Ying Pan

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.

Electrochemical immunosensor detection of urinary lactoferrin in clinical samples for urinary tract infection diagnosis

Urine is the most abundant and easily accessible of all body fluids and provides an ideal route for non-invasive diagnosis of human diseases, particularly of the urinary tract. Electrochemical biosensors are well suited for urinary diagnostics due to their excellent sensitivity, low-cost, and ability to detect a wide variety of target molecules including nucleic acids and protein biomarkers. We report the development of an electrochemical immunosensor for direct detection of the urinary tract infection (UTI) biomarker lactoferrin from infected clinical samples. An electrochemical biosensor array with alkanethiolate self-assembled monolayer (SAM) was used. Electrochemical impedance spectroscopy was used to characterize the mixed SAM, consisted of 11-mercaptoundecanoic acid and 6-mercapto-1-hexanol. A sandwich amperometric immunoassay was developed for detection of lactoferrin from urine, with a detection limit of 145 pg/ml. We validated lactoferrin as a biomarker of pyuria (presence of white blood cells in urine), an important hallmark of UTI, in 111 patient-derived urine samples. Finally, we demonstrated multiplex detection of urinary pathogens and lactoferrin through simultaneous detection of bacterial nucleic acid (16S rRNA) and host immune response protein (lactoferrin) on a single sensor array. Our results represent first integrated sensor platform capable of quantitative pathogen identification and measurement of host immune response, potentially providing clinical diagnosis that is not only more expeditious but also more informative than the current standard.

Hedgehog Signaling Restrains Bladder Cancer Progression by Eliciting Stromal Production of Urothelial Differentiation Factors

Hedgehog (Hh) pathway inhibitors are clinically effective in treatment of basal cell carcinoma and medulloblastoma, but fail therapeutically or accelerate progression in treatment of endodermally derived colon and pancreatic cancers. In bladder, another organ of endodermal origin, we find that despite its initial presence in the cancer cell of origin Sonic hedgehog (Shh) expression is invariably lost during progression to invasive urothelial carcinoma. Genetic blockade of stromal response to Shh furthermore dramatically accelerates progression and decreases survival time. This cancer-restraining effect of Hh pathway activity is associated with stromal expression of BMP signals, which stimulate urothelial differentiation. Progression is dramatically reduced by pharmacological activation of BMP pathway activity with low-dose FK506, suggesting an approach to management of human bladder cancer.

Endoscopic molecular imaging of human bladder cancer using a CD47 antibody

Fluorescently labeled CD47 antibody is an endoscopic molecular imaging agent to diagnose human bladder cancer. Lighting Up Bladder Cancer Lesions Molecular imaging of bladder cancer could greatly improve on current methods of diagnosis, which rely on white light–based imaging that looks for superficial tissue changes, such as color and texture. To this end, Pan et al. targeted the cancer-specific marker CD47 that is present on the surface of solid tumors, including in the bladder. The authors attached a brightly fluorescent tag, called a quantum dot, to a CD47 antibody. The fluorescent antibody was instilled into human bladders with muscle and nonmuscle invasive cancer that had been recently removed from patients. Overall, 119 bladder regions were analyzed using the fluorescent antibody and blue light cystoscopy. The authors reported a sensitivity of 82.9% and a specificity of 90.5%. They further correctly identified five of six carcinoma in situ lesions—a diagnostic challenge for white light imaging. Because this approach only requires topical administration of a fluorescent antibody and the use of already available clinical tools, it is hoped that this molecular imaging approach to diagnosing various bladder cancers will translate after further optimization. A combination of optical imaging technologies with cancer-specific molecular imaging agents is a potentially powerful strategy to improve cancer detection and enable image-guided surgery. Bladder cancer is primarily managed endoscopically by white light cystoscopy with suboptimal diagnostic accuracy. Emerging optical imaging technologies hold great potential for improved diagnostic accuracy but lack imaging agents for molecular specificity. Using fluorescently labeled CD47 antibody (anti-CD47) as molecular imaging agent, we demonstrated consistent identification of bladder cancer with clinical grade fluorescence imaging systems, confocal endomicroscopy, and blue light cystoscopy in fresh surgically removed human bladders. With blue light cystoscopy, the sensitivity and specificity for CD47-targeted imaging were 82.9 and 90.5%, respectively. We detected variants of bladder cancers, which are diagnostic challenges, including carcinoma in situ, residual carcinoma in tumor resection bed, recurrent carcinoma following prior intravesical immunotherapy with Bacillus Calmette-Guérin (BCG), and excluded cancer from benign but suspicious-appearing mucosa. CD47-targeted molecular imaging could improve diagnosis and resection thoroughness for bladder cancer.

Interobserver agreement of confocal laser endomicroscopy for bladder cancer.

BACKGROUND AND PURPOSE Emerging optical imaging technologies such as confocal laser endomicroscopy (CLE) hold promise in improving bladder cancer diagnosis. The purpose of this study was to determine the interobserver agreement of image interpretation using CLE for bladder cancer. METHODS Experienced CLE urologists (n=2), novice CLE urologists (n=6), pathologists (n=4), and nonclinical researchers (n=5) were recruited to participate in a 2-hour computer-based training consisting of a teaching and validation set of intraoperative white light cystoscopy (WLC) and CLE video sequences from patients undergoing transurethral resection of bladder tumor. Interobserver agreement was determined using the κ statistic. RESULTS Of the 31 bladder regions analyzed, 19 were cancer and 12 were benign. For cancer diagnosis, experienced CLE urologists had substantial agreement for both CLE and WLC+CLE (90%, κ 0.80) compared with moderate agreement for WLC alone (74%, κ 0.46), while novice CLE urologists had moderate agreement for CLE (77%, κ 0.55), WLC (78%, κ 0.54), and WLC+CLE (80%, κ 0.59). Pathologists had substantial agreement for CLE (81%, κ 0.61), and nonclinical researchers had moderate agreement (77%, κ 0.49) in cancer diagnosis. For cancer grading, experienced CLE urologists had fair to moderate agreement for CLE (68%, κ 0.64), WLC (74%, κ 0.67), and WLC+CLE (53%, κ 0.33), as did novice CLE urologists for CLE (53%, κ 0.39), WLC (66%, κ 0.50), and WLC+CLE (61%, κ 0.49). Pathologists (65%, κ 0.55) and nonclinical researchers (61%, κ 0.56) both had moderate agreement for CLE in cancer grading. CONCLUSIONS CLE is an adoptable technology for cancer diagnosis in novice CLE observers after a short training with moderate interobserver agreement and diagnostic accuracy similar to WLC alone. Experienced CLE observers may be capable of achieving substantial levels of agreement for cancer diagnosis that is higher than with WLC alone.

Clinical Validation of Integrated Nucleic Acid and Protein Detection on an Electrochemical Biosensor Array for Urinary Tract Infection Diagnosis

Background Urinary tract infection (UTI) is a common infection that poses a substantial healthcare burden, yet its definitive diagnosis can be challenging. There is a need for a rapid, sensitive and reliable analytical method that could allow early detection of UTI and reduce unnecessary antibiotics. Pathogen identification along with quantitative detection of lactoferrin, a measure of pyuria, may provide useful information towards the overall diagnosis of UTI. Here, we report an integrated biosensor platform capable of simultaneous pathogen identification and detection of urinary biomarker that could aid the effectiveness of the treatment and clinical management. Methodology/Principal Findings The integrated pathogen 16S rRNA and host lactoferrin detection using the biosensor array was performed on 113 clinical urine samples collected from patients at risk for complicated UTI. For pathogen detection, the biosensor used sandwich hybridization of capture and detector oligonucleotides to the target analyte, bacterial 16S rRNA. For detection of the protein biomarker, the biosensor used an analogous electrochemical sandwich assay based on capture and detector antibodies. For this assay, a set of oligonucleotide probes optimized for hybridization at 37°C to facilitate integration with the immunoassay was developed. This probe set targeted common uropathogens including E. coli, P. mirabilis, P. aeruginosa and Enterococcus spp. as well as less common uropathogens including Serratia, Providencia, Morganella and Staphylococcus spp. The biosensor assay for pathogen detection had a specificity of 97% and a sensitivity of 89%. A significant correlation was found between LTF concentration measured by the biosensor and WBC and leukocyte esterase (p<0.001 for both). Conclusion/Significance We successfully demonstrate simultaneous detection of nucleic acid and host immune marker on a single biosensor array in clinical samples. This platform can be used for multiplexed detection of nucleic acid and protein as the next generation of urinary tract infection diagnostics.

In vivo biodistribution and toxicity of intravesical administration of quantum dots for optical molecular imaging of bladder cancer

Optical molecular imaging holds the potential to improve cancer diagnosis. Fluorescent nanoparticles such as quantum dots (QD) offer superior optical characteristics compared to organic dyes, but their in vivo application is limited by potential toxicity from systemic administration. Topical administration provides an attractive route for targeted nanoparticles with the possibility of minimizing exposure and reduced dose. Previously, we demonstrated successful ex vivo endoscopic imaging of human bladder cancer by topical (i.e. intravesical) administration of QD-conjugated anti-CD47. Herein we investigate in vivo biodistribution and toxicity of intravesically instilled free QD and anti-CD47-QD in mice. In vivo biodistribution of anti-CD47-QD was assessed with inductively coupled plasma mass spectrometry. Local and systemic toxicity was assessed using blood tests, organ weights, and histology. On average, there was no significant accumulation of QD outside of the bladder, although in some mice we detected extravesical biodistribution of QD suggesting a route for systemic exposure under some conditions. There were no indications of acute toxicity up to 7 days after instillation. Intravesical administration of targeted nanoparticles can reduce systemic exposure, but for clinical use, nanoparticles with established biosafety profiles should be used to decrease long-term toxicity in cases where systemic exposure occurs.

Molecular Imaging in Urology

Imaging plays an integral role in urology by providing anatomical detail and diagnostic insight of urologic diseases. Cross-sectional imaging technologies including computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound (US) are used primarily for initial diagnosis, surgical planning, and disease surveillance. In the operating room setting, optical imaging based on white light has provided the illumination for endourologic procedures of the upper and lower urinary tracts, as well as complex abdominal and pelvic surgeries increasingly performed through the laparoscopic/robotic approach. Nevertheless, current imaging technologies still carry limitations such as inadequate sensitivity in early detection of metastases (CT and MRI) and suboptimal diagnostic accuracy (e.g., white-light cystoscopy). Molecular imaging offers the possibility of improved early detection, intraoperative surgical guidance with molecular specificity, and therapeutic monitoring. This review focuses on this exciting emerging field within urology.

Next generation of optical diagnostics for bladder cancer using probe-based confocal laser endomicroscopy

Real-time imaging with confocal laser endomicroscopy (CLE) probes that fit in standard endoscopes has emerged as a clinically feasible technology for optical biopsy of bladder cancer. Confocal images of normal, inflammatory, and neoplastic urothelium obtained with intravesical fluorescein can be differentiated by morphologic characteristics. We compiled a confocal atlas of the urinary tract using these diagnostic criteria to be used in a prospective diagnostic accuracy study. Patients scheduled to undergo transurethral resection of bladder tumor underwent white light cystoscopy (WLC), followed by CLE, and histologic confirmation of resected tissue. Areas that appeared normal by WLC were imaged and biopsied as controls. We imaged and prospectively analyzed 135 areas in 57 patients. We show that CLE improves the diagnostic accuracy of WLC for diagnosing benign tissue, low and high grade cancer. Interobserver studies showed a moderate level of agreement by urologists and nonclinical researchers. Despite morphologic differences between inflammation and cancer, real-time differentiation can still be challenging. Identification of bladder cancer-specific contrast agents could provide molecular specificity to CLE. By using fluorescently-labeled antibodies or peptides that bind to proteins expressed in bladder cancer, we have identified putative molecular contrast agents for targeted imaging with CLE. We describe one candidate agent - anti-CD47 - that was instilled into bladder specimens. The tumor and normal urothelium were imaged with CLE, with increased fluorescent signal demonstrated in areas of tumor compared to normal areas. Thus, cancer-specificity can be achieved using molecular contrast agents ex vivo in conjunction with CLE.

Abstract 4595: Molecular imaging of urothelial cancer using EGFR-binding peptides

INTRODUCTION White light cystoscopy (WLC), the current standard for bladder cancer diagnosis, has recognized shortcomings in differentiating non-papillary urothelial carcinoma from inflammation. Confocal laser endomicroscopy (CLE) with fluorescein (a non-specific contrast agent) can provide in vivo microscopy of the bladder as an adjunct to WLC. Targeted CLE with fluorescently labeled peptides against tumor surface markers may further enhance the utility of CLE for real time endoscopic imaging. Epidermal growth factor receptor (EGFR) is over-expressed in most bladder cancer cells, but absent in the superficial layer of normal urothelium, making it an attractive target for cancer-specific imaging. We report our efforts to identify a non-activating, EGFR-binding peptide labeled with FITC as a cancer-specific contrast agent for ex vivo bladder imaging with CLE. METHODS To screen for EGFR binding peptides, fragments of the EGFR-binding motif of human EGF (EGF1∼4) were displayed on phage surface. Binding affinity of the phage to recombinant EGFR and EGFR-expressing bladder cancer cell line was measured by ELISA. Peptides with the highest EGFR affinity were chemically synthesized and FITC labeled. Potential for activation of EGFR by the peptides was assessed in two EGFR-expressing cell lines by Western blot. For ex vivo imaging of urothelial cancer, fresh human bladder (n=1) or ureter (n=2) specimens were instilled and incubated with FITC-EGFR binding peptide, or a random sequence negative control peptide. Excess peptide was drained, specimens were opened and suspicious and normal appearing mucosa were imaged with CLE. Imaged tissue sections were excised for histopathologic comparison. The bladder and one ureter were first imaged with negative control peptide followed by imaging with the EGF peptide. RESULTS All phage displaying EGF peptides showed greater affinity to recombinant EGFR and EGFR-expressing cells compared to wild type phage. The two phage with highest affinity for EGFR in both ELISA assays were synthesized. Cell stimulation with both peptides did not induce phosphorylation of EGFR or downstream signaling molecules. In bladder and ureter specimens, greater fluorescence intensity was detected in areas of tumor compared to normal urothelium on CLE with EGF4-FITC. Histopathology confirmed that the suspicious regions of the bladder and ureter imaged were urothelial carcinoma. The random peptide showed minimal fluorescent signal on both normal and tumor areas imaged, and subsequent imaging of the same areas with EGF4-FITC demonstrated abundant fluorescent signal in tumor areas only. CONCLUSION We identified peptides derived from the EGF sequence that specifically bind, but do not activate EGFR. Ex vivo imaging of urothelial cancer with EGF4 peptide suggests that it can be used for cancer-specific molecular imaging. Future studies will be aimed at in vivo validation of EGF4 peptide on animal models for urothelial cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4595. doi:1538-7445.AM2012-4595