Functional Genomic mRNA Profiling of Colorectal Adenomas : Identification and Validation of CD44 and Splice Variant CD44v6 as Molecular Imaging Targets

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths worldwide. High adenoma miss rates, especially seen in high-risk patients, demand for better endoscopic detection. By fluorescently \u27highlighting\u27 specific molecular characteristics, endoscopic molecular imaging has great potential to fulfill this need. To implement this technique effectively, target proteins that distinguish adenomas from normal tissue must be identified. In this study we applied in silico Functional Genomic mRNA (FGmRNA) profiling, which is a recently developed method that results in an enhanced view on the downstream effects of genomic alterations occurring in adenomas on gene expression levels. FGmRNA profiles of sporadic adenomas were compared to normal colon tissue to identify overexpressed genes. We validated the protein expression of the top identified genes, AXIN2, CEMIP, CD44 and JUN, in sporadic adenoma patient samples via immunohistochemistry (IHC). CD44 was identified as the most attractive target protein for imaging purposes and we proved its relevance in high-risk patients by demonstrating CD44 protein overexpression in Lynch lesions. Subsequently, we show that the epithelial splice variant CD44V6 is highly overexpressed in our patient samples and we demonstrated the feasibility of visualizing adenomas in ApcMin/+ mice in vivo by using a fluorescently labeled CD44v6 targeting peptide. In conclusion, via in silico functional genomics and ex vivo protein validation, this study identified CD44 as an attractive molecular target for both sporadic and high-risk Lynch adenomas, and demonstrates the in vivo applicability of a small peptide drug directed against splice variant CD44v6 for adenoma imaging

Functional Genomic mRNA Profiling of Colorectal Adenomas:Identification and in vivo Validation of CD44 and Splice Variant CD44v6 as Molecular Imaging Targets

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths worldwide. High adenoma miss rates, especially seen in high-risk patients, demand for better endoscopic detection. By fluorescently \u27highlighting\u27 specific molecular characteristics, endoscopic molecular imaging has great potential to fulfill this need. To implement this technique effectively, target proteins that distinguish adenomas from normal tissue must be identified. In this study we applied in silico Functional Genomic mRNA (FGmRNA) profiling, which is a recently developed method that results in an enhanced view on the downstream effects of genomic alterations occurring in adenomas on gene expression levels. FGmRNA profiles of sporadic adenomas were compared to normal colon tissue to identify overexpressed genes. We validated the protein expression of the top identified genes, AXIN2, CEMIP, CD44 and JUN, in sporadic adenoma patient samples via immunohistochemistry (IHC). CD44 was identified as the most attractive target protein for imaging purposes and we proved its relevance in high-risk patients by demonstrating CD44 protein overexpression in Lynch lesions. Subsequently, we show that the epithelial splice variant CD44V6 is highly overexpressed in our patient samples and we demonstrated the feasibility of visualizing adenomas in ApcMin/+ mice in vivo by using a fluorescently labeled CD44v6 targeting peptide. In conclusion, via in silico functional genomics and ex vivo protein validation, this study identified CD44 as an attractive molecular target for both sporadic and high-risk Lynch adenomas, and demonstrates the in vivo applicability of a small peptide drug directed against splice variant CD44v6 for adenoma imaging

Potential red-flag identification of colorectal adenomas with wide-field fluorescence molecular endoscopy

Adenoma miss rates in colonoscopy are unacceptably high, especially for sessile serrated adenomas / polyps (SSA/Ps) and in high-risk populations, such as patients with Lynch syndrome. Detection rates may be improved by fluorescence molecular endoscopy (FME), which allows morphological visualization of lesions with high-definition white-light imaging as well as fluorescence-guided identification of lesions with a specific molecular marker. In a clinical proof-of-principal study, we investigated FME for colorectal adenoma detection, using a fluorescently labelled antibody (bevacizumab-800CW) against vascular endothelial growth factor A (VEGFA), which is highly upregulated in colorectal adenomas. Methods: Patients with familial adenomatous polyposis (n = 17), received an intravenous injection with 4.5, 10 or 25 mg of bevacizumab-800CW. Three days later, they received NIR-FME. Results: VEGFA-targeted NIR-FME detected colorectal adenomas at all doses. Best results were achieved in the highest (25 mg) cohort, which even detected small adenomas ( < 3 mm). Spectroscopy analyses of freshly excised specimen demonstrated the highest adenoma-to-normal ratio of 1.84 for the 25 mg cohort, with a calculated median tracer concentration in adenomas of 6.43 nmol/mL. Ex vivo signal analyses demonstrated NIR fluorescence within the dysplastic areas of the adenomas. Conclusion: These results suggest that NIR-FME is clinically feasible as a real-time, red-flag technique for detection of colorectal adenomas

Potential Red-Flag Identification of Colorectal Adenomas with Wide-Field Fluorescence Molecular Endoscopy

Adenoma miss rates in colonoscopy are unacceptably high, especially for sessile serrated adenomas/polyps (SSA/Ps) and in high-risk populations, such as patients with Lynch syndrome. Detection rates may be improved by fluorescence molecular endoscopy (FME), which allows morphological visualization of lesions with high-definition white-light imaging as well as fluorescence-guided identification of lesions with a specific molecular marker. In a clinical proof-of-principal study, we investigated FME for colorectal adenoma detection, using a fluorescently labelled antibody (bevacizumab-800CW) against vascular endothelial growth factor A (VEGFA), which is highly upregulated in colorectal adenomas. Methods: Patients with familial adenomatous polyposis (n = 17), received an intravenous injection with 4.5, 10 or 25 mg of bevacizumab-800CW. Three days later, they received NIR-FME. Results: VEGFA-targeted NIR-FME detected colorectal adenomas at all doses. Best results were achieved in the highest (25 mg) cohort, which even detected small adenomas ( Conclusion: These results suggest that NIR-FME is clinically feasible as a real-time, red-flag technique for detection of colorectal adenomas

EGFR & CD44 as potential targets for detection of (pre)malignant lesions with the use offluorescent Near Infrared (NIR) endoscopy

Background Colorectal carcinoma (CRC) represents one of the leading causes of cancer related deaths worldwide. Early diagnosis of (pre)malignant colorectal lesions is the key factor in reducing CRC related morbidity and mortality. Unfortunately, conventional white-light endoscopy appears to be insufficient. For this reason a progressive dual technique is being developed, combining white-light endoscopy with a targeted fluorescent Near Infrared (NIR) modality. With this, a red-flagged imaging method based on a wide-field overview will be accomplished, which enables immediate tissue differentiation and proper small lesion detection. To carry out this fluorescent NIR-technique, cancer-specific NIR-targets need to be identified first. Based on literature, membrane receptors EGFR and CD44 potentially fulfill such a role, but their successful application for identifying (pre)malignant lesions by using the NIR-modality has yet to be investigated. Methods Polyps from Lynch patients (LS), a hereditary colon cancer syndrome hallmarked by small sized colorectal lesions, were included and immunohistochemically (IHC) stained for the presence of EGFR and CD44. Polyps of different dysplasia stages, namely low grade dysplasia (LGD), high grade dysplasia (HGD), carcinoma (CA) tissue and surrounding normal crypts were separately scored on a 4-point scale (0-3) for staining intensity and percentage of positively stained dysplastic cells per tissue sample. In addition, non-hereditary rectal cancer (RC) material was stained and scored for EGFR to serve as a comparison. Results For EGFR we found high receptor expression; respectively 85% for LGD (n=64), 74% for HGD (n=65) and 85% for CA (n=34). The surrounding normal crypts showed significantly less EGFR-expression (all P<0.05; overall sensitivity 80%/specificity 71%). Furthermore, our data suggest a significant trend of increasing EGFR-intensity following progression within the adenoma-carcinoma sequence (P=0.001). Moreover, we did not find measurable difference between the hereditary lynch carcinoma tissue and the non-hereditary rectal carcinoma material (P=0.71). For CD44 we also found high receptor expression; respectively 88% for LGD (n=74), 73% for HGD (n=60) and 85% for CA (n=33). Interestingly, we found hardly any CD44 expression within the normal crypts (all P<0.001), which makes this an extremely specific target in identifying (pre)malignant lesions (overall sensitivity 82%/specificity 99%). Additionally, throughout the tissue of all three dysplasia stages, we established an overall heterogeneous distribution pattern of both receptors. This emphasizes the need for a wide-field overview and real-time tissue differentiation. Conclusion Our data suggest that both EGFR and CD44 could serve as potential NIR-targets for future diagnostic purposes in identifying (pre)malignant lesions using the fluorescent targeted NIR-endoscopic technique.

Fluorescence molecular endoscopy: A new frontier in the field of gastroenterology

Wit-licht endoscopie is de huidige standaard voor het inspecteren en detecteren van afwijkingen in het maagdarmkanaal. Deze endoscopische techniek heeft echter als groot nadeel dat het alleen in staat is afwijkingen te onderscheiden op basis van – vaak subtiele – morfologische kenmerken en veranderingen. Moleculaire beeldvorming is in staat om – op een minimaal invasieve manier – een afwijking te onderscheiden en te identificeren op basis van bepaalde eiwitten op bijvoorbeeld het celmembraan. Gezien moleculaire veranderingen al in een vroeg stadium van de ziekte kunnen optreden, zelfs voordat morfologische veranderingen zichtbaar worden, biedt deze techniek de mogelijkheid om afwijkingen al in een zeer vroeg stadium op het spoor te komen. Door een dergelijke moleculaire toepassing te incorporeren in een standaard endoscopische wit-licht techniek ontstaat een nieuwe techniek: Moleculaire Fluorescentie-endoscopie (FME). Het principe van deze moleculaire fluorescentie techniek berust op het fluorescent ‘labelen’ van specifieke – tot overexpressie komende – eiwitten en op die manier kwaadaardige afwijkingen en diens premaligne voorlopercellen letterlijk te doen oplichten; zo is het mogelijk om afwijkingen beter te identificeren en, tijdens de scopie, te kunnen onderscheiden van goedaardige poliepen. Het doel van dit proefschrift is om de door ons ontwikkelde nabij infrarode (NIR) FME techniek te vertalen naar de klinische praktijk. Dit hebben we gedaan door de toepassing van de techniek te valideren in een preklinisch diermodel, alsmede door de klinische toepassing in patiënten met vroege afwijkingen in zowel de dikke darm als slokdarm te evalueren. Daarnaast beschrijft het proefschrift de zoektocht naar nieuwe ‘target’- eiwitten, evenals meer experimentele toepassingen van NIR-FME

Tyrosine kinase inhibitor induced growth factor receptor upregulation enhances the efficacy of near-infrared targeted photodynamic therapy in esophageal adenocarcinoma cell lines

Esophageal carcinoma (EC) is a global health problem, with disappointing 5-year survival rates of only 15-25%. Near-infrared targeted photodynamic therapy (NIR-tPDT) is a novel strategy in which cancer-targeted phototoxicity is able to selectively treat malignant cells. In this in vitro report we demonstrate the applicability of antibody-based NIR-tPDT in esophageal adenocarcinoma (EAC), using the phototoxic compounds cetuximab-IRDye700DX and trastuzumab-IRDye700DX, targeting respectively epidermal growth factor receptor 1 (EGFR) and 2 (HER2). Furthermore, we demonstrate that NIR-tPDT can be made more effective by tyrosine kinase inhibitor (TKI) induced growth receptor upregulation. Together, these results unveil a novel strategy for non-invasive EAC treatment, and by pretreatment-induced receptor upregulation its future clinical application may be optimized

Data-Driven Prioritization and Review of Targets for Molecular-Based Theranostic Approaches in Pancreatic Cancer

Molecularly targeted therapeutic and imaging strategies directed at aberrant signaling pathways in pancreatic tumor cells may improve the poor outcome of pancreatic ductal adenocarcinoma (PDA). Therefore, relevant molecular targets need to be identified.  Methods: We collected publicly available expression profiles of patient-derived normal pancreatic tissue (n = 77) and PDA samples (n = 103). Functional genomic messenger RNA profiling was applied to predict target upregulation on the protein level. We prioritized these targets based on current status of preclinical therapeutic and imaging evaluation in PDA.  Results: We identified 213 significantly upregulated proteins in PDA compared with normal pancreatic tissue. We prioritized mucin-1, mesothelin, g-glutamyltransferase 5, and cathepsin-E as the most interesting targets, because studies already demonstrated their potential for both therapeutic and imaging strategies in literature.  Conclusion: This study can assist clinicians and drug developers in deciding which theranostic targets should be taken for further clinical evaluation in PDA