Fluorescence-guided surgery using cetuximab-800CW in patients with penile carcinoma.

OBJECTIVE: To investigate the feasibility of fluorescence molecular imaging (FMI), using cetuximab-800CW, as an intraoperative tool to determine surgical margins in penile squamous cell carcinoma (PSCC). PATIENTS AND METHODS: A total of 11 patients with PSCC received 75 mg cetuximab followed by 15 mg cetuximab-800CW 2 days before surgery. FMI of the whole excision specimen and tissue slices was performed. Fluorescence visualisation was correlated to histopathology. Based on tumour and healthy tissue regions of interest, mean fluorescence intensity was calculated for each individual patient. RESULTS: Significant differences between tumour and healthy mean fluorescence intensity were found with tumour-to-background ratios of a median (IQR) of 1.51 (0.99) and a mean (SD) of 1.51 (0.32) in the excision specimen and tissue slices, respectively. One patient showed a high relative fluorescence intensity with a signal-to-background ratio of 1.79, corresponding to a tumour-positive margin on fresh frozen sectioning. CONCLUSION: In this Phase I study we showed that cetuximab-800CW seems suitable to discriminate PSCC from background tissue. The tracer was well tolerated, and no false positive spots were seen.

Potential Receptors for Targeted Imaging of Lymph Node Metastases in Penile Cancer.

Imaging modalities using tumor-directed monoclonal antibodies may be of value to improve the pre- and intraoperative detection and resection of lymph node (LN) metastatic disease in penile squamous cell carcinoma (PSCC). We investigated the expression of prostate-specific membrane antigen (PSMA), vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR) and epithelial cell adhesion molecule (EpCAM) to analyze their potency for diagnostic applications. Antigen expression was determined in primary tumors and LNs with and without metastases of 22 patients with PSCC. The total immunostaining score (TIS, 0-12) was determined as the product of a proportion score (PS, 0-4) and an intensity score (IS, 0-3). EGFR and VEGF expression were high in primary tumor (median TIS 8) and LN metastases (median TIS 6 and 8, respectively). No EGFR expression was seen in LNs without metastases. However, LNs without metastases did show VEGF expression (median TIS 6). No EpCAM or PSMA expression was seen in PSCC. This study shows that VEGF and EGFR expression is moderate to high in LN metastases of PSCC. Both VEGF and EGFR warrant further clinical evaluation to determine their value as a target for pre- and intraoperative imaging modalities in the detection of LN metastases in PSCC.

EpCAM Expression in Lymph Node Metastases of Urothelial Cell Carcinoma of the Bladder: A Pilot Study.

In this retrospective pilot study, the feasibility of the epithelial cell adhesion molecule (EpCAM) as an imaging target for lymph node (LN) metastatic disease of urothelial cell carcinoma (UCC) of the bladder was investigated. LN metastases and LNs without metastases of patients who underwent pelvic lymph node dissection because of muscle invasive bladder cancer (MIBC) were used. Primary tumors of the same patients were used from cystectomy specimen, transurethral resections, and biopsies. A pathologist, blinded to clinical data, scored EpCAM immunoreactivity. This method determines a total immunostaining score, which is the product of a proportion score and an intensity score. EpCAM expression was observed in 19/20 (95%) LNs with UCC metastases and in 11/12 (92%) of the primary tumors. EpCAM expression was absent in 14/14 (100%) LNs without metastases. Median EpCAM expression (TIS) in LN metastases was 5 (IQR 2.0-8.0) and in the primary tumors 6 (IQR 2.3-11.0). Based on the absence of staining in LNs without metastases, EpCAM show high tumor distinctiveness. EpCAM seems to be a feasible imaging target in LN metastases of UCC of the bladder. Pre- and perioperative visualization of these metastases will improve disease staging and improve the complete resection of LN metastases in MIBC.

Immuno-based detection of extracellular vesicles in urine as diagnostic marker for prostate cancer.

Extracellular vesicles (including the subclass exosomes) secreted by cells contain specific proteins and RNA that could be of interest in determining new markers. Isolation/characterization of PCa-derived exosomes from bodily fluids enables us to discover new markers for this disease. Unfortunately, isolation with current techniques (ultracentrifugation) is labor intensive and other techniques are still under development. The goal of our study was to develop a highly sensitive time-resolved fluorescence immunoassay (TR-FIA) for capture/detection of PCa-derived exosomes. In our assay, biotinylated capture antibodies against human CD9 or CD63 were incubated on streptavidin-coated wells. After application of exosomes, Europium-labeled detection antibodies (CD9 or CD63) were added. Cell medium from 37 cell lines was taken to validate this TR-FIA. Urine was collected (after digital rectal exam) from patients with PCa (n = 67), men without PCa (n = 76). As a control, urine was collected from men after radical prostatectomy (n = 13), women (n = 16) and patients with prostate cancer without digital rectal exam (n = 16). Signal intensities were corrected for urinary PSA and creatinine. This TR-FIA can measure purified exosomes with high sensitivity and minimal background signals. Exosomes can be measured in medium from 37 cell lines and in urine. DRE resulted in a pronounced increase in CD63 signals. After DRE and correction for urinary PSA, CD9 and CD63 were significantly higher in men with PCa. This TR-FIA enabled us to measure exosomes with high sensitivity directly from urine and cell medium. This TR-FIA forms the basis for testing different antibodies directed against exosome membrane markers to generate disease-specific detection assays.