Katherine Wu

Dynamic real-time microscopy of the urinary tract using confocal laser endomicroscopy.

OBJECTIVES To develop the diagnostic criteria for benign and neoplastic conditions of the urinary tract using probe-based confocal laser endomicroscopy (pCLE), a new technology for dynamic, in vivo imaging with micron-scale resolution. The suggested diagnostic criteria will formulate a guide for pCLE image interpretation in urology. METHODS Patients scheduled for transurethral resection of bladder tumor (TURBT) or nephrectomy were recruited. After white-light cystoscopy (WLC), fluorescein was administered as contrast. Different areas of the urinary tract were imaged with pCLE via direct contact between the confocal probe and the area of interest. Confocal images were subsequently compared with standard hematoxylin and eosin analysis. RESULTS pCLE images were collected from 66 participants, including 2 patients who underwent nephrectomy. We identified key features associated with different anatomic landmarks of the urinary tract, including the kidney, ureter, bladder, prostate, and urethra. In vivo pCLE of the bladder demonstrated distinct differences between normal mucosa and neoplastic tissue. Using mosaicing, a post hoc image-processing algorithm, individual image frames were juxtaposed to form wide-angle views to better evaluate tissue microarchitecture. CONCLUSIONS In contrast to standard pathologic analysis of fixed tissue with hematoxylin and eosin, pCLE provides real time microscopy of the urinary tract to enable dynamic interrogation of benign and neoplastic tissues in vivo. The diagnostic criteria developed in this study will facilitate adaptation of pCLE for use in conjunction with WLC to expedite diagnosis of urinary tract pathology, particularly bladder cancer.

Comparison of 2.6- and 1.4-mm imaging probes for confocal laser endomicroscopy of the urinary tract.

INTRODUCTION Probe-based confocal laser endomicroscopy (pCLE) is an emerging technology for dynamic, in vivo imaging of the urinary tract with micron-scale resolution. We conducted a comparative analysis of pCLE with a 2.6-mm probe and a 1.4-mm probe that is compatible with flexible endoscopes. MATERIALS AND METHODS Sixty-seven patients scheduled for bladder tumor resection were recruited. pCLE imaging was performed using 2.6- and 1.4-mm probes. Image quality with the different probes was examined and further compared with standard histopathology. RESULTS Images with the 2.6-mm probe have better resolution of cell morphology. The 1.4-mm probe has a wider field of view and better view of microarchitecture. While image quality with the 2.6-mm probe is superior, the 1.4-mm probe is compatible with flexible cystoscopy and maneuverability is maintained, enabling imaging of areas of the bladder that were previously challenging to access with the larger probe. CONCLUSIONS The optical specifications of the 2.6-mm probe are more suitable for distinguishing urinary tract histopathology. Further design optimization to improve resolution and additional validation of the diagnostic accuracy of the smaller probe are needed to help extend application of pCLE for optical biopsy of the upper and lower urinary tract.

Real time diagnosis of bladder cancer with probe-based confocal laser endomicroscopy

Probe-based confocal laser endomicroscopy (pCLE) is an emerging technology for in vivo optical imaging of the urinary tract. Particularly for bladder cancer, real time optical biopsy of suspected lesions will likely lead to improved management of bladder cancer. With pCLE, micron scale resolution is achieved with sterilizable imaging probes (1.4 or 2.6 mm diameter), which are compatible with standard cystoscopes and resectoscopes. Based on our initial experience to date (n = 66 patients), we have demonstrated the safety profile of intravesical fluorescein administration and established objective diagnostic criteria to differentiate between normal, benign, and neoplastic urothelium. Confocal images of normal bladder showed organized layers of umbrella cells, intermediate cells, and lamina propria. Low grade bladder cancer is characterized by densely packed monomorphic cells with central fibrovascular cores, whereas high grade cancer consists of highly disorganized microarchitecture and pleomorphic cells with indistinct cell borders. Currently, we are conducting a diagnostic accuracy study of pCLE for bladder cancer diagnosis. Patients scheduled to undergo transurethral resection of bladder tumor are recruited. Patients undergo first white light cystocopy (WLC), followed by pCLE, and finally histologic confirmation of the resected tissues. The diagnostic accuracy is determined both in real time by the operative surgeon and offline after additional image processing. Using histology as the standard, the sensitivity, specificity, positive and negative predictive value of WLC and WLC + pCLE are calculated. With additional validation, pCLE may prove to be a valuable adjunct to WLC for real time diagnosis of bladder cancer.

Abstract 2233: Phage display selection of cancer-specific peptides on human bladder for molecular imaging of bladder cancer

Introduction and Objectives: Molecular imaging could transform cancer diagnosis by coupling existing imaging modalities with labeled tumor-specific antibodies or peptides. Current diagnosis of bladder cancer is by white light cystoscopy, which has suboptimal specificity for differentiating non-papillary cancer from inflammation. To identify bladder cancer-specific peptides, we applied phage display, an unbiased, combinatorial strategy in which up to 10⁁9 foreign peptides or proteins are individually expressed on bacteriophage to screen against cellular targets (biopanning). We report our effort to identify bladder cancer-specific peptides via biopanning with cystectomy specimens. Methods: An M13 phage library displaying random linear heptapeptides was instilled into fresh cystectomy specimens (n=2). Non-specific phage were washed away and tumor was excised to extract tumor-bound phage, which were validated by bladder cancer cell line-based ELISA, flow cytometry, and immunofluorescence. Results: Both bladders had high-grade urothelial carcinoma on pathology. 148 phage were obtained after 2 rounds of selection. 77 of these showed preferential binding to 2 bladder cancer cell lines (HT-1376, T24) by ELISA compared to negative control (insertless phage). 7 phage with >10-fold signals over the negative control were further validated with flow cytometry and immunofluorescence. By detecting cell-bound phage with fluorescently labeled M13 phage antibody, increased staining of T24 cells by all candidate phage compared to negative control was demonstrated by both flow cytometry (2-3-fold) and immunofluorescence. Peptides were sequenced for additional validation of tumor specificity with primary normal human urothelial and bladder tumor cells. Conclusions: We have developed a technique for efficient biopanning of bladder cancer-specific peptides using whole cystectomy specimens. These peptides could serve as molecular contrast agents for targeted imaging with confocal laser endomicroscopy or fluorescent cystocsopy, and could potentially be utilized for targeted therapy. Compared to prior reports, which utilized cell lines and xenografts as biopanning targets, our approach offers a more clinically-relevant, efficient approach to identify peptides for cancer-specific imaging and therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2233. doi:10.1158/1538-7445.AM2011-2233