Comparison of Signal Intensity in Bladder Urothelial Carcinomas Using pHLIP® Molecular Probes
Borivoj Golijanin, BS1, Ali Amin, MD1, Michael DuPont, BS2, Anna Moshnikova, PhD2, Ohad Kott, MD3, Yana K. Reshetnyak, PhD2, Oleg A. Andreev, PhD2, Dragan J. Golijanin, MD3.
1Department of Pathology & Laboratory Medicine, The Miriam Hospital; The Warren Alpert Medical School of Brown University, Providence, RI, USA, 2Physics Department, University of Rhode Island, Kingston, RI, USA, 3The Minimally Invasive Urology Institute, The Miriam Hospital; The Warren Alpert Medical School of Brown University, Providence, RI, USA.
BACKGROUND: Urothelial carcinomas (UC) are a heterogeneous malignancy with an acidic microenvironment. pH low insertion peptides (pHLIPs) are a class of pH specific transmembrane peptides that target the acidic microenvironment of cancer cells. pHLIP variant-3 (Var3-pHLIP) was conjugated to a near infrared fluorescent (NIRF) dye to evaluate specificity and sensitivity as a tumor targeting molecular imaging probe. METHODS: After incubation for 15 minutes with Var3-pHLIP conjugated to indocyanine green (ICG) or IRDye® 800CW, 38 ex-vivo bladder specimens from patients undergoing robotic assisted laparoscopic radical cystectomy for bladder cancer were placed under NIRF capable laser to excite the fluorophore. Peak signal intensity and the corrected mean intensity of malignant and nonmalignant cases were analyzed and compared using paired samples t-test. The number of lesions visible under NIRF was compared with the number identified under white light using paired samples t-test. Expression of NIRF signal and identification of lesions under white light by urologic oncology pathologist were compared to histopathology for specificity and sensitivity calculations. RESULTS: Of 58 lesions processed for histopathological evaluation, 47 (81%) were seen under white light and 57 (98.3%) were seen with Var3=pHLIP, representing an improved diagnosis by 17.3% (p=0.003). In NIRF, Var3-pHLIP demonstrated an average peak signal intensity of 116.4 relative fluorescent units (RFU) in malignant cases, and nonmalignant cases had average peak of 44.3 RFU (p<0.001). Corrected average signal intensity of malignant cases demonstrated an average of 52.9 RFU/µm2 and nonmalignant cases showed an average of 25 RFU/µm2 (p<0.001). Var3-pHLIP demonstrated 98% sensitivity and 100% specificity in identification of UC. CONCLUSIONS: Var3-pHLIP NIRF enhanced visualization and improved diagnosis of UC. Categorizing into broader categories of malignant and nonmalignant, the unique Var3-pHLIP NIRF signal can be used to differentiate between the two groups based on both peak signal intensity and mean intensity per unit area of lesion. NIRF Var3-pHLIP identified UC irrespective of subtype, previous treatment, and stage. All CIS cases missed by white light cystoscopy were diagnosed using Var3-pHLIP NIRF imaging. Additional work into digital, automated analysis of Var3-pHLIP NIRF signal and development of a fluorescent signal analysis capable cystoscope can lead to diagnosis of bladder cancer at the time of cystoscopy.
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