Confocal laser endomicroscopy: a novel method for prediction of relapse in Crohn's disease.

BACKGROUND AND STUDY AIMS: Confocal laser endomicroscopy (CLE) has been shown to predict relapse in ulcerative colitis in remission, but little is currently known about its role in Crohn's disease. The aim of this study was to identify reproducible CLE features in patients with Crohn's disease and to examine whether these are risk factors for relapse. PATIENTS AND METHODS: This was a single-center prospective feasibility study of CLE imaging in patients with Crohn's disease. CLE imaging was performed in the terminal ileum and four colorectal sites, and was correlated with histopathology and macroscopic appearance. Clinical relapse, defined as the need for treatment escalation or surgical intervention, was recorded during follow-up. RESULTS: The study included 50 patients: 39 with Crohn's disease (20 in remission), and 11 controls. Ileal fluorescein leakage and microerosions were significantly more frequent in patients with endoscopically active Crohn's disease compared with patients with inactive Crohn's disease and controls (P = 0.005 and (P = 0.006, respectively). The same applied to colorectal fluorescein leakage and vascular alterations ((P = 0.043 and (P = 0.034, respectively). During a 12-month follow-up period, ileal fluorescein leakage and microerosions were significant risk factors for relapse in the subgroup of patients in remission (log rank (P = 0.009 and (P = 0.007, respectively) as well as in the entire group of patients with Crohn's disease (log rank (P = 0.006 and (P = 0.01, respectively). Inter- and intraobserver reproducibility was almost perfect (κ > 0.80) or substantial (κ > 0.60) for the majority of CLE parameters. CONCLUSIONS: CLE can identify reproducible microscopic changes in the terminal ileum that are risk factors for relapse in patients with otherwise inactive Crohn's disease. TRIAL REGISTRATION: ClinicalTrials.gov (NCT01738529).

Endoscopic ultrasound guided needle-based confocal laser endomicroscopy in solid pancreatic masses - a prospective validation study.

BACKGROUND AND STUDY AIMS: Endoscopic ultrasound fine-needle aspiration (EUS-FNA) is a keystone in diagnosing and staging of pancreatic masses. Recently, a microfiber that can pass through a 19-gauge needle has been introduced for confocal laser endomicroscopy (nCLE). The aims of this study were to evaluate the diagnostic value and the reproducibility of nCLE criteria for solid malignant lesions. PATIENTS AND METHODS: This prospective dual-center study included patients with pancreatic masses suspicious of malignancy referred for EUS-FNA. Endomicroscopic imaging was performed under EUS-guidance until organ-specific structures were obtained. Afterwards, standard cytology was obtained and patients were followed for up to 12 months. All nCLE parameters included in former studies were correlated with the final diagnosis (dark lobular structures/normal acinar cells, dark cell aggregates > 40 µm, dilated irregular vessels with fluorescein leakage, fine white fibrous bands, small black cell movements, pseudoglandular structures). Finally, three CLE novices and three CLE experts assessed the unedited movies from all patients. RESULTS: Twenty-eight patients were enrolled in the study. A final diagnosis was obtained in 24 patients (86 %). One patient (3 %) died before a diagnosis was obtained, while 3 were lost to follow-up (11 %). In 18/24 patients (74 %) the diagnosis was malignant. The mean sensitivity, specificity, and accuracy for the nCLE parameters ranged from 19 - 93 %, 0 - 56 %, 26 - 69 %, respectively. The inter-observer values ranged from κ = 0.20 - 0.41 for novices and κ = -0.02 - 0.38 for experts. CONCLUSIONS: The diagnostic value of nCLE in solid pancreatic masses is questionable and the inter-observer agreement for both novices and CLE experts appears limited.

Tumor neoangiogenesis detection by confocal laser endomicroscopy and anti-CD105 antibody: Pilot study.

AIM: To evaluate neoangiogenesis in patients with colon cancer by two fluorescently labeled antibodies on fresh biopsy samples imaged with confocal laser endomicroscopy (CLE). METHODS: CLE is an imaging technique for gastrointestinal endoscopy providing in vivo microscopy at subcellular resolution. An important question in validating tumor angiogenesis is what proportion of the tumor vascular network is represented by pre-existing parent tissue vessels and newly formed vessels. CD105 (endoglin) represents a proliferation-associated endothelial cell adhesion molecule. In contrast to pan-endothelial markers, such as CD31, CD105 is preferentially expressed in activated endothelial cells that participate in neovascularization. Thus, we evaluated CD105 and CD31 expression from samples of ten patients with primary rectal adenocarcinoma, using a dedicated endomicroscopy system. A imaging software was used to obtain the Z projection of the confocal serial images from each biopsy sample previously combined into stacks. Vascular density and vessel diameters were measured within two 50 µm x 475 µm rectangular regions of interest centered in the middle of each image in the horizontal and vertical direction. The results were averaged over all the patients and were expressed as the mean ± SE. RESULTS: The use of an anti-CD105 antibody was found to be suitable for the detection of blood vessels in colon cancer. Whereas anti-CD31 antibodies stained blood vessels in both normal and pathologic colon equally, CD105 expression was observed primarily in malignant lesions, with little or no expression in the vessels of the normal mucosa (244.21 ± 130.7 vessels/mm(3) in only four patients). The average diameter of anti-CD105 stained vessels was 10.97 ± 0.6 µm in tumor tissue, and the vessel density was 2787.40 ± 134.8 vessels/mm(3). When using the anti-CD31 antibody, the average diameter of vessels in the normal colon tissue was 7.67 ± 0.5 µm and the vessel density was 3191.60 ± 387.8 vessels/mm(3), while in the tumors we obtained an average diameter of 10.88 ± 0.8 µm and a vessel density of 4707.30 ± 448.85 vessels/mm(3). Thus, there were more vessels stained with CD31 than CD105 (P < 0.05). The average vessel diameter was similar for both CD31 and CD105 staining. A qualitative comparison between CLE vs immunohistochemistry lead to similar results. CONCLUSION: Specific imaging and quantification of tumor microvessels are feasible in human rectal cancer using CLE examination and CD105 immunostaining of fresh tissue samples.

CD133/CD166/Ki-67 triple immunofluorescence assessment for putative cancer stem cells in colon carcinoma.

BACKGROUND & AIMS: Colorectal cancer represents the third most common malignancy and the fourth most common cause of cancer death worldwide. The existence of drug-resistant colon cancer stem cells is thought to be one of the most important reasons behind treatment failure in colon cancer, their existence putatively leading to metastasis and recurrences. The aim of our study was to investigate the immunoexpression patterns of CD133 and CD166 in colon carcinoma, both individually and in combination, assessing their significance as prognostic markers. METHODS: A total of 45 retrospective colon adenocarcinoma cases were investigated by enzymatic and multiple fluorescence immunohistochemistry for their CD133 and CD166 expression and colocalization. RESULTS: Both CD133 and CD166 were expressed to different extents in all cancer specimens, with a predominant cytoplasmic pattern for CD133 and a more obvious membranous-like pattern for CD166. Overall, when comparing their reactivity for the tumoral tissue, CD166 expression areas seemed to be smaller than those of CD133. However, there was a direct correlation between CD133 and CD166 expression levels throughout the entire spectrum of lesions, with higher values for dysplastic lesions. Colocalization of CD133/CD166 was obvious at the level of cells membranes, with higher coefficients in high grade dysplasia, followed by well and moderate differentiated tumours. : CD133/CD166 colocalization is an early event occurring in colon tumorigenesis, with the highest coefficients recorded for patients with high grade dysplasia, followed by well differentiated tumours. Thus, we consider that the coexpression of these two markers could be useful for further prognostic and therapeutically stratification of patients with colon cancer.

Evaluation of new morphometric parameters of neoangiogenesis in human colorectal cancer using confocal laser endomicroscopy (CLE) and targeted panendothelial markers.

The tumor microcirculation is characterized by an abnormal vascular network with dilated, tortuous and saccular vessels. Therefore, imaging the tumor vasculature and determining its morphometric characteristics represent a critical goal for optimizing the cancer treatment that targets the blood vessels (i.e. antiangiogenesis therapy). The aim of this study was to evaluate new vascular morphometric parameters in colorectal cancer, difficult to achieve through conventional immunohistochemistry, by using the confocal laser endomicroscopy method. Fresh biopsies from tumor and normal tissue were collected during colonoscopy from five patients with T3 colorectal carcinoma without metastasis and were marked with fluorescently labeled anti-CD31 antibodies. A series of optical slices spanning 250 µm inside the tissue were immediately collected for each sample using a confocal laser endomicroscope. All measurements were expressed as the mean ± standard error. The mean diameter of tumor vessels was significantly larger than the normal vessels (9.46±0.4 µm vs. 7.60±0.3 µm, p = 0.0166). The vessel density was also significantly higher in the cancer vs. normal tissue samples (5541.05±262.81 vs. 3755.79±194.96 vessels/mm3, p = 0.0006). These results were confirmed by immunohistochemistry. In addition, the tortuosity index and vessel lengths were not significantly different (1.05±0.016 and 28.30±3.27 µm in normal tissue, vs. 1.07±0.008 and 26.49±3.18 µm in tumor tissue respectively, p = 0.5357 and p = 0.7033). The daughter/mother ratio (ratio of the sum of the squares of daughter vessel radii over the square of the mother vessel radius) was 1.15±0.09 in normal tissue, and 1.21±0.08 in tumor tissue (p = 0.6531). The confocal laser endomicroscopy is feasible for measuring more vascular parameters from fresh tumor biopsies than conventional immunohistochemistry alone. Provided new contrast agents will be clinically available, future in vivo use of CLE could lead to identification of novel biomarkers based on the morphometric characteristics of tumor vasculature.

Confocal laser endomicroscopy for the morphometric evaluation of microvessels in human colorectal cancer using targeted anti-CD31 antibodies.

INTRODUCTION: Numerous anti-angiogenic agents are currently developed to limit tumor growth and metastasis. While these drugs offer hope for cancer patients, their transient effect on tumor vasculature is difficult to assess in clinical settings. Confocal laser endomicroscopy (CLE) is a novel endoscopic imaging technology that enables histological examination of the gastrointestinal mucosa. The aim of the present study was to evaluate the feasibility of using CLE to image the vascular network in fresh biopsies of human colorectal tissue. For this purpose we have imaged normal and malignant biopsy tissue samples and compared the vascular network parameters obtained with CLE with established histopathology techniques. MATERIALS AND METHODS: Fresh non-fixed biopsy samples of both normal and malignant colorectal mucosa were stained with fluorescently labeled anti-CD31 antibodies and imaged by CLE using a dedicated endomicroscopy system. Corresponding biopsy samples underwent immunohistochemical staining for CD31, assessing the microvessel density (MVD) and vascular areas for comparison with CLE data, which were measured offline using specific software. RESULTS: The vessels were imaged by CLE in both normal and tumor samples. The average diameter of normal vessels was 8.5±0.9 µm whereas in tumor samples it was 13.5±0.7 µm (p = 0.0049). Vascular density was 188.7±24.9 vessels/mm(2) in the normal tissue vs. 242.4±16.1 vessels/mm(2) in the colorectal cancer samples (p = 0.1201). In the immunohistochemistry samples, the MVD was 211.2±42.9/mm(2) and 351.3±39.6/mm(2) for normal and malignant mucosa, respectively. The vascular area was 2.9±0.5% of total tissue area for the normal mucosa and 8.5±2.1% for primary colorectal cancer tissue. CONCLUSION: Selective imaging of blood vessels with CLE is feasible in normal and tumor colorectal tissue by using fluorescently labeled antibodies targeted against an endothelial marker. The method could be translated into the clinical setting for monitoring of anti-angiogenic therapy.

Confocal laser endomicroscopy and immunoendoscopy for real-time assessment of vascularization in gastrointestinal malignancies.

Gastrointestinal cancers represent a major cause of morbidity and mortality, with incomplete response to chemotherapy in the advanced stages and poor prognosis. Angiogenesis plays a crucial part in tumor growth and metastasis, with most gastrointestinal cancers depending strictly on the development of a new and devoted capillary network. Confocal laser endomicroscopy is a new technology which allows in vivo microscopic analysis of the gastrointestinal mucosa and its microvascularization during ongoing endoscopy by using topically or systemically administered contrast agents. Targeting markers of angiogenesis in association with confocal laser endomicroscopic examination (immunoendoscopy), as a future challenge, will add functional analysis to the morphological aspect of the neoplastic process. This review describes previous experience in endomicroscopic examination of the upper and lower digestive tract with emphasis on vascularization, resulting in a broad spectrum of potential clinical applications, and also preclinical research that could be translated to human studies.