Date Published: June 30, 2017
Publisher: Public Library of Science
Author(s): Giovanni Domenico De Palma, Irene Colavita, Gerardo Zambrano, Mariano Cesare Giglio, Francesco Maione, Gaetano Luglio, Giovanni Sarnelli, Antonio Rispo, Pietro Schettino, Francesco Paolo D’Armiento, Fatima Domenica Elisa De Palma, Valeria D’Argenio, Francesco Salvatore, Valli De Re.
Targeted molecular probes have been used to detect sporadic colonic dysplasia during confocal laser endomicroscopy (CLE) with promising results. This is a feasibility pilot study aiming to assess the potential role of CLE combined with a fluorescent-labeled peptide to stain and detect dysplasia associated with Ulcerative Colitis.
A phage-derived heptapeptide with predicted high binding affinity for dysplastic tissue, was synthesized and labeled with fluorescein. Eleven lesions with suspected dysplasia at endoscopy were excised from nine patients with long-standing ulcerative colitis. Specimens were sprayed with the peptide and examined by CLE. The CLE images were then compared to the corresponding histological sections.
At definitive histology, 4 lesions were diagnosed as inflammatory polyps, 6 as dysplastic lesions and one as invasive cancer. In inflammatory polyps, the fluorescence signal came from peri-cryptal spaces and crypt lumen due to passive accumulation of the peptide in these areas. Dysplasia was associated with active binding of the peptide to dysplastic colonocytes.
Ex vivo staining of ulcerative colitis-associated dysplasia using a fluorescent labeled molecular probe and CLE is feasible. In vivo studies on larger populations are required to evaluate the safety and the effective contribution of molecular probes in cancer surveillance of ulcerative colitis.
Patients with long-standing ulcerative colitis (UC) have an increased risk of developing colorectal cancer (CRC). This neoplasia has a cumulative incidence of 2%, 8% and 18% after 10, 20 and 30 years of disease, respectively . Patients affected by long-standing UC are therefore candidates for surveillance colonoscopy to detect dysplasia [2,3]. Other than in association with polypoid lesions, dysplasia in UC can also develop in non-polypoid lesions or even within unremarkable mucosa (invisible dysplasia) . Therefore, enhanced optical imaging techniques have been used to facilitate detection of dysplasia in these patients.
Nine patients were enrolled in the study. All patients had long-standing UC (> 10 years) and presented quiescent UC  at surveillance colonoscopy. A total of 11 lesions were obtained from the enrolled patients. In particular, 7 polypoid lesions and 4 non-polypoid were detected at endoscopy and eventually resected. At definitive histology, 4 lesions were diagnosed as inflammatory psudo-polyps, 6 as dysplastic lesions (low-grade dysplasia) and one as invasive cancer (SM3). Table 1 shows the histological characteristics of the collected lesions.
Several studies have assessed the feasibility of detecting colonic dysplasia using targeted fluorescent probes . Antibodies against specific antigens or peptides showing binding affinity for dysplastic tissue have been tested in animal models of CRC [11,20] and in patients with sporadic adenomas or CRC [13,15]. In particular, in vivo studies have yielded encouraging results in terms of sensitivity and specificity . In this promising context, the possibility of detecting neoplastic changes associated to IBD using molecular CLE has remained unexplored to date. To our knowledge, this is the first study to assess and confirm the possibility to detect UC-associated dysplasia at ex-vivo CLE examination using a molecular staining probe. In particular, we used the fluorescein-conjugated VRPMPLQ peptide as a molecular probe. This is a phage-derived heptapeptide that has been reported to highlight sporadic colonic dysplasia in vivo during CLE.15In this study, for the first time, the VRPMPLQ peptide has demonstrated to be able to detect also the dysplasia associated with Ulcerative Colitis. We believe this result is far from being obvious. Indeed, VRPMPLQ was originally selected because of its binding affinity to sporadic adenomatous polyps . Like most sporadic cancers, sporadic adenomatous polyps are usually sustained by a chromosomal instabilitypathway ,while neoplastic changes in UC are more frequently associated to the microsatellite instability pathway . These different genetic pathways result in different phenotypes  that might result in different affinity for this probe, due to differential expression of the molecular target of the VRPMPLQ peptide, that is unknown yet .