Surgery is the cornerstone of oncologic therapy with curative intent. However, identification of tumor cells in the resection margins is difficult, resulting in nonradical resections, increased ...cancer recurrence and subsequent decreased patient survival. Novel imaging techniques that aid in demarcating tumor margins during surgery are needed. Overexpression of carcinoembryonic antigen (CEA) is found in the majority of gastrointestinal carcinomas, including colorectal and pancreas. We developed ssSM3E/800CW, a novel CEA‐targeted near‐infrared fluorescent (NIRF) tracer, based on a disulfide‐stabilized single‐chain antibody fragment (ssScFv), to visualize colorectal and pancreatic tumors in a clinically translatable setting. The applicability of the tracer was tested for cell and tissue binding characteristics and dosing using immunohistochemistry, flow cytometry, cell‐based plate assays and orthotopic colorectal (HT‐29, well differentiated) and pancreatic (BXPC‐3, poorly differentiated) xenogeneic human–mouse models. NIRF signals were visualized using the clinically compatible FLARE™ imaging system. Calculated clinically relevant doses of ssSM3E/800CW selectively accumulated in colorectal and pancreatic tumors/cells, with highest tumor‐to‐background ratios of 5.1 ± 0.6 at 72 hr postinjection, which proved suitable for intraoperative detection and delineation of tumor boarders and small (residual) tumor nodules in mice, between 8 and 96 hr postinjection. Ex vivo fluorescence imaging and pathologic examination confirmed tumor specificity and the distribution of the tracer. Our results indicate that ssSM3E/800CW shows promise as a diagnostic tool to recognize colorectal and pancreatic cancers for fluorescent‐guided surgery applications. If successfully translated clinically, this tracer could help improve the completeness of surgery and thus survival.
What's new?
The use of fluorescent tracers to identify cancerous cells at tumor margins could improve outcomes for surgery with curative intent. This study describes a newly developed near‐infrared fluorescent tracer targeted to carcinoembryonic antigen (CEA), a glycoprotein overexpressed in gastrointestinal cancers. In mice, the fluorescent agent, ssSM3E/800CW, accumulated specifically in tumor cells, enabling the detection and delineation of tumor borders on a clinically relevant timescale. If successfully translated into the clinic, ssSM3E/800CW could facilitate the visualization of cancerous tissue and improve the completeness of surgical resection.
Purpose
The purpose of this study was to identify suitable molecular targets for tumor-specific imaging of pancreatic adenocarcinoma.
Procedures
The expression of eight potential imaging targets was ...assessed by the target selection criteria (TASC)—score and immunohistochemical analysis in normal pancreatic tissue (
n
= 9), pancreatic (
n
= 137), and periampullary (
n
= 28) adenocarcinoma.
Results
Integrin α
v
β
6
, carcinoembryonic antigen (CEA), epithelial growth factor receptor (EGFR), and urokinase plasminogen activator receptor (uPAR) showed a significantly higher (all
p
< 0.001) expression in pancreatic adenocarcinoma compared to normal pancreatic tissue and were confirmed by the TASC score as promising imaging targets. Furthermore, these biomarkers were expressed in respectively 88 %, 71 %, 69 %, and 67 % of the pancreatic adenocarcinoma patients.
Conclusions
The results of this study show that integrin α
v
β
6
, CEA, EGFR, and uPAR are suitable targets for tumor-specific imaging of pancreatic adenocarcinoma.
A crucial point for the management of pancreatic ductal adenocarcinoma (PDAC) is the decrease of R1 resections. Our aim was to evaluate the combination of multispectral optoacoustic tomography (MSOT) ...with fluorescence guided surgery (FGS) for diagnosis and perioperative detection of tumor nodules and resection margins in a xenotransplant mouse model of human pancreatic cancer. The peptide cRGD, conjugated with the near infrared fluorescent (NIRF) dye IRDye800CW and with a trans‐cyclooctene (TCO) tag for future click chemistry (cRGD‐800CW‐TCO), was applied to PDAC bearing immunodeficient nude mice; 27 days after orthotopic transplantation of human AsPC‐1 cells into the head of the pancreas, mice were injected with cRGD‐800CW‐TCO and imaged with fluorescence‐ and optoacoustic devices before and 2, 6 and 24 hr after injection, before they were sacrificed and dissected with a guidance of FGS imaging system. Fluorescence imaging of cRGD‐800CW‐TCO allowed detection of the tumor area but without information about the depth, whereas MSOT allowed high resolution 3 D identification of the tumor area, in particular of small tumor nodules. Highly sensitive delineation of tumor burden was achieved during FGS in all mice. Imaging of whole‐mouse cryosections, histopathological analysis and NIRF microscopy confirmed the localization of cRGD‐800CW‐TCO within the tumor tissue. In principle, all imaging modalities applied here were able to detect PDAC in vivo. However, the combination of MSOT and FGS provided detailed spatial information of the signal and achieved a complete overview of the distribution and localization of cRGD‐800CW‐TCO within the tumor before and during surgical intervention.
What's new?
Pancreatic cancer has poor prognosis, because it initially causes few symptoms, and it's very aggressive. When tumors are surgically removed, microscopic remnants frequently remain, enabling the cancer to return. Advanced imaging techniques, such as fluorescence guided surgery, help surgeons see the boundary between healthy and tumor tissue. Here, the authors demonstrate the effectiveness of multispectral imaging tomography (MSOT) in combination with an integrin‐targeting fluorescent probe to collect high resolution 3 D images of pancreatic tumors to be excised. This is the first study showing the unique ability of MSOT to provide detailed information about widespread pancreatic tumors prior to surgery.
Intraoperative identification of rectal cancer (RC) can be challenging, especially because of fibrosis after treatment with preoperative chemo- and radiotherapy (CRT). Tumor-targeted fluorescence ...imaging can enhance the contrast between tumor and normal tissue during surgery. Promising targets for RC imaging are carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM) and the tyrosine-kinase receptor Met (c-Met). The effect of CRT on their expression determines their applicability for imaging. Therefore, we investigated whether CRT modifies expression patterns in tumors, lymph node (LN) metastases and adjacent normal rectal tissues.
Preoperative biopsies, primary tumor specimens and metastatic LNs were collected from 38 RC patients who did not receive CRT (cohort 1) and 34 patients who did (cohort 2). CEA, EpCAM and c-Met expression was determined using immunohistochemical staining and was semiquantified by a total immunostaining score (TIS), consisting of the percentage and intensity of stained tumor cells (0-12).
In both cohorts CEA, EpCAM and c-Met were significantly highly expressed in >60% of tumor tissues compared with adjacent normal epithelium (T/N ratio,
<0.01). EpCAM showed the most homogenous expression in tumors, whereas CEA showed the highest T/N ratio. Most importantly, CEA and EpCAM expression did not significantly change in normal or neoplastic RC tissue after CRT, whereas levels of c-Met changed (
=0.02). Tissues of eight patients with a pathological complete response after CRT showed expression of all biomarkers with TIS close to normal epithelium.
Histological evaluation shows that CEA, EpCAM and c-Met are suitable targets for RC imaging, because all three are significantly enhanced in cancer tissue from primary tumors or LN metastases compared with normal adjacent tissue. Furthermore, the expression of CEA and EpCAM is not significantly changed after CRT. These data underscore the applicability of c-Met and especially, CEA and EpCAM as targets for image-guided RC surgery, both before and after CRT.
Targeted image-guided oncologic surgery (IGOS) relies on the recognition of cell surface-associated proteins, which should be abundantly present on tumor cells but preferably absent on cells in ...surrounding healthy tissue. The transmembrane receptor tyrosine kinase EphA2, a member of the A class of the Eph receptor family, has been reported to be highly overexpressed in several tumor types including breast, lung, brain, prostate, and colon cancer and is considered amongst the most promising cell membrane-associated tumor antigens by the NIH. Another member of the Eph receptor family belonging to the B class, EphB4, has also been found to be upregulated in multiple cancer types. In this study, EphA2 and EphB4 are evaluated as targets for IGOS of colorectal cancer by immunohistochemistry (IHC) using a tissue microarray (TMA) consisting of 168 pairs of tumor and normal tissue. The IHC sections were scored for staining intensity and percentage of cells stained. The results show a significantly enhanced staining intensity and more widespread distribution in tumor tissue compared with adjacent normal tissue for EphA2 as well as EphB4. Based on its more consistently higher score in colorectal tumor tissue compared to normal tissue, EphB4 appears to be a promising candidate for IGOS of colorectal cancer. In vitro experiments using antibodies on human colon cancer cells confirmed the possibility of EphB4 as target for imaging.
Folate receptor alpha (FRα) is known to be upregulated in a variety of cancers, including non-small cell lung cancer (NSCLC) and breast cancer. To ensure reliable implementation of diagnostic- and ...therapeutic agents, concordance of FRα expression between biopsy, primary tumor and metastases is important. Using immunohistochemistry (Mab 26B3.F2) these concordances were investigated in 60 NSCLC and 40 breast cancer patients. False positivity of FRα expression on breast and lung cancer biopsies was limited to less than 5%. In NSCLC, FRα expression was shown in 21/34 adenocarcinomas and 4/26 squamous cell carcinomas (SCC). Concordance of FRα expression between biopsy and primary tumor was achieved in respectively 83% and 91% of adenocarcinomas and SCCs. Approximately 80% of all local and distant metastases of NSCLC patients showed concordant FRα expression as their corresponding primary tumor. In breast cancer, FRα positivity was shown in 12/40 biopsies, 20/40 lumpectomies and 6/20 LN metastases, with concordance of 68% between biopsy and primary tumor and 60% between primary tumor and LN metastases. In conclusion, this study shows high concordance rates of FRα expression between biopsies and metastases compared to primary NSCLC and breast cancers, underscoring the applicability of FRα-targeted agents in these patients.
Background. Uveal melanoma is the most common primary intraocular tumor in adults, and up to 50% of patients will develop liver metastases. Complete surgical resection of these metastases can improve ...5-year survival, but only a few patients are eligible for radical surgical treatment. The aim of this study was to introduce a near-infrared (NIR) fluorescence laparoscope during minimally invasive surgery for intraoperative identification of uveal melanoma hepatic metastases and to use it to provide guidance during resection. Methods. Three patients diagnosed with one solitary liver metastasis from uveal melanoma are presented. Patients received 10 mg indocyanine green (ICG) intravenously 24 hours before surgery. A NIR fluorescence laparoscope was used to detect malignant liver lesions. Results. In all 3 patients, laparoscopic NIR fluorescence imaging using ICG successfully identified uveal melanoma metastases. In 2 patients, multiple additional lesions were identified by inspection and NIR fluorescence imaging, which were not identified by preoperative conventional imaging. In one patient, one additional lesion, not identified by computed tomography, magnetic resonance imaging, laparoscopic ultrasonography, and inspection, was observed with NIR fluorescence imaging only. Importantly, NIR fluorescence imaging provided guidance during resection of these metastases. Conclusions. We describe the successful use of laparoscopic identification and resection of uveal melanoma liver metastases using NIR fluorescence imaging and ICG. This procedure is minimally invasive and should be used as complementary to conventional techniques for the detection and resection of liver metastases.
Pre- and intraoperative diagnostic techniques facilitating tumor staging are of paramount importance in colorectal cancer surgery. The urokinase receptor (uPAR) plays an important role in the ...development of cancer, tumor invasion, angiogenesis, and metastasis and over-expression is found in the majority of carcinomas. This study aims to develop the first clinically relevant anti-uPAR antibody-based imaging agent that combines nuclear (111In) and real-time near-infrared (NIR) fluorescent imaging (ZW800-1). Conjugation and binding capacities were investigated and validated in vitro using spectrophotometry and cell-based assays. In vivo, three human colorectal xenograft models were used including an orthotopic peritoneal carcinomatosis model to image small tumors. Nuclear and NIR fluorescent signals showed clear tumor delineation between 24h and 72h post-injection, with highest tumor-to-background ratios of 5.0 ± 1.3 at 72h using fluorescence and 4.2 ± 0.1 at 24h with radioactivity. 1-2 mm sized tumors could be clearly recognized by their fluorescent rim. This study showed the feasibility of an uPAR-recognizing multimodal agent to visualize tumors during image-guided resections using NIR fluorescence, whereas its nuclear component assisted in the pre-operative non-invasive recognition of tumors using SPECT imaging. This strategy can assist in surgical planning and subsequent precision surgery to reduce the number of incomplete resections.
The receptor for urokinase-type plasminogen activator (uPAR) is associated with cancer development and progression. Within the tumor microenvironment uPAR is expressed by malignant cells as well as ...tumor-associated stromal cells. However, the contribution of uPAR expression in these stromal cells to malignancy and patient survival in colorectal cancer is still unclear. This study compares the association of uPAR expression in both colorectal tumor-associated stromal cells and neoplastic cells with clinico-pathological characteristics and patient survival using tissue micro arrays (TMA).
Immunohistochemical staining of uPAR expression was performed on tumor tissue from 262 colorectal cancer patients. Kaplan-Meier, log rank, and uni- and multivariate Cox's regression analyses were used to calculate associations between uPAR expression and patient survival.
In the colorectal tumor-associated stromal microenvironment, uPAR is expressed in macrophages, (neoangiogenic) endothelial cells and myofibroblasts. uPAR expression in tumor-associated stromal cells and neoplastic cells (and both combined) were negatively associated with overall survival (OS) and Disease Free Survival (DFS). Uni- and multivariate Cox's regression analysis for combined uPAR expression in tumor-associated stromal and neoplastic cells showed significant and independent negative associations with OS and DFS. Only uPAR expression in tumor-associated stromal cells showed independent significance in the uni- and multivariate analysis for DFS.
This study demonstrates a significant independent negative association between colorectal cancer patient survival and uPAR expression in especially tumor-associated stromal cells.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Tumor-specific fluorescent imaging agents are moving towards the clinic, supporting surgeons with real-time intraoperative feedback about tumor locations. The epithelial cell adhesion molecule ...(EpCAM) is considered as one of the most promising tumor-specific proteins due its high overexpression on epithelial-derived cancers. This study describes the development and evaluation of EpCAM-F800, a novel fluorescent anti-EpCAM antibody fragment, for intraoperative tumor imaging. Fab production, conjugation to the fluorophore IRDye 800CW, and binding capacities were determined and validated using HPLC, spectrophotometry and cell-based assays. In vivo, dose escalation-, blocking-, pharmacokinetic- and biodistribution studies (using both fluorescence and radioactivity) were performed, next to imaging of clinically relevant orthotopic xenografts for breast and colorectal cancer. EpCAM-F800 targets EpCAM with high specificity in vitro, which was validated using in vivo blocking experiments with a 10x higher dose of unlabeled Fab. The optimal dose range for fluorescence tumor detection in mice was 1–5 nmol (52–260 μg), which corresponds to a human equivalent dose of 0.2–0.8 mg/kg. Biodistribution showed high accumulation of EpCAM-F800 in tumors and metabolizing organs. Breast and colorectal tumors could clearly be visualized within 8 h post-injection and up to 96 h, while the agent already showed homogenous tumor distribution within 4 h. The blood half-life was 4.5 h. This study describes the development and evaluation of a novel EpCAM-targeting agent and the feasibility to visualize breast and colorectal tumors by fluorescence imaging during resections. EpCAM-F800 will be translated for clinical use, considering its abundance in a broad range of tumor types.
•EpCAM-F800 is a fluorescent anti-EpCAM Fab for intraoperative tumor imaging.•EpCAM-F800 targets EpCAM with high specificity in vitro and in vivo .•Breast and colorectal tumors could be clearly detected using EpCAM-F800 .•The optimal dose range for fluorescence tumor detection in mice was 1–5 nmol.•EpCAM-F800 will be translated for clinical use and enter the clinic shortly.
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