CEA, EpCAM, alpha v beta 6 and uPAR expression in rectal cancer patients with a pathological complete response after neoadjuvant therapy

Rectal cancer patients with a complete response after neoadjuvant therapy can be monitored with a watch-and-wait strategy. However, regrowth rates indicate that identification of patients with a pathological complete response (pCR) remains challenging. Targeted near-infrared fluorescence endoscopy is a potential tool to improve response evaluation. Promising tumor targets include carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), integrin alpha v beta 6, and urokinase-type plasminogen activator receptor (uPAR). To investigate the applicability of these targets, we analyzed protein expression by immunohistochemistry and quantified these by a total immunostaining score (TIS) in tissue of rectal cancer patients with a pCR. CEA, EpCAM, alpha v beta 6, and uPAR expression in the diagnostic biopsy was high (TIS > 6) in, respectively, 100%, 100%, 33%, and 46% of cases. CEA and EpCAM expressions were significantly higher in the diagnostic biopsy compared with the corresponding tumor bed (p < 0.01). CEA, EpCAM, alpha v beta 6, and uPAR expressions were low (TIS < 6) in the tumor bed in, respectively, 93%, 95%, 85%, and 62.5% of cases. Immunohistochemical evaluation shows that CEA and EpCAM could be suitable targets for response evaluation after neoadjuvant treatment, since expression of these targets in the primary tumor bed is low compared with the diagnostic biopsy and adjacent pre-existent rectal mucosa in more than 90% of patients with a pCR.Surgical oncolog

Orthotopic Breast Cancer Model to Investigate the Therapeutic Efficacy of Nanobody-Targeted Photodynamic Therapy

Photodynamic therapy (PDT) is characterized by the local application of laser light, which activates a photosensitizer to lead to the formation of singlet oxygen and other toxic reactive oxygen species, to finally kill cells. Recently, photosensitizers have been conjugated to nanobodies to render PDT more selective to cancer cells. Nanobodies are the smallest naturally derived antibody fragments from heavy-chain antibodies that exist in animals of the Camelidae family. Indeed, we have shown that nanobody-targeted PDT can lead to extensive and selective tumor damage, and thus the subsequent step is to assess whether this damage can delay or even inhibit tumor growth in vivo. To evaluate the therapeutic efficacy of PDT, mouse models are mostly employed in which human tumors are grown subcutaneously in the flank of the animals. Although very useful, it has been suggested that these tumors are further away from their natural environment and that tumors developed in the organ or tissue of origin would be closer to the natural situation. Thus, this chapter describes the development of an orthotopic model of breast cancer and the application of nanobody-targeted PDT, for the assessment of the therapeutic efficacy

Orthotopic Breast Cancer Model to Investigate the Therapeutic Efficacy of Nanobody-Targeted Photodynamic Therapy

Photodynamic therapy (PDT) is characterized by the local application of laser light, which activates a photosensitizer to lead to the formation of singlet oxygen and other toxic reactive oxygen species, to finally kill cells. Recently, photosensitizers have been conjugated to nanobodies to render PDT more selective to cancer cells. Nanobodies are the smallest naturally derived antibody fragments from heavy-chain antibodies that exist in animals of the Camelidae family. Indeed, we have shown that nanobody-targeted PDT can lead to extensive and selective tumor damage, and thus the subsequent step is to assess whether this damage can delay or even inhibit tumor growth in vivo. To evaluate the therapeutic efficacy of PDT, mouse models are mostly employed in which human tumors are grown subcutaneously in the flank of the animals. Although very useful, it has been suggested that these tumors are further away from their natural environment and that tumors developed in the organ or tissue of origin would be closer to the natural situation. Thus, this chapter describes the development of an orthotopic model of breast cancer and the application of nanobody-targeted PDT, for the assessment of the therapeutic efficacy

Detecting tumour-positive resection margins after oral cancer surgery by spraying a fluorescent tracer activated by gamma-glutamyltranspeptidase

Surgical oncolog

Side-by-Side Comparison of uPAR-Targeting Optical Imaging Antibodies and Antibody Fragments for Fluorescence-Guided Surgery of Solid Tumors

Purpose: Radical resection is paramount for curative oncological surgery. Fluorescence-guided surgery (FGS) aids in intraoperative identification of tumor-positive resection margins. This study aims to assess the feasibility of urokinase plasminogen activator receptor (uPAR) targeting antibody fragments for FGS in a direct comparison with their parent IgG in various relevant in vivo models. Procedures: Humanized anti-uPAR monoclonal antibody MNPR-101 (uIgG) was proteolytically digested into F(ab’)2 and Fab fragments named uFab2 and uFab. Surface plasmon resonance (SPR) and cell assays were used to determine in vitro binding before and after fluorescent labeling with IRDye800CW. Mice bearing subcutaneous HT-29 human colonic cancer cells were imaged serially for up to 120 h after fluorescent tracer administration. Imaging characteristics and ex vivo organ biodistribution were further compared in orthotopic pancreatic ductal adenocarcinoma (BxPc-3-luc2), head-and-neck squamous cell carcinoma (OSC-19-luc2-GFP), and peritoneal carcinomatosis (HT29-luc2) models using the clinical Artemis fluorescence imaging system. Results: Unconjugated and conjugated uIgG, uFab2, and uFab specifically recognized uPAR in the nanomolar range as determined by SPR and cell assays. Subcutaneous tumors were clearly identifiable with tumor-to-background ratios (TBRs) > 2 after 72 h for uIgG-800F and 24 h for uFab2-800F and uFab-800F. For the latter two, mean fluorescence intensities (MFIs) dipped below predetermined threshold after 72 h and 36 h, respectively. Tumors were easily identified in the orthotopic models with uIgG-800F consistently having the highest MFIs and uFab2-800F and uFab-800F having similar values. In biodistribution studies, kidney and liver fluorescence approached tumor fluorescence after uIgG-800F administration and surpassed tumor fluorescence after uFab2-800F or uFab-800F administration, resulting in interference in the abdominal orthotopic mouse models. Conclusions: In a side-by-side comparison, FGS with uPAR-targeting antibody fragments compared with the parent IgG resulted in earlier tumor visualization at the expense of peak fluorescence intensity

Data-Driven Identification of Targets for Fluorescence-Guided Surgery in Non-Small Cell Lung Cancer

Purpose: Intraoperative identification of lung tumors can be challenging. Tumor-targeted fluorescence-guided surgery can provide surgeons with a tool for real-time intraoperative tumor detection. This study evaluated cell surface biomarkers, partially selected via data-driven selection software, as potential targets for fluorescence-guided surgery in non-small cell lung cancers: adenocarcinomas (ADC), adenocarcinomas in situ (AIS), and squamous cell carcinomas (SCC).  Procedures: Formalin-fixed paraffin-embedded tissue slides of resection specimens from 15 patients with ADC and 15 patients with SCC were used and compared to healthy tissue. Molecular targets were selected based on two strategies: (1) a data-driven selection using > 275 multi-omics databases, literature, and experimental evidence; and (2) the availability of a fluorescent targeting ligand in advanced stages of clinical development. The selected targets were carbonic anhydrase 9 (CAIX), collagen type XVII alpha 1 chain (collagen XVII), glucose transporter 1 (GLUT1), G protein-coupled receptor 87 (GPR87), transmembrane protease serine 4 (TMPRSS4), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), folate receptor alpha (FRα), integrin αvβ6 (αvβ6), and urokinase-type plasminogen activator receptor (uPAR). Tumor expression of these targets was assessed by immunohistochemical staining. A total immunostaining score (TIS, range 0–12), combining the percentage and intensity of stained cells, was calculated. The most promising targets in ADC were explored in six AIS tissue slides to explore its potential in non-palpable lesions.  Results: Statistically significant differences in TIS between healthy lung and tumor tissue for ADC samples were found for CEA, EpCAM, FRα, αvβ6, CAIX, collagen XVII, GLUT-1, and TMPRSS4, and of these, CEA, CAIX, and collagen XVII were also found in AIS. For SCC, EpCAM, uPAR, CAIX, collagen XVII, and GLUT-1 were found to be overexpressed.  Conclusions: EpCAM, CAIX, and Collagen XVII were identified using concomitant use of data-driven selection software and clinical evidence as promising targets for intraoperative fluorescence imaging for both major subtypes of non-small cell lung carcinomas

Dose-Finding Study of a CEA-Targeting Agent, SGM-101, for Intraoperative Fluorescence Imaging of Colorectal Cancer

Background: Carcinoembryonic antigen is overexpressed in colorectal cancer (CRC), making it an optimal target for fluorescence imaging. A phase I/II study was designed to determine the optimal imaging dose of SGM-101 for intraoperative fluorescence imaging of primary and recurrent CRC. Methods: Patients were included and received a single dose of SGM-101 at least 24 h before surgery. Patients who received routine anticancer therapy (i.e., radiotherapy or chemotherapy) also were eligible. A dedicated near-infrared imaging system was used for real-time fluorescence imaging during surgery. Safety assessments were performed and SGM-101 efficacy was evaluated per dose level to determine the most optimal imaging dose. Results: Thirty-seven patients with CRC were included in the analysis. Fluorescence was visible in all primary and recurrent tumors. In seven patients, no fluorescence was seen; all were confirmed as pathological complete responses after neoadjuvant therapy. Two tumors showed false-positive fluorescence. In the 37 patients, a total of 97 lesions were excised. The highest mean intraoperative tumor-to-background ratio (TBR) of 1.9 (p = 0.019) was seen in the 10-mg dose. This dose showed a sensitivity of 96%, specificity of 63%, and negative predictive value of 94%. Nine patients (24%) had a surgical plan alteration based on fluorescence, with additional malignant lesions detected in six patients. Conclusions: The optimal imaging dose was established at 10 mg 4 days before surgery. The results accentuate the potential of SGM-101 and designated a promising base for the multinational phase III study, which enrolled the first patients in June 2019

Molecular targets for diagnostic and intraoperative imaging of pancreatic ductal adenocarcinoma after neoadjuvant FOLFIRINOX treatment

Neoadjuvant systemic treatment is increasingly being integrated in the standard treatment of pancreatic ductal adenocarcinoma (PDAC) patients to improve oncological outcomes. Current available imaging techniques remain unreliable in assessing response to therapies, as they cannot distinguish between (vital) tumor tissue and therapy induced fibrosis (TIF). Consequently, resections with tumor positive margins and subsequent early post-operative recurrences occur and patients eligible for potential radical resection could be missed. To optimize patient selection and monitor results of neoadjuvant treatment, PDAC-specific diagnostic and intraoperative molecular imaging methods are required. This study aims to evaluate molecular imaging targets for PDAC after neoadjuvant FOLFIRINOX treatment. Expression of integrin αvβ6, carcinoembryonic antigen cell adhesion molecule 5 (CEACAM5), mesothelin, prostate-specific membrane antigen (PSMA), urokinase-type plasminogen activator receptor, fibroblast activating receptor, integrin α5 subunit and epidermal growth factor receptor was evaluated using immunohistochemistry. Immunoreactivity was determined using the semiquantitative H-score. Resection specimens from patients after neoadjuvant FOLFIRINOX treatment containing PDAC (n = 32), tumor associated pancreatitis (TAP) and TIF (n = 15), normal pancreas parenchyma (NPP) (n = 32) and tumor positive (n = 24) and negative (n = 56) lymph nodes were included. Integrin αvβ6, CEACAM5, mesothelin and PSMA stainings showed significantly higher expression in PDAC compared to TAP and NPP. No expression of αvβ6, CEACAM5 and mesothelin was observed in TIF. Integrin αvβ6 and CEACAM5 allow for accurate metastatic lymph node detection. Targeting integrin αvβ6, CEA, mesothelin and PSMA has the potential to distinguish vital PDAC from fibrotic tissue after neoadjuvant FOLFIRINOX treatment. Integrin αvβ6 and CEACAM5 detect primary tumors and tumor positive lymph nodes

A multimodal molecular imaging approach targeting urokinase plasminogen activator receptor for the diagnosis, resection and surveillance of urothelial cell carcinoma

With a 5-year recurrence rate of 30–78%, urothelial cell carcinoma (UCC) rates amongst the highest of all solid malignancies. Consequently, after transurethral resection, patients are subjugated to life-long endoscopic surveillance. A multimodal near-infrared (NIR) fluorescence-based imaging strategy can improve diagnosis, resection and surveillance, hence increasing quality of life. Methods: Expression of urokinase plasminogen activator receptor (uPAR) and epithelial cell adhesion molecule (EpCAM) are determined on paraffin-embedded human UCC using immunohistochemistry and on UCC cell lines by flow cytometry. MNPR-101, a humanised monoclonal antibody targeting uPAR is conjugated to IRDye800CW and binding is validated in vitro using surface plasmon resonance and cell-based binding assays. In vivo NIR fluorescence and photoacoustic three-dimensional (3D) imaging are performed with subcutaneously growing human UM-UC-3luc2 cells in BALB/c-nude mice. The translational potential is confirmed in a metastasising UM-UC-3luc2 orthotopic mouse model. Infliximab-IRDye800CW and rituximab-IRDye800CW are used as controls. Results: UCCs show prominent uPAR expression at the tumour-stroma interface and EpCAM on epithelial cells. uPAR and EpCAM are expressed by 6/7 and 4/7 UCC cell lines, respectively. In vitro, MNPR-101-IRDye800CW has a picomolar affinity for domain 2-3 of uPAR. In vivo fluorescence imaging with MNPR-101-IRDye800CW, specifically delineates both subcutaneous and orthotopic tumours with tumour-to-background ratios reaching as high as 6.8, differing significantly from controls (p &lt; 0.0001). Photoacoustic 3D in depth imaging confirms the homogenous distribution of MNPR-101-IRDye800CW through the tumour. Conclusions: MNPR-101-IRDye800CW is suitable for multimodal imaging of UCC, awaiting clinical translation