Fluorescence-guided surgery (FGS) can play a key role in improving radical resection rates by assisting surgeons to gain adequate visualization of malignant tissue intraoperatively. Designed... Show moreFluorescence-guided surgery (FGS) can play a key role in improving radical resection rates by assisting surgeons to gain adequate visualization of malignant tissue intraoperatively. Designed ankyrin repeat proteins (DARPins) possess optimal pharmacokinetic and other properties for in vivo imaging. This study aims to evaluate the preclinical potential of epithelial cell adhesion molecule (EpCAM)-binding DARPins as targeting moieties for near-infrared fluorescence (NIRF) and photoacoustic (PA) imaging of cancer. EpCAM-binding DARPins Ac2, Ec4.1, and non-binding control DARPin Off7 were conjugated to IRDye 800CW and their binding efficacy was evaluated on EpCAM-positive HT-29 and EpCAM-negative COLO-320 human colon cancer cell lines. Thereafter, NIRF and PA imaging of all three conjugates were performed in HT-29_luc2 tumor-bearing mice. At 24 h post-injection, tumors and organs were resected and tracer biodistributions were analyzed. Ac2-800CW and Ec4.1-800CW specifically bound to HT-29 cells, but not to COLO-320 cells. Next, 6 nmol and 24 h were established as the optimal in vivo dose and imaging time point for both DARPin tracers. At 24 h post-injection, mean tumor-to-background ratios of 2.60 ± 0.3 and 3.1 ± 0.3 were observed for Ac2-800CW and Ec4.1-800CW, respectively, allowing clear tumor delineation using the clinical Artemis NIRF imager. Biodistribution analyses in non-neoplastic tissue solely showed high fluorescence signal in the liver and kidney, which reflects the clearance of the DARPin tracers. Our encouraging results show that EpCAM-binding DARPins are a promising class of targeting moieties for pan-carcinoma targeting, providing clear tumor delineation at 24 h post-injection. The work described provides the preclinical foundation for DARPin-based bimodal NIRF/PA imaging of cancer. Show less
PurposeFluorescence-guided surgery (FGS) can play a key role in improving radical resection rates by assisting surgeons to gain adequate visualization of malignant tissue intraoperatively. Designed... Show morePurposeFluorescence-guided surgery (FGS) can play a key role in improving radical resection rates by assisting surgeons to gain adequate visualization of malignant tissue intraoperatively. Designed ankyrin repeat proteins (DARPins) possess optimal pharmacokinetic and other properties for in vivo imaging. This study aims to evaluate the preclinical potential of epithelial cell adhesion molecule (EpCAM)-binding DARPins as targeting moieties for near-infrared fluorescence (NIRF) and photoacoustic (PA) imaging of cancer.MethodsEpCAM-binding DARPins Ac2, Ec4.1, and non-binding control DARPin Off7 were conjugated to IRDye 800CW and their binding efficacy was evaluated on EpCAM-positive HT-29 and EpCAM-negative COLO-320 human colon cancer cell lines. Thereafter, NIRF and PA imaging of all three conjugates were performed in HT-29_luc2 tumor-bearing mice. At 24 h post-injection, tumors and organs were resected and tracer biodistributions were analyzed.ResultsAc2-800CW and Ec4.1-800CW specifically bound to HT-29 cells, but not to COLO-320 cells. Next, 6 nmol and 24 h were established as the optimal in vivo dose and imaging time point for both DARPin tracers. At 24 h post-injection, mean tumor-to-background ratios of 2.60 & PLUSMN; 0.3 and 3.1 & PLUSMN; 0.3 were observed for Ac2-800CW and Ec4.1-800CW, respectively, allowing clear tumor delineation using the clinical Artemis NIRF imager. Biodistribution analyses in non-neoplastic tissue solely showed high fluorescence signal in the liver and kidney, which reflects the clearance of the DARPin tracers.ConclusionOur encouraging results show that EpCAM-binding DARPins are a promising class of targeting moieties for pan-carcinoma targeting, providing clear tumor delineation at 24 h post-injection. The work described provides the preclinical foundation for DARPin-based bimodal NIRF/PA imaging of cancer. Show less
Jeremiasse, B.; Rijs, Z.; Angoelal, K.R.; Hiemcke-Jiwa, L.S.; Boed, E.A. de; Kuppen, P.J.K.; ... ; Steeg, A.F.W. van der 2023
Simple Summary The only cure for children with Ewing sarcoma (ES) is surgery. Unfortunately, surgeons are often not able to differentiate healthy from malignant tissue. Fluorescent imaging during... Show moreSimple Summary The only cure for children with Ewing sarcoma (ES) is surgery. Unfortunately, surgeons are often not able to differentiate healthy from malignant tissue. Fluorescent imaging during the operation will facilitate recognition of malignant cells, but unfortunately there are no ES specific tracers available yet. We searched for proteins on ES cells that could be used as a target against which specific tracers could be developed. The most promising proteins, CD99, CD117, and GD2, were found in paraffin-embedded tissue samples collected from ES patients. Tracers against CD99 and CD117, consisting of monoclonal antibodies attached with a fluorescent dye, showed positive signals on cultured ES cells. In a proof-of-concept study, these tracers were topically applied on fresh ES tissue, showing a signal in the tumor. Our results indicate the applicability for fluorescence-guided surgery of ES-based tracers, but these data have to be confirmed in a larger cohort of pediatric ES patients. Fluorescence-guided surgery (FGS), based on fluorescent tracers binding to tumor-specific biomarkers, could assist surgeons to achieve complete tumor resections. This study evaluated potential biomarkers for FGS in pediatric Ewing sarcoma (ES). Immunohistochemistry (IHC) was performed to assess CD99, CXCR4, CD117, NPY-R-Y1, and IGF-1R expression in ES biopsies and resection specimens. LINGO-1 and GD2 evaluation did not work on the acquired tissue. Based on the immunoreactive scores, anti-CD99 and anti-CD117 were evaluated for binding specificity using flow cytometry and immunofluorescence microscopy. Anti-GD2, a tracer in the developmental phase, was also tested. These three tracers were topically applied to a freshly resected ES tumor and adjacent healthy tissue. IHC demonstrated moderate/strong CD99 and CD117 expression in ES tumor samples, while adjacent healthy tissue had limited expression. Flow cytometry and immunofluorescence microscopy confirmed high CD99 expression, along with low/moderate CD117 and low GD2 expression, in ES cell lines. Topical anti-CD99 and anti-GD2 application on ES tumor showed fluorescence, while anti-CD117 did not show fluorescence for this patient. In conclusion, CD99-targeting tracers hold promise for FGS of ES. CD117 and GD2 tracers could be potential alternatives. The next step towards development of ES-specific FGS tracers could be ex vivo topical application experiments on a large cohort of ES patients. Show less
Huisman, B.W.; Cankat, M.; Bosse, T.; Vahrmeijer, A.L.; Rissmann, R.; Burggraaf, J.; ... ; Poelgeest, M.I.E. van 2023
BackgroundDifferentiating high-grade dysplasia (HGD) and T1 colorectal cancer (T1CRC) from low-grade dysplasia (LGD) in colorectal polyps can be challenging. Incorrect recognition of HGD or T1CRC... Show moreBackgroundDifferentiating high-grade dysplasia (HGD) and T1 colorectal cancer (T1CRC) from low-grade dysplasia (LGD) in colorectal polyps can be challenging. Incorrect recognition of HGD or T1CRC foci can lead to a need for additional treatment after local resection, which might not have been necessary if it was recognized correctly. Tumor-targeted fluorescence-guided endoscopy might help to improve recognition.ObjectiveSelecting the most suitable HGD and T1CRC-specific imaging target from a panel of well-established biomarkers: carcinoembryonic antigen (CEA), c-mesenchymal-epithelial transition factor (c-MET), epithelial cell adhesion molecule (EpCAM), folate receptor alpha (FRα), and integrin alpha-v beta-6 (αvβ6).MethodsEn bloc resection specimens of colorectal polyps harboring HGD or T1CRC were selected. Immunohistochemistry on paraffin sections was used to determine the biomarker expression in normal epithelium, LGD, HGD, and T1CRC (scores of 0–12). The differential expression in HGD-T1CRC components compared to surrounding LGD and normal components was assessed, just as the sensitivity and specificity of each marker.Results60 specimens were included (21 HGD, 39 T1CRC). Positive expression (score >1) of HGD-T1CRC components was found in 73.3%, 78.3%, and 100% of cases for CEA, c-MET, and EpCAM, respectively, and in <40% for FRα and αvβ6. Negative expression (score 0–1) of the LGD component occurred more frequently for CEA (66.1%) than c-MET (31.6%) and EpCAM (0%). The differential expression in the HGD-T1CRC component compared to the surrounding LGD component was found for CEA in 66.7%, for c-MET in 43.1%, for EpCAM in 17.2%, for FRα in 22.4%, and for αvβ6 in 15.5% of the cases. Moreover, CEA showed the highest combined sensitivity (65.0%) and specificity (75.0%) for the detection of an HGD-T1CRC component in colorectal polyps.ConclusionOf the tested targets, CEA appears the most suitable to specifically detect HGD and T1 cancer foci in colorectal polyps. An in vivo study using tumor-targeted fluorescence-guided endoscopy should confirm these findings. Show less
Introduction: Myxofibrosarcoma (MFS) is the most common soft-tissue sarcoma subtype in elderly patients. Local recurrence (LR) remains a major concern as the lack of intraoperative guidance and an... Show moreIntroduction: Myxofibrosarcoma (MFS) is the most common soft-tissue sarcoma subtype in elderly patients. Local recurrence (LR) remains a major concern as the lack of intraoperative guidance and an infiltrative growth pattern with long, slender tails hamper surgeons' ability to achieve adequate resection margins for MFS. Fluorescence-guided surgery (FGS) could overcome this concern by delineating tumor tissue during surgery. One of the most important steps to successful FGS is to define a tumor-specific biomarker that is highly overexpressed in tumor tissue while low or absent in adjacent healthy tissue. The aim of this study is to evaluate the expression of eight previously selected promising biomarkers for FGS in MFS tissue samples with adjacent healthy tissue using immunohistochemistry (IHC). Methods: The following eight biomarkers were stained in seventeen paraffin-embedded MFS samples: tumor endothelial marker-1 (TEM-1, also known as endosialin/CD248), vascular endothelial growth factor receptor-1 (VEGFR-1, also known as Flt-1), vascular endothelial growth factor receptor-2 (VEGFR-2, also known as Flk1), vascular endothelial growth factor-A (VEGF-A), epidermal growth factor receptor (EGFR), insulin-like growth factor-1 receptor (IGF-1R), platelet derived growth factor receptor-alpha (PDGFR-alpha), and cluster of differentiation 40 (CD40, also known as TNFRSF5). A pathologist specializing in sarcoma annotated the margin between the tumor and adjacent healthy tissue in each MFS tissue sample. Subsequently, we used an objective IHC scoring method to assess and compare the difference in staining intensity between the tumor and adjacent healthy tissue, which is crucial for the use of FGS. Results: TEM-1, VEGF-A, and PDGFR-alpha stained all MFS tumors, while the other biomarkers did not show expression in all MFS tumors. Ultimately, TEM-1 was identified as the most suitable biomarker for FGS in MFS based on higher tumor-to-background (TBR) staining intensity compared to VEGF-A and PDGFR-alpha, regardless of preoperative therapy. Conclusion: TEM-1-targeted FGS tracers should be further investigated to optimize MFS treatment. Show less
Houvast, R.D.; Duijvenvoorde, M. van; Chua, J.; Vankemmelbeke, M.; Durrant, L.G.; Inderson, A.; ... ; Kuppen, P.J.K. 2023
Background: Targeted molecular imaging may improve tumor cell identification during diagnosis and resection of pancreatic ductal adenocarcinoma (PDAC). Although many molecular imaging biomarkers... Show moreBackground: Targeted molecular imaging may improve tumor cell identification during diagnosis and resection of pancreatic ductal adenocarcinoma (PDAC). Although many molecular imaging biomarkers are (over)expressed in PDAC, intertumoral heterogeneity of biomarker expression hampers universal tracer administration. Preoperative, patient-specific screening and selection of the most optimal biomarker could therefore improve tumor delineation. Objective: This study evaluated whether fine-needle biopsy (FNB) specimens could be used to preoperatively predict biomarker expression in the corresponding primary PDAC specimen. Methods: Expression of previously identified PDAC biomarkers alpha(v)beta(6), CEACAM5, EGFR, mesothelin, Le(a/c/x), and sdi-Le(a) on FNB and corresponding primary tumor (PT) specimens (n = 45) was evaluated using immunohistochemistry and quantified using a semi-automated image analysis workflow. Results: Biomarker expression on FNB and PT tissues showed high concordance ( increment H-score <= 50), i.e. was present in 62% of cases for alpha(v)beta(6), 61% for CEACAM5, 85% for EGFR, 69% for mesothelin, 76% for Le(a/c/x), and 79% for sdi-Le(a), indicating high concordance. Except for alpha(v)beta(6), biomarker expression on FNB tissues was positively correlated with PT expression for all biomarkers. Subgroup analyses showed that neoadjuvant therapy (NAT) had no major and/or significant effect on concordance, expression difference and, except for mesothelin, correlation of biomarker expression between FNB and PT tissues. Conclusion: This study demonstrated that biomarker expression in FNB tissues is predictive for PT expression, irrespective of the application of NAT. These findings thereby provide the foundation for the clinical application of an FNB-based biomarker-screening workflow, eventually facilitating a patient-specific approach of molecular imaging tracer administration in PDAC. Show less
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.... Show morePurpose: 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 (FR alpha), integrin alpha v beta 6 (alpha v beta 6), 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, FR alpha, alpha v beta 6, 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. Show less
Simple Summary Patients diagnosed with pancreatic cancer have a poor prognosis at time of diagnosis, with a 5-year survival rate of merely 10%. The only treatment with curative intent is surgical... Show moreSimple Summary Patients diagnosed with pancreatic cancer have a poor prognosis at time of diagnosis, with a 5-year survival rate of merely 10%. The only treatment with curative intent is surgical resection of the tumor and adjacent tumor-containing lymph nodes. To improve surgical outcome and survival, additional (imaging) tools are needed that support complete surgical tumor resection. Firstly, more accurate monitoring of tumor response to neoadjuvant treatment and subsequent determination of resectability is needed. Secondly, an imaging tool is needed for intraoperative guidance allowing accurate identification, delineation, and complete resection of the tumor and suspected lymph nodes. Therefore, both tumor-targeted PET/CT before surgery and real time fluorescence-guidance during surgery could be helpful to improve patient outcome. This review focusses on literature considering tumor-targeted PET/CT and near-infrared fluorescence (NIRF) imaging. Several tumor-targeted agents are under clinical evaluation, and several other promising agents are currently tested preclinically, both with promising results. Their additional diagnostic value and feasibility for future implementation in standard clinical care of PDAC has yet to be established in phase III clinical trials. Background: Despite recent advances in the multimodal treatment of pancreatic ductal adenocarcinoma (PDAC), overall survival remains poor with a 5-year cumulative survival of approximately 10%. Neoadjuvant (chemo- and/or radio-) therapy is increasingly incorporated in treatment strategies for patients with (borderline) resectable and locally advanced disease. Neoadjuvant therapy aims to improve radical resection rates by reducing tumor mass and (partial) encasement of important vascular structures, as well as eradicating occult micrometastases. Results from recent multicenter clinical trials evaluating this approach demonstrate prolonged survival and increased complete surgical resection rates (R0). Currently, tumor response to neoadjuvant therapy is monitored using computed tomography (CT) following the RECIST 1.1 criteria. Accurate assessment of neoadjuvant treatment response and tumor resectability is considered a major challenge, as current conventional imaging modalities provide limited accuracy and specificity for discrimination between necrosis, fibrosis, and remaining vital tumor tissue. As a consequence, resections with tumor-positive margins and subsequent early locoregional tumor recurrences are observed in a substantial number of patients following surgical resection with curative intent. Of these patients, up to 80% are diagnosed with recurrent disease after a median disease-free interval of merely 8 months. These numbers underline the urgent need to improve imaging modalities for more accurate assessment of therapy response and subsequent re-staging of disease, thereby aiming to optimize individual patient's treatment strategy. In cases of curative intent resection, additional intra-operative real-time guidance could aid surgeons during complex procedures and potentially reduce the rate of incomplete resections and early (locoregional) tumor recurrences. In recent years intraoperative imaging in cancer has made a shift towards tumor-specific molecular targeting. Several important molecular targets have been identified that show overexpression in PDAC, for example: CA19.9, CEA, EGFR, VEGFR/VEGF-A, uPA/uPAR, and various integrins.Tumor-targeted PET/CT combined with intraoperative fluorescence imaging, could provide valuable information for tumor detection and staging, therapy response evaluation with re-staging of disease and intraoperative guidance during surgical resection of PDAC. Methods: A literature search in the PubMed database and (inter)national trial registers was conducted, focusing on studies published over the last 15 years. Data and information of eligible articles regarding PET/CT as well as fluorescence imaging in PDAC were reviewed. Areas covered: This review covers the current strategies, obstacles, challenges, and developments in targeted tumor imaging, focusing on the feasibility and value of PET/CT and fluorescence imaging for integration in the work-up and treatment of PDAC. An overview is given of identified targets and their characteristics, as well as the available literature of conducted and ongoing clinical and preclinical trials evaluating PDAC-targeted nuclear and fluorescent tracers. Show less
Huisman, B.W.; Cankat, M.; Bosse, T.; Vahrmeijer, A.L.; Rissmann, R.; Burggraaf, J.; ... ; Poelgeest, M.I.E. van 2021
Simple Summary: Vulvar tumors are sometimes difficult to distinguish from adjacent healthy tissue during surgery, causing recurrence rates of up to 40% and co-morbidity. Fluorescence-guided surgery... Show moreSimple Summary: Vulvar tumors are sometimes difficult to distinguish from adjacent healthy tissue during surgery, causing recurrence rates of up to 40% and co-morbidity. Fluorescence-guided surgery illuminating neoplastic tissue is increasingly being used to assist surgeons for various types of cancers. As no suitable tracers are known yet for vulvar tumors, we have evaluated which proteins could be targeted for fluorescence-guided surgery. Immunohistochemistry was used to study the distribution of nine membrane proteins in healthy and (pre)malignant tissues that were consequently analyzed using quantitative image analysis. Integrin alpha v beta 6 allowed for the most robust discrimination of VSCCs and adjacent premalignant lesions compared with surrounding healthy tissue. The use of an alpha v beta 6 targeted near-infrared fluorescent probe for FGS of vulvar (pre)malignancies should be evaluated in future studies.Surgical removal of vulvar squamous cell carcinoma (VSCC) is associated with significant morbidity and high recurrence rates. This is at least partially related to the limited visual ability to distinguish (pre)malignant from normal vulvar tissue. Illumination of neoplastic tissue based on fluorescent tracers, known as fluorescence-guided surgery (FGS), could help resect involved tissue and decrease ancillary mutilation. To evaluate potential targets for FGS in VSCC, immunohistochemistry was performed on paraffin-embedded premalignant (high grade squamous intraepithelial lesion and differentiated vulvar intraepithelial neoplasia) and VSCC (human papillomavirus (HPV)-dependent and -independent) tissue sections with healthy vulvar skin as controls. Sections were stained for integrin alpha v beta 6, CAIX, CD44v6, EGFR, EpCAM, FR alpha, MRP1, MUC1 and uPAR. The expression of each marker was quantified using digital image analysis. H-scores were calculated and percentages positive cells, expression pattern, and biomarker localization were assessed. In addition, tumor-to-background ratios were established, which were highest for (pre)malignant vulvar tissues stained for integrin alpha v beta 6. In conclusion, integrin alpha v beta 6 allowed for the most robust discrimination of VSCCs and adjacent premalignant lesions compared to surrounding healthy tissue in immunohistochemically stained tissue sections. The use of an alpha v beta 6 targeted near-infrared fluorescent probe for FGS of vulvar (pre)malignancies should be evaluated in future studies. Show less
Huisman, B.W.; Cankat, M.; Bosse, T.; Vahrmeijer, A.L.; Rissmann, R.; Burggraaf, J.; ... ; Poelgeest, M.I.E. van 2021
Surgical removal of vulvar squamous cell carcinoma (VSCC) is associated with significant morbidity and high recurrence rates. This is at least partially related to the limited visual ability to... Show moreSurgical removal of vulvar squamous cell carcinoma (VSCC) is associated with significant morbidity and high recurrence rates. This is at least partially related to the limited visual ability to distinguish (pre)malignant from normal vulvar tissue. Illumination of neoplastic tissue based on fluorescent tracers, known as fluorescence-guided surgery (FGS), could help resect involved tissue and decrease ancillary mutilation. To evaluate potential targets for FGS in VSCC, immunohistochemistry was performed on paraffin-embedded premalignant (high grade squamous intraepithelial lesion and differentiated vulvar intraepithelial neoplasia) and VSCC (human papillomavirus (HPV)-dependent and -independent) tissue sections with healthy vulvar skin as controls. Sections were stained for integrin αvβ6, CAIX, CD44v6, EGFR, EpCAM, FRα, MRP1, MUC1 and uPAR. The expression of each marker was quantified using digital image analysis. H-scores were calculated and percentages positive cells, expression pattern, and biomarker localization were assessed. In addition, tumor-to-background ratios were established, which were highest for (pre)malignant vulvar tissues stained for integrin αvβ6. In conclusion, integrin αvβ6 allowed for the most robust discrimination of VSCCs and adjacent premalignant lesions compared to surrounding healthy tissue in immunohistochemically stained tissue sections. The use of an αvβ6 targeted near-infrared fluorescent probe for FGS of vulvar (pre)malignancies should be evaluated in future studies. Show less
Simple Summary: Distinguishing pancreatic cancer from healthy tissue before and during surgery can be enhanced by using molecular tracers directed at molecules on tumor cells allowing high-contrast... Show moreSimple Summary: Distinguishing pancreatic cancer from healthy tissue before and during surgery can be enhanced by using molecular tracers directed at molecules on tumor cells allowing high-contrast visualization of tumor tissue, eventually improving diagnosis and surgical removal. Albeit sugar molecules and proteins carrying a large amount of sugars-mucins- have gained significant interest as tumor-specific targets, their relative presence on structures surrounding tumor tissues and lymph node metastases is unknown. The current study shows that the presence of several, but not all, investigated sugar molecules and mucins on pancreatic cancer cells is higher compared to surrounding tissues. Moreover, given their abundance on tumor cells in lymph nodes and their absence on normal lymph nodes, all investigated targets are high-potential targets for visualization of lymph node metastases. This study paves the way for the development of molecular tracers against the targets evaluated herein to allow improvement of pancreatic cancer treatment.Targeted molecular imaging may overcome current challenges in the preoperative and intraoperative delineation of pancreatic ductal adenocarcinoma (PDAC). Tumor-associated glycans Le(a/c/x), sdi-Le(a), sLe(a), sLe(x), sTn as well as mucin-1 (MUC1) and mucin-5AC (MU5AC) have gained significant interest as targets for PDAC imaging. To evaluate their PDAC molecular imaging potential, biomarker expression was determined using immunohistochemistry on PDAC, (surrounding) chronic pancreatitis (CP), healthy pancreatic, duodenum, positive (LN+) and negative lymph node (LN-) tissues, and quantified using a semi-automated digital image analysis workflow. Positive expression on PDAC tissues was found on 83% for Le(a/c/x), 94% for sdi-Le(a), 98% for sLe(a), 90% for sLe(x), 88% for sTn, 96% for MUC1 and 67% for MUC5AC, where all were not affected by the application of neoadjuvant therapy. Compared to PDAC, all biomarkers were significantly lower expressed on CP, healthy pancreatic and duodenal tissues, except for sTn and MUC1, which showed a strong expression on duodenum (sTn tumor:duodenum ratio: 0.6, p < 0.0001) and healthy pancreatic tissues (MUC1 tumor:pancreas ratio: 1.0, p > 0.9999), respectively. All biomarkers are suitable targets for correct identification of LN+, as well as the distinction of LN+ from LN- tissues. To conclude, this study paves the way for the development and evaluation of Le(a/c/x)-, sdi-Le(a)-, sLe(a)-, sLe(x)- and MUC5AC-specific tracers for molecular imaging of PDAC imaging and their subsequent introduction into the clinic. Show less
Baart, V.M.; Manen, L. van; Bhairosingh, S.S.; Vuijk, F.A.; Iamele, L.; Jonge, H. de; ... ; Sier, C.F.M. 2021
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... Show morePurpose 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. Show less
Rijs, Z.; Jeremiasse, B.; Shifai, N.; Gelderblom, H.; Sier, C.F.M.; Vahrmeijer, A.L.; ... ; Sande, M.A.J. van de 2021
Sarcomas are a rare heterogeneous group of malignant neoplasms of mesenchymal origin which represent approximately 13% of all cancers in pediatric patients. The most prevalent pediatric bone... Show moreSarcomas are a rare heterogeneous group of malignant neoplasms of mesenchymal origin which represent approximately 13% of all cancers in pediatric patients. The most prevalent pediatric bone sarcomas are osteosarcoma (OS) and Ewing sarcoma (ES). Rhabdomyosarcoma (RMS) is the most frequently occurring pediatric soft tissue sarcoma. The median age of OS and ES is approximately 17 years, so this disease is also commonly seen in adults while non-pleiomorphic RMS is rare in the adult population. The mainstay of all treatment regimens is multimodal treatment containing chemotherapy, surgical resection, and sometimes (neo)adjuvant radiotherapy. A clear resection margin improves both local control and overall survival and should be the goal during surgery with a curative intent. Real-time intraoperative fluorescence-guided imaging could facilitate complete resections by visualizing tumor tissue during surgery. This review evaluates whether non-targeted and targeted fluorescence-guided surgery (FGS) could be beneficial for pediatric OS, ES, and RMS patients. Necessities for clinical implementation, current literature, and the positive as well as negative aspects of non-targeted FGS using the NIR dye Indocyanine Green (ICG) were evaluated. In addition, we provide an overview of targets that could potentially be used for FGS in OS, ES, and RMS. Then, due to the time- and cost-efficient translational perspective, we elaborate on the use of antibody-based tracers as well as their disadvantages and alternatives. Finally, we conclude with recommendations for the experiments needed before FGS can be implemented for pediatric OS, ES, and RMS patients. Show less
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... Show moreWith 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-31uc2 cells in BALB/c-nude mice. The translational potential is confirmed in a metastasising UM-UC-31uc2 orthotopic mouse model. InfliximabIRDye800CW 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 < 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. (C) 2021 The Author(s). Published by Elsevier Ltd. Show less
Sier, V.Q.; Vries, M.R. de; Vorst, J.R. van der; Vahrmeijer, A.L.; Kooten, C. van; Cruz, L.J.; ... ; Muthana, M. 2021
Surgeons rely almost completely on their own vision and palpation to recognize affected tissues during surgery. Consequently, they are often unable to distinguish between different cells and tissue... Show moreSurgeons rely almost completely on their own vision and palpation to recognize affected tissues during surgery. Consequently, they are often unable to distinguish between different cells and tissue types. This makes accurate and complete resection cumbersome. Targeted image-guided surgery (IGS) provides a solution by enabling real-time tissue recognition. Most current targeting agents (tracers) consist of antibodies or peptides equipped with a radiolabel for Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT), magnetic resonance imaging (MRI) labels, or a near-infrared fluorescent (NIRF) dye. These tracers are preoperatively administered to patients, home in on targeted cells or tissues, and are visualized in the operating room via dedicated imaging systems. Instead of using these 'passive' tracers, there are other, more 'active' approaches of probe delivery conceivable by using living cells (macrophages/monocytes, neutrophils, T cells, mesenchymal stromal cells), cell(-derived) fragments (platelets, extracellular vesicles (exosomes)), and microorganisms (bacteria, viruses) or, alternatively, 'humanized' nanoparticles. Compared with current tracers, these active contrast agents might be more efficient for the specific targeting of tumors or other pathological tissues (e.g., atherosclerotic plaques). This review provides an overview of the arsenal of possibilities applicable for the concept of cell-based tracers for IGS. Show less
Purpose of review Observational studies have shown that serum 25-OH vitamin D [25(OH)D] is inversely associated with overall cancer risk in many malignancies. We performed a systematic literature... Show morePurpose of review Observational studies have shown that serum 25-OH vitamin D [25(OH)D] is inversely associated with overall cancer risk in many malignancies. We performed a systematic literature review to determine whether vitamin D deficiency is related to head and neck cancer (HNC) etiology and outcome. Recent findings The search yielded five prospective studies reporting 25(OH)D levels prior to cancer diagnosis and their effect on the risk of HNC. Eight studies were cross-sectional or case-control studies, in which 25(OH)D levels were only measured after cancer diagnosis. Two studies found an inverse association between 25(OH)D level and HNC risk, while two other prospective cohort studies demonstrated no connection between 25(OH)D and HNC risk. Several studies reported cancer patients to have significantly lower 25(OH)D levels than controls. Associations between 25(OH)D and prognosis and mortality were variable. The link between vitamin D and HNC has so far only been investigated in a few observational, prospective, and case-control studies. Vitamin D deficiency may be more common in HNC patients than in the healthy population. There is no evidence for a causal relationship. Further studies are needed to evaluate whether low 25(OH)D concentrations play a role in the development or outcome of HNCs. Show less
Assessing lymph node (LN) status during tumor resection is fundamental for the staging of colorectal cancer. Current guidelines require a minimum of 12 LNs to be harvested during resection and... Show moreAssessing lymph node (LN) status during tumor resection is fundamental for the staging of colorectal cancer. Current guidelines require a minimum of 12 LNs to be harvested during resection and ultra-staging regional lymph nodes by sentinel lymph node (SLN) assessment is being extensively investigated. The current study presents novel near-infrared (NIR) fluorescent dyes for simultaneous pan lymph node (PanLN; regional) and SLN mapping. PanLN-Forte was intravenously injected in mice and assessed for accumulation in regional LNs. SLN800 was injected intradermally in mice, after which the collection and retention of fluorescence in SLNs were measured using indocyanine green (ICG) and its precursor, SLN700, as references. LNs in the cervical, inguinal, jejunal, iliac, and thoracic basins could clearly be distinguished after a low dose intravenous injection of PanLN-Forte. Background fluorescence was significantly lower compared to the parent compound ZW800-3A (p < 0.001). SLN700 and SLN800 specifically targeted SLNs with fluorescence being retained over 40-fold longer than the current clinically used agent ICG. Using SLN700 and SLN800, absolute fluorescence in SLN was at least 10 times higher than ICG in second-tier nodes, even at 1 hour post-injection. Histologically, the fluorescent signal localized in the LN medulla (PanLN-Forte) or sinus entry (SLN700/SLN800). PanLN-Forte and SLN800 appear to be optimal for real-time NIR fluorescence imaging of regional and SLNs, respectively. Show less
