection was planned in accordance with the evaluative results of the previous colonoscopic examination. Study was divided into2stages.
Stage Ⅰ: pilot study
In order to establish the proper antibody concentration and incubation time, the first3participants with previously confirmed CRC were recruited for a pilot study. After topical application of5ml of Alexa Fluor488labeled anti-EGFR antibody to the CRC at concentrations of1:500,1:100, or1:50, CLE (Pentax EC-3870K) imaging was performed after a10and15min incubation. Targeted biopsy was taken from the observed site. Molecular imaging with high quality was achieved at a dilution of1:50and incubation time of10min.
Stage Ⅱ:prospective study
Thirty-seven patients with known colorectal neoplasia were enrolled in the prospective study. After fully rinsing, EGFR antibody at the concentration of1:50was topically sprayed to the neoplastic tissue. Molecular imaging was conducted by CLE after10min incubation. Confocal imaging of normal mucosa adjacent to the neoplastic lesions from the same patients was also performed in10cases. Targeted biopsy was taken at all observed sites for immunohistochemistry (IHC) and histology. Fluorescence intensity of confocal images was quantified offline using Image J. Three ROIs of60*60μm with the strongest fluorescence in the representative image were selected for calculation. The grading of H&E results was performed by an experienced gastrointestinal pathologist in a blinded manner according to the modified Vienna classification. Serum samples from4patients were taken4-6weeks after molecular imaging and were tested for human anti-mouse antibodies (HAMAs).
Part Two: Functional imaging of epithelial gaps in small intestine of inflammatory bowel disease using confocal laser endomicroscopy. Study was divided into2stages, clinical study and animal study.
Stage Ⅰ: clinical study
The study group included outpatient with known or suspected to have a diagnosis of IBD. Asymptomatic individuals undergoing colonoscopy for health surveillance or follow up after polypectomy were included as controls. After thorough bowel preparation and conscious sedation, confocal laser endoscopy (Pentax EC3870CIK, Tokyo, Japan) was carried out with the patients. After successful intubation into the terminal ileum,6ml of fluoresceince sodium was injected intravenously. A minimum of5different, normal-appearing sites in terminal ileum were imaged using CLE. CLE images were collected and stored for further evaluation of gaps density.
Stage Ⅱ: animal study
Rats were randomly assigned to3groups: IBD group, normal control group, and IBD treated with VSL#3group. Confocal images was carried out by rigid confocal mini-microscope FIVE1in terminal ileum of rats for gaps density (number of gaps per1000cells). Biopsy samples were taken in each animal group for ex vivo IHC of occludin and ZO-1and Enzyme-linked Immunosorbent Assay (ELISA) of IL-1β. Effect of IL-1β on epithelial gaps formation was evaluated by CLE imaging after intraperitoneal injection of IL-1(3in doses of5and10μg/kg in rats.
Results:
Part One: In vivo molecular imaging of colorectal neoplasia by confocal laser endomicroscopy.
A total of37patients (21men,16women) with colorectal neoplastic lesions were enrolled in prospective study. After topical application of the labeled anti-EGFR antibody to all lesions, a specific EGFR fluorescence signal could be detected in18(94.7%) of the19CRC and in12(66.7%) of the18colorectal adenomas. Eight CLE-positive carcinomas included8well-differentiated carcinomas (8/8),10moderately differentiated (10/11), and0poorly differentiated (0/0). Six out of10high-grade intraepithelial neoplasia (6/10) and six out of eight low-grade intraepithelial neoplasia (6/8) were CLE positive. The mean fluorescence intensity was52.84±5.73for CRC and44.31±4.86for colorectal adenomas (p=0.329). Normal mucosa showed no specific fluorescence signal in7cases and weak fluorescence signal in3cases. The mean fluorescence signal intensity of normal mucosa was36.23±7.90compared with53.78±7.06in neoplastic tissues of the same10patients (p<0.001). The kappa value demonstrated a substantial agreement between in vivo EGFR imaging by CLE and ex vivo EGFR staining by IHC (κ=0.788). None of the4serum samples were found to be increased for HAMAs titers.
Part Two:Functional imaging of epithelial gaps in small intestine of inflammatory bowel disease using confocal laser endomicroscopy.
A total of43participants (19UC patients,4CD patients, and20controls) were enrolled. Compared with controls (4.45gaps/1000cells), the mean gap density in the terminal ileum of IBD patients was significantly increased (52.74gaps/1000cells, p <0.001). Compared with normal rats (14.17±4.17gaps/1000cells)., the mean gap density for the IBD ratswas significantly higher (47.83±7.57gaps/1000cells, p<0.001). For IBD treated with VSL#3rats, the mean gap density was significantly lower compared with IBD rats (19.33±4.84gaps/1000cells, p<0.001). Compared with controls, the mean gap densities in rats intraperitoneally injected with IL-1β in the dosages of10and5μg/kg were both significantly increased (205.00±23.48gaps/1000cells and157.67±28.36gaps/1000cells). Compared with normal control group, the expressions level of occludin and ZO-1were increased, and the expression of IL-1β was decreased in IBD treated with VSL#3group (p=0.028, p<0.001, p<0.001).
Conclusions:
1. CLE is a novel technique that can be used in molecular imaging in vivo with specific EGFR molecular probe in patients with colorectal neoplasia. This new technique shows a promising imaging approach for targeted therapies of CRC.
2. Current study demonstrates that CLE can be used in functional imaging of epithelial gaps in vivo. Patients with IBD show increased epithelial gap than control group observed using CLE. Our study shows firstly that probiotics VSL#3could reduce the epithelial gap density in terminal ileum of IBD rat model by decreasing proinflammatory cytokine, and increasing tight junction protein expression.
Significance:
This study demonstrates, for the first time, that molecular imaging of EGFR is feasible in vivo using a fluorescent-labeled EGFR antibody in combination with CLE imaging in patients with colorectal neoplasia. Specific cellular signal could be observed by CLE molecular imaging. A substantial agreement between in vivo EGFR imaging by CLE and ex vivo EGFR staining by IHC was achieved. Calculation of mean grey scale of the CLE images could differentiate colorectal neoplasia and normal colorecatal mucosa. This new imaging technique could contribute to early detection, differentiatial diagnosis and targeted individual therapy of CRC. Moreover, CLE can be used in functional imaging of epithelial gaps in vivo. Our findings also demonstrate firstly that probiotics VSL#3could decrease the epithelial gap density observed by CLE in terminal ileum of IBD rat model by reducing proinflammatory cytokine, and increasing tight junction protein expression. This study shows new evidences for probiotics treatment in IBD patients.
结直肠癌(colorectal cancer, CRC)是目前消化道最常见的恶性肿瘤之一。迄今为止,公认的结直肠癌发病机制为“腺瘤—癌”顺序模式。对结直肠癌及其癌前病变的早期发现、早期诊断和鉴别诊断能够显著提高患者的生存率。炎症性肠病(inflammatory bowel disease, IBD)是一组病因尚未完全清楚肠道免疫系统调节紊乱的非特异性慢性肠道炎症性疾病。虽然其发展成为结直肠癌的概率比较低,但炎症性肠病癌变长期以来却被认为是其最严重的并发症。对疾病的早期评估和干预是成功的预防策略之一,能够显著改善患者的预后。
迄今为止,内镜检查和活检诊断仍是许多下消化道疾病包括结直肠瘤变和炎症性肠病的诊断金标准。共聚焦激光显微内镜(confocal laser endomicroscopy,CLE)的研发为下消化道疾病的早期诊断提供了强有力的技术支持。共聚焦显微内镜是一种新的显微内镜成像技术,可使图像放大1000倍,实现了在体内实时显示胃肠道组织、细胞和亚细胞结构,能够准确鉴别正常、增生或瘤变的黏膜,被誉为“光学活检”,使在体研究胃肠道生理、病理分子机制成为可能。近年来,共聚焦显微内镜分子成像及功能成像方面的研究正逐渐成为研究热点。
分子成像能够检测肿瘤细胞的特征性分子表达,显微内镜分子成像实现了分子成像从体外至体内的重大突破,它结合了分子探针和显微内镜技术,实现在体实时检测肿瘤分子的表达水平。表皮生长因子受体(epidermal growth factor receptor, EGFR)是目前研究较为深入的肿瘤血管新生相关分子标记物。目前已经有利用共聚焦内镜在体对人结直肠癌动物菏瘤鼠肿瘤组织EGFR分子成像的相关报道,首次实现了结直肠癌的EGFR体内分子成像,该研究还证实了共聚焦激光显微内镜分子成像技术可体内实时鉴别不同类别种植瘤的EGFR表达水平差异。此外,作者还通过体外对人新鲜结直肠癌组织标本表面喷洒荧光标记的EGFR抗体探针的方式,利用FIVE1小型共聚焦探针行EGFR分子成像,结果表明共聚焦系统可检测肿瘤组织和非肿瘤组织的EGFR表达差异。该实验为EGFR分子成像由动物实验向人体应用过渡提供了理论依据。
依据形态学和功能学诊断标准,显微内镜功能成像能够对细胞、组织的病理生理动态过程进行在体实时成像,如细胞脱落、细胞迁移、坏死和凋亡及组织的血流灌注及其改变。上皮细胞的不断更新(细胞脱落,gaps)是一个可能导致胃肠道上皮屏障破坏的潜在因素。研究表明在炎症环境下,上皮增生更新加快,上皮屏障功能破坏的危险性则变得复杂化。Kiesslich等人的研究显示,动物腹腔注射TNF-α诱导小肠炎症时有20%的gaps存在通透性改变。一项研究利用CLE观察IBD患者及对照组静脉注射荧光素钠后gaps的密度,发现IBD患者回肠末端黏膜上皮细胞gaps的密度明显高于正常对照组。还有研究显示,IBD患者回肠末端gaps密度升高能够预测疾病的复发。既往的研究发现,益生菌能够通过多种机制通过促进紧密连接蛋白成熟等多种方式保护紧密连接蛋白,继而降低肠道上皮通透性、维持肠道上皮的屏障功能；此外,研究证实,多种益生菌及其蛋白成分能有效的减少炎性因子诱导或坏死性小肠结肠炎的上皮细胞凋亡。因此,我们推测益生菌可能能够修复炎症性肠病回肠末端黏膜上皮细胞gaps。然而,迄今为止,尚无在体观察益生菌对肠道上皮gaps的影响及其机制的研究报道。
本研究拟围绕共聚焦激光显微内镜在下消化道病变分子成像和功能成像中的应用展开研究,研究目的包括：1.评估共聚焦显微内镜在体对结直肠癌和腺瘤患者行EGFR分子成像的价值,并比较在体共聚焦显微内镜分子成像技术和体外活检组织免疫组化技术在评价EGFR表达水平上的价值；2.利用共聚焦显微内镜对炎症性肠病患者回肠末端上皮细胞脱落(gaps)行功能成像并检测其密度,探索益生菌对上皮gaps的修复作用及其可能的机制。
研究方法：
第一部分：共聚焦显微内镜对结直肠瘤变在体分子成像的研究
自2011年1月至2011年6月,于我院就诊的40例已知结直肠瘤变的连续门诊或住院患者入组研究,所有的患者均依据术前的结肠镜检查安排了外科手术或内镜下切除手术。研究分为两个阶段：
第一阶段,先期研究,为了探索合适的分子探针浓度和孵育时间,共聚焦显微结肠镜(Pentax EC-3870K)白光模式下,用喷洒管对3名已知结直肠癌的患者肿瘤组织表面喷洒5ml Alexa Fluor488绿色荧光标记的EGFR抗体探针(探针浓度分别为1：50,1：100和1：500),10min及15min后开启共聚焦显微内镜模式行分子成像,随后于分子成像部位行靶向活检。结果显示,分子探针浓度在1：50、孵育时间在10min时即可达到较好的分子成像质量。
第二阶段,前瞻性研究,37例已知结直肠瘤变患者入组研究,充分冲洗组织表面后,在体对患者肿瘤组织或腺瘤组织表面喷洒1：50浓度的EGFR抗体探针,10min后利用共聚焦显微结肠镜行在体EGFR分子成像。此外,对10例结直肠瘤变患者,我们除对瘤变组织行分子成像外,同时对邻近的正常黏膜组织进行了EGFR在体分子成像。所有在体分子成像的部位均行靶向活检行体外EGFR免疫组织化学检查和HE病理检查。共聚焦在体分子成像的图像均予以事后分析,每位患者选取荧光强度最强的1张图片,选取该图片3个60*601μm荧光强度最强的感兴趣区域(region of interest, ROI),用ImageJ软件进行灰度值分析。名有经验的消化病理医师按维也纳分型对病理结果做出盲法诊断。此外,4名参与研究的患者于内镜结束后的4-6周后采取外周血检测血人抗小鼠抗体变化。
第二部分：共聚焦显微内镜对炎症性肠病回肠gaps功能成像的研究
研究分为两个步骤,分别为临床研究和动物研究。
第一部分,临床研究,纳入已知或怀疑炎症性肠病来我院就诊的门诊患者,对照组为健康查体者及息肉切除术后复查患者。在充分的肠道准备后在静脉全麻下对所有入组患者行共聚焦显微结肠镜(Pentax EC-3870K)检查,所有患者在进镜至回肠末端,静脉注射6ml荧光素钠,开启共聚焦模式对至少5个白光模式下正常的黏膜部位上皮细胞gaps进行观察并采集图像。事后采用盲法对采集的图像进行分析,计数患者回肠末端gaps密度。
第二部分,动物研究,动物模型分为TNBS造模IBD大鼠组、益生菌VSL#3治疗IBD大鼠组和正常对照大鼠组三组,利用小型动物活体共聚焦成像系统FIVE1(Optiscan公司)在体观察三组动物模型回肠末端上皮gaps密度(每1000个上皮细胞中gaps的数目),对大鼠回肠末端黏膜活检组织行免疫组化检测紧密连接蛋白Occludin和ZO-1的表达量,利用酶联免疫吸附试验(ELISA)检测黏膜炎症因子IL-1β表达,研究益生菌治疗对IBD大鼠回肠末端gaps的修复作用。此外,我们还对大鼠腹腔注射不同剂量的IL-1β后观察大鼠回肠末端上皮gaps密度的变化,探讨炎症因子IL-1β对上皮细胞gaps的影响。
研究结果：
第一部分：共聚焦显微内镜对结直肠瘤变在体分子成像的研究
前瞻性研究共纳入37例结直肠瘤变患者(21例男性,16例女性),表面喷洒AF488标记EGFR抗体后利用共聚焦内镜行分子成像显示,18例结直肠癌(18/19,94.7%)和12例结直肠腺瘤(12/18,66.7%)可检测到EGFR特异性荧光信号。18例EGFR分子成像阳性的结直肠癌组织中包括8例高分化(8/8)、10例中分化(10/11)和0例低分化(0/0)。12例分子成像阳性的结直肠腺瘤组织中包括6例高级别上皮内瘤变(6/10)和6例低级别上皮内瘤变(6/8)。对共聚焦图像灰度值分析显示,结直肠癌EGFR分子成像平均灰度值为52.84±5.73,结直肠腺瘤为44.31±4.86(p=0.329)。而对正常结肠黏膜行EGFR分子成像显示,仅有3例呈弱阳性,其余7例均为阴性。10例正常黏膜分子成像的平均灰度值为36.23±7.90,对应的10例结直肠瘤变组织平均灰度值为53.78±7.06(p<0.001)。体外组织EGFR免疫组化和体内EGFR分子成像的一致性的Kappa值较高,为0.788。4-6周后采集的4例血清样本显示,所有样本HAMA滴度值均未有明显升高。
第二部分：共聚焦显微内镜对炎症性肠病回肠gaps功能成像的研究
共有43例患者入组临床研究,其中,19例溃疡性结肠炎患者,4例克罗恩病患者与20例正常对照患者。与正常对照组(1.50gaps/1000细胞)相比,IBD患者的回肠末端上皮细胞gaps密度显著增加(38.00gaps/1000细胞,p<0.001)。动物研究中,与正常对照组大鼠(14.17±4.17gaps/1000细胞)相比,IBD组大鼠回肠末端上皮gaps密度(47.83±7.57gaps/1000细胞)显著升高(p<0.001)。益生菌VSL#3治疗IBD组大鼠回肠末端gaps密度较IBD组大鼠显著降低(19.33±4.84gaps/1000细胞,p<0.001)。腹腔注射10或5μg/kg IL-1β后,大鼠回肠末端gaps密度均显著升高(205.00±23.48gaps/1000细胞和157.67±28.36gaps/1000细胞)。益生菌治疗IBD组大鼠回肠末端黏膜紧密连接蛋白Occludin和ZO-1表达升高,炎症因子IL-1β表达下降。
研究结论：
1.本研究首次证实了利用共聚焦显微内镜和荧光标记EGFR抗体分子探针可实现对结直肠瘤变患者的在EGFR分子成像。这种新型体内分子成像技术为结直肠癌的分子靶向治疗提供了理论依据。
2.共聚焦内镜可以对细胞脱落gaps行在体功能成像,共聚焦内镜观察下,IBD患者回肠末端gaps密度显著升高,在体对IBD动物模型行功能成像表明,益生菌可能通过降低炎症因子表达,提高紧密连接蛋白表达的方式修复IBD动物模型回肠末端黏膜gaps。
研究意义：
本研究第一次利用共聚焦显微内镜,通过表面喷洒荧光标记EGFR抗体探针的方式实现了对结直肠瘤变患者在体EGFR分子成像。共聚焦显微内镜能够检测出靶向EGFR分子的特异性细胞荧光信号。对成像部位行体外EGFR免疫组化检测与在体EGFR分子成像一致性较高。通过计算共聚焦分子成像灰度值的方式能够区分结直肠瘤变和正常结直肠黏膜。EGFR显微内镜分子成像的实现,将有助于早期发现结直肠癌,并对其行分型诊断和指导分子靶向治疗。此外,共聚焦显微内镜能够对IBD患者回肠末端gaps行功能成像,研究还首次证实了益生菌能够通过降低炎症因子和提高紧密连接蛋白的表达等多种机制修复上皮细胞gaps,这可能是益生菌治疗IBD的机制之一。
Background and aims:
Colorectal cancer (CRC) is one of the most common malignant tumors of gastrointestinal tract. The well-accepted pathogenetic framework for colorectal tumorigenesis is an adenoma-carcinoma sequence. Early detection, diagnosis and differential diagnosis of colorectal cancer and precancerous lesions could significantly increase the overall survival rate of patients. Inflammatory bowel disease (IBD) is a kind of nonspecific chronic bowel inflammatory disease with unclear pathogenesis and characterized by intestinal immune system disorder. Although the probability of IBD become colorectal cancer is low, colorectal neoplasia is recognized as the most severe complication of IBD. Early assessment and intervention for IBD is one of the successful prevention strategies, improving the prognosis of IBD patients.
To date, endoscopy and pathology are the gold standard of many lower gastrointestinal tract diease, including colorectal neoplasia and inflammatory bowel diease. With the advances in in optical instruments, e.g. confocal laser endomicroscopy (CLE), the early detection of lower gastrointestinal tract diease became possible. Confocal endomicroscopy is new endomicroscopic technique with a1000fold magnification, could observe the gastrointestinal tract in vivo at tissue, cellular and subcellular level, and differetiate normal, hyperplastic and neoplastic mucosa, which is named as "optical biopsy". With CLE, we can investigate the physiological and pathological mechanism gastrointestinal trat in vivo. Molecular imaging and functional imaging using CLE are rapidly advanced subjects of gastrointestinal endoscopy.
The specific molecular expression of tumor cells can be detected by molecular imaging. Molecular imaging using CLE realized the breakthrough of molecular imaging from ex vivo to in vivo, combining molecular probe and endomicroscopic technique, detecting the molecular expression of tumor cells in vivo. Epidermal growth factor receptor (EGFR) is one of the molecular markers of tumor angiogenesis. A study has reported in vivo molecular imaging of EGFR in human CRC mice model, firstly achieving in vivo molecular imaging of EGFR in CRC. The study demonstrated that molecular imaging using CLE could differentiate CRC with different EGFR expression. Moreover, molecular imaging was also carried out in fresh CRC tissue by topically applied with fluorescein labelled EGFR antibody. This study provided a theoratical basis for the transition of molecular imaging from animal experiment to clinical use.
Based on the morphological and functional diagnostic criteria, functional imaging using CLE could observe the pathophysiological process of tissue and cell in vivo, e.g. cell shedding, cell migration, cell necrosis and apoptosis and tissue perfusion. Cell shedding (gaps) is a potential factor that might cause gastrointestinal epithelial barrier dysfunction. Studies have shown that inflammation could drive the epithelial cells to increased shedding. A study by Kiesslich et al has demonstrated20%of gaps were permeable in animal intraperitoneally injected with TNF-α. Other studies have shown that the gaps density in the termial ileum of IBD patients was significantly higher than controls by CLE imaging, and the increased epithelial gaps could predict the relapse of disease. Probiotics could maintain intestinal epithelial barrier by increasing tight junction protein expression, decrease epithelial cell apoptosis induced by inflammatory factors and necrotizing enterocolitis, and decrease the intestinal permeability. However, to date, no study has investigate the influence of probiotics on intestinal epithelial gaps by in vivo observation and the possible mechanisms.
The aims of the current study were:
(1) to evaluate the use of CLE for in vivo molecular imaging of EGFR in patients with colorectal neoplasia and to perform a comparative analysis of in vitro detection of EGFR expression with IHC and in vivo EGFR molecular imaging using CLE;
(2) to investigate the gaps density in patients with IBD by CLE, and to evaluate the function of probiotics application to the epithelial gaps in IBD rat model using CLE, and discuss the possible mechanisms.
Methods:
Part One: In vivo molecular imaging of colorectal neoplasia by confocal laser endomicroscopy.
From January2011to June2011,40consecutive patients known to have colorectal neoplasia from previous examinations as outpatients or inpatients at our hospital were enrolled in our study. In all patients, surgical or endoscopic resection was planned in accordance with the evaluative results of the previous colonoscopic examination. Study was divided into2stages.
Stage Ⅰ: pilot study
In order to establish the proper antibody concentration and incubation time, the first3participants with previously confirmed CRC were recruited for a pilot study. After topical application of5ml of Alexa Fluor488labeled anti-EGFR antibody to the CRC at concentrations of1:500,1:100, or1:50, CLE (Pentax EC-3870K) imaging was performed after a10and15min incubation. Targeted biopsy was taken from the observed site. Molecular imaging with high quality was achieved at a dilution of1:50and incubation time of10min.
Stage Ⅱ:prospective study
Thirty-seven patients with known colorectal neoplasia were enrolled in the prospective study. After fully rinsing, EGFR antibody at the concentration of1:50was topically sprayed to the neoplastic tissue. Molecular imaging was conducted by CLE after10min incubation. Confocal imaging of normal mucosa adjacent to the neoplastic lesions from the same patients was also performed in10cases. Targeted biopsy was taken at all observed sites for immunohistochemistry (IHC) and histology. Fluorescence intensity of confocal images was quantified offline using Image J. Three ROIs of60*60μm with the strongest fluorescence in the representative image were selected for calculation. The grading of H&E results was performed by an experienced gastrointestinal pathologist in a blinded manner according to the modified Vienna classification. Serum samples from4patients were taken4-6weeks after molecular imaging and were tested for human anti-mouse antibodies (HAMAs).
Part Two: Functional imaging of epithelial gaps in small intestine of inflammatory bowel disease using confocal laser endomicroscopy. Study was divided into2stages, clinical study and animal study.
Stage Ⅰ: clinical study
The study group included outpatient with known or suspected to have a diagnosis of IBD. Asymptomatic individuals undergoing colonoscopy for health surveillance or follow up after polypectomy were included as controls. After thorough bowel preparation and conscious sedation, confocal laser endoscopy (Pentax EC3870CIK, Tokyo, Japan) was carried out with the patients. After successful intubation into the terminal ileum,6ml of fluoresceince sodium was injected intravenously. A minimum of5different, normal-appearing sites in terminal ileum were imaged using CLE. CLE images were collected and stored for further evaluation of gaps density.
Stage Ⅱ: animal study
Rats were randomly assigned to3groups: IBD group, normal control group, and IBD treated with VSL#3group. Confocal images was carried out by rigid confocal mini-microscope FIVE1in terminal ileum of rats for gaps density (number of gaps per1000cells). Biopsy samples were taken in each animal group for ex vivo IHC of occludin and ZO-1and Enzyme-linked Immunosorbent Assay (ELISA) of IL-1β. Effect of IL-1β on epithelial gaps formation was evaluated by CLE imaging after intraperitoneal injection of IL-1(3in doses of5and10μg/kg in rats.
Results:
Part One: In vivo molecular imaging of colorectal neoplasia by confocal laser endomicroscopy.
A total of37patients (21men,16women) with colorectal neoplastic lesions were enrolled in prospective study. After topical application of the labeled anti-EGFR antibody to all lesions, a specific EGFR fluorescence signal could be detected in18(94.7%) of the19CRC and in12(66.7%) of the18colorectal adenomas. Eight CLE-positive carcinomas included8well-differentiated carcinomas (8/8),10moderately differentiated (10/11), and0poorly differentiated (0/0). Six out of10high-grade intraepithelial neoplasia (6/10) and six out of eight low-grade intraepithelial neoplasia (6/8) were CLE positive. The mean fluorescence intensity was52.84±5.73for CRC and44.31±4.86for colorectal adenomas (p=0.329). Normal mucosa showed no specific fluorescence signal in7cases and weak fluorescence signal in3cases. The mean fluorescence signal intensity of normal mucosa was36.23±7.90compared with53.78±7.06in neoplastic tissues of the same10patients (p<0.001). The kappa value demonstrated a substantial agreement between in vivo EGFR imaging by CLE and ex vivo EGFR staining by IHC (κ=0.788). None of the4serum samples were found to be increased for HAMAs titers.
Part Two:Functional imaging of epithelial gaps in small intestine of inflammatory bowel disease using confocal laser endomicroscopy.
A total of43participants (19UC patients,4CD patients, and20controls) were enrolled. Compared with controls (4.45gaps/1000cells), the mean gap density in the terminal ileum of IBD patients was significantly increased (52.74gaps/1000cells, p <0.001). Compared with normal rats (14.17±4.17gaps/1000cells)., the mean gap density for the IBD ratswas significantly higher (47.83±7.57gaps/1000cells, p<0.001). For IBD treated with VSL#3rats, the mean gap density was significantly lower compared with IBD rats (19.33±4.84gaps/1000cells, p<0.001). Compared with controls, the mean gap densities in rats intraperitoneally injected with IL-1β in the dosages of10and5μg/kg were both significantly increased (205.00±23.48gaps/1000cells and157.67±28.36gaps/1000cells). Compared with normal control group, the expressions level of occludin and ZO-1were increased, and the expression of IL-1β was decreased in IBD treated with VSL#3group (p=0.028, p<0.001, p<0.001).
Conclusions:
1. CLE is a novel technique that can be used in molecular imaging in vivo with specific EGFR molecular probe in patients with colorectal neoplasia. This new technique shows a promising imaging approach for targeted therapies of CRC.
2. Current study demonstrates that CLE can be used in functional imaging of epithelial gaps in vivo. Patients with IBD show increased epithelial gap than control group observed using CLE. Our study shows firstly that probiotics VSL#3could reduce the epithelial gap density in terminal ileum of IBD rat model by decreasing proinflammatory cytokine, and increasing tight junction protein expression.
Significance:
This study demonstrates, for the first time, that molecular imaging of EGFR is feasible in vivo using a fluorescent-labeled EGFR antibody in combination with CLE imaging in patients with colorectal neoplasia. Specific cellular signal could be observed by CLE molecular imaging. A substantial agreement between in vivo EGFR imaging by CLE and ex vivo EGFR staining by IHC was achieved. Calculation of mean grey scale of the CLE images could differentiate colorectal neoplasia and normal colorecatal mucosa. This new imaging technique could contribute to early detection, differentiatial diagnosis and targeted individual therapy of CRC. Moreover, CLE can be used in functional imaging of epithelial gaps in vivo. Our findings also demonstrate firstly that probiotics VSL#3could decrease the epithelial gap density observed by CLE in terminal ileum of IBD rat model by reducing proinflammatory cytokine, and increasing tight junction protein expression. This study shows new evidences for probiotics treatment in IBD patients.
引文
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9. Goetz M, Wang TD. Molecular imaging in gastrointestinal endoscopy. Gastroenterology,2010,138 (3):828-833.
10. Goetz M, Ansems JV, Galle PR, Schuchmann M, Kiesslich R. In vivo real-time imaging of the liver with confocal endomicroscopy permits visualization of the temporospatial patterns of hepatocyte apoptosis. Am J Physiol Gastrointest Liver Physiol.2011 Nov;301(5):G764-72
11. Li CQ, Xie XJ, Yu T, et al. Classification of inflammation activity in ulcerative colitis by confocal laser endomicroscopy. Am J Gastroenterol 2010;105:1391-1396.
12. Kiesslich R, Goetz M, Angus EM, et al. Identification of Epithelial gaps in Human Small and Large Intestine by Confocal Endomicroscopy. Gastroenterology. 2007;133(6):1769-78.
13. Liu JJ, Madsen KL, Boulanger P, et al. Mind The gaps:Confocal Endomicroscopy Showed Increased Density of Small Bowel Epithelial gaps in Inflammatory Bowel Disease. J Clin Gastroenterol.2011;45(3):240-5.
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16. Goetz M, Watson A, Kiesslich R. Confocal laser endomicroscopy in gastrointestinal diseases. J Biophotonics.2011 Aug;4(7-8):498-508.
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6. Shih DQ, Targan SR. Insights into IBD Pathogenesis. Curr Gastroenterol Rep, 2009,11(6):473-480.
7. Saltz L. Epidermal growth factor receptor-negative colorectal cancer: is there truly such an entity? Clin Colorectal Cancer.2005 Nov;5 Suppl 2:S98-100.
8. Barrett T, Koyama Y, Hama Y, et al. In vivo diagnosis of epidermal growth factor receptor expression using molecular imaging with a cocktail of optically labeled monoclonal antibodies. Clin Cancer Res,2007,13 (22 Pt 1):6639-6648.
9. Goetz M, Wang TD. Molecular imaging in gastrointestinal endoscopy. Gastroenterology,2010,138 (3):828-833.
10. Goetz M, Ansems JV, Galle PR, Schuchmann M, Kiesslich R. In vivo real-time imaging of the liver with confocal endomicroscopy permits visualization of the temporospatial patterns of hepatocyte apoptosis. Am J Physiol Gastrointest Liver Physiol.2011 Nov;301(5):G764-72
11. Li CQ, Xie XJ, Yu T, et al. Classification of inflammation activity in ulcerative colitis by confocal laser endomicroscopy. Am J Gastroenterol 2010;105:1391-1396.
12. Kiesslich R, Goetz M, Angus EM, et al. Identification of Epithelial gaps in Human Small and Large Intestine by Confocal Endomicroscopy. Gastroenterology. 2007;133(6):1769-78.
13. Liu JJ, Madsen KL, Boulanger P, et al. Mind The gaps:Confocal Endomicroscopy Showed Increased Density of Small Bowel Epithelial gaps in Inflammatory Bowel Disease. J Clin Gastroenterol.2011;45(3):240-5.
14. Watson AJ, Chu S, Sieck L, et al. Epithelial Barrier Function In Vivo Is Sustained Despite gaps in Epithelial Layers. Gastroenterology.2005;129(3):902-12.
15. Kiesslich R, Duckworth CA, Moussata D, et al. Local barrier dysfunction identified by confocal laser endomicroscopy predicts relapse in inflammatory bowel disease. Gut.2011 Nov 24.
16. Goetz M, Watson A, Kiesslich R. Confocal laser endomicroscopy in gastrointestinal diseases. J Biophotonics.2011 Aug;4(7-8):498-508.
1. Cowley GP, Smith JA, Gusterson BA. Increased EGF receptors on human squamous carcinoma cell lines. Br J Cancer.1986 Feb;53(2):223-9.
2. Huang SM, Harari PM. Epidermal growth factor receptor inhibition in cancer therapy:biology, rationale and preliminary clinical results. Invest New Drugs,1999, 17 (3):259-269.
3. Ciardiello F, Tortora G. A novel approach in the treatment of cancer: targeting the epidermal growth factor receptor. Clin Cancer Res.2001 Oct;7(10):2958-70.
4. Jaszewski R, Levi E, Sochacki P, et al. Expression of epidermal growth factor-receptor related protein (ERRP) in human colorectal carcinogenesis. Cancer Lett.2004 Sep 30;213(2):249-55.
5. Kaklamanis L, Gatter KC, Mortensen N, et al. Interleukin-4 receptor and epidermal growth factor receptor expression in colorectal cancer. Br J Cancer.1992 Oct;66(4):712-6.
6. Han YD, Hong YK, Kang JG, et al. Relation of the expression of cyclooxygenase-2 in colorectal adenomas and adenocarcinomas to angiogenesis and prognosis. J Korean Soc Coloproctol.2010 Oct;26(5):339-46.
7. Porebska I, Harlozinska A, Bojarowski T. Expression of the tyrosine kinase activity growth factor receptors (EGFR, ERB B2, ERB B3) in colorectal adenocarcinomas and adenomas. Tumour Biol.2000 Mar-Apr;21(2):105-15.
8. Salomon DS, Brandt R, Ciardiello F, et al. Epidermal growth factor-related peptides and their receptors in human malignancies. Crit Rev Oncol Hematol,1995,19 (3): 183-232.
9. Normanno N, De Luca A, Bianco C, et al. Epidermal growth factor receptor (EGFR) signaling in cancer. Gene,2006,366 (1):2-16.
10. Ciardiello F, Tortora G. EGFR antagonists in cancer treatment. N Engl J Med, 2008,358 (11):1160-74.
11. Hemming AW, Davis NL, Kluftinger A, et al. Prognostic markers of colorectal cancer: an evaluation of DNA content, epidermal growth factor receptor, and Ki-67. J Surg Oncol.1992 Nov;51(3):147-52.
12. Cunningham D, Humblet Y, Siena S, et al. Cetuximab monotherapy and cetuximab plus irinotecan in irinotecan-refractory metastatic colorectal cancer. N Engl J Med.2004 Jul 22;351(4):337-45.
13. Chung KY, Shia J, Kemeny NE, et al. Cetuximab shows activity in colorectal cancer patients with tumors that do not express the epidermal growth factor receptor by immunohistochemistry. J Clin Oncol.2005 Mar 20;23(9):1803-10.
14. Saltz L. Epidermal growth factor receptor-negative colorectal cancer: is there truly such an entity? Clin Colorectal Cancer.2005 Nov;5 Suppl 2:S98-100.
15. Hamilton SR. Targeted therapy of cancer: new roles for pathologists in colorectal cancer. Mod Pathol.2008 May;21 Suppl 2:S23-30.
16. Rex DK, Cutler CS, Lemmel GT, et al. Colonoscopic miss rates of adenomas determined by back-to-back colonoscopies. Gastroenterology.1997 Jan;112(1):24-8.
17. Li CQ, Xie XJ, Yu T, et al. Classification of inflammation activity in ulcerative colitis by confocal laser endomicroscopy. Am J Gastroenterol.2010 Jun; 105(6):1391-6.
18. Neumann H, Vieth M, Atreya R, et al. Assessment of Crohn's disease activity by confocal laser endomicroscopy. Inflamm Bowel Dis.2012 Dec;18(12):2261-9.
19. Zambelli A, Villanacci V, Buscarini E, et al. Collagenous colitis: a case series with confocal laser microscopy and histology correlation. Endoscopy.2008 Jul;40(7):606-8
20. Li Z, Yu T, Zuo XL, et al. Confocal laser endomicroscopy for in vivo diagnosis of gastric intraepithelial neoplasia: a feasibility study. Gastrointest Endosc.2010 Dec;72(6):1146-53.
21. Wallace MB, Sharma P, Lightdale C, et al. Preliminary accuracy and interobserver agreement for the detection of intraepithelial neoplasia in Barrett's esophagus with probe-based confocal laser endomicroscopy. Gastrointest Endosc.2010 Jul;72(1):19-24.
22. Hurlstone DP, Baraza W, Brown S, et al. In vivo real-time confocal laser scanning endomicroscopic colonoscopy for the detection and characterization of colorectal neoplasia. Br J Surg.2008 May;95(5):636-45.
23. Goetz M, Wang TD. Molecular imaging in gastrointestinal endoscopy. Gastroenterology.2010 Mar;138(3):828-33.
24. Goetz M, Kiesslich R. Advances of endomicroscopy for gastrointestinal physiology and diseases. Am J Physiol Gastrointest Liver Physiol,2010,298 (6): G797-G806.
25. 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 Feb;138(2):435-46.
26. Foersch S, Kiesslich R, Waldner MJ, et al. Molecular imaging of VEGF in gastrointestinal cancer in vivo using confocal laser endomicroscopy. Gut.2010 Aug;59(8):1046-55.
27. Hsiung PL, Hardy J, Friedland S, et al. Detection of colonic dysplasia in vivo using a targeted heptapeptide and confocal microendoscopy. Nat Med.2008 Apr; 14(4):454-8.
28. Nakai Y, Shinoura S, Ahluwalia A, et al. Molecular imaging of epidermal growth factor-receptor and survivin in vivo in porcine esophageal and gastric mucosae using probe-based confocal laser-induced endomicroscopy: proof of concept. J Physiol Pharmacol.2012 Jun;63(3):303-7.
29. Schlemper RJ, Riddell RH, Kato Y, et al. The Vienna classification of gastrointestinal epithelial neoplasia. Gut.2000 Aug;47(2):251-5.
30. Mariotte D, Dupont B, Gervais R, et al. Anti-cetuximab IgE ELISA for identification of patients at a high risk of cetuximab-induced anaphylaxis. MAbs. 2011 Jul-Aug;3(4):396-401.
31. Jonker DJ, O'Callaghan CJ, Karapetis CS, et al. Cetuximab for the treatment of colorectal cancer. N Engl J Med.2007 Nov 15;357(20):2040-8.
32. Atkins D, Reiffen KA, Tegtmeier CL, et al. Immunohistochemical detection of EGFR in paraffin-embedded tumor tissues: variation in staining intensity due to choice of fixative and storage time of tissue sections. J Histochem Cytochem.2004 Jul;52(7):893-901.
33. Goldstein NS, Armin M. Epidermal growth factor receptor immunohistochemical reactivity in patients with American Joint Committee on Cancer Stage IV colon adenocarcinoma: implications for a standardized scoring system. Cancer.2001 Sep 1;92(5):1331-46.
34. Atreya R, Waldner MJ, Neurath MF. Molecular imaging: interaction between basic and clinical science. Gastroenterol Clin North Am.2010 Dec;39(4):911-22.
35. Keller R, Winde G, Terpe HJ, et al. Fluorescence endoscopy using a fluorescein-labeled monoclonal antibody against carcinoembryonic antigen in patients with colorectal carcinoma and adenoma. Endoscopy.2002 Oct;34(10):801-7.
36. Alencar H, Funovics MA, Figueiredo J, et al. Colonic adenocarcinomas: near-infrared microcatheter imaging of smart probes for early detection-study in mice. Radiology.2007 Jul;244(1):232-8.
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