Clinical Translation of Ex Vivo Sentinel Lymph Node Mapping for Colorectal Cancer Using Invisible Near-Infrared Fluorescence Light

BACKGROUND: Sentinel lymph node (SLN) mapping in colorectal cancer may have prognostic and therapeutic significance; however, currently available techniques are not optimal. We hypothesized that the combination of invisible near-infrared (NIR) fluorescent light and ex vivo injection could solve remaining problems of SLN mapping in colorectal cancer. METHODS: The FLARE imaging system was used for real-time identification of SLNs after injection of the NIR lymphatic tracer HSA800 in the colon and rectum of (n = 4) pigs. A total of 32 SLN mappings were performed in vivo and ex vivo after oncologic resection using an identical injection technique. Guided by these results, SLN mappings were performed in ex vivo tissue specimens of 24 consecutive colorectal cancer patients undergoing resection. RESULTS: Lymph flow could be followed in real-time from the injection site to the SLN using NIR fluorescence. In pigs, the SLN was identified in 32 of 32 (100%) of SLN mappings under both in vivo and ex vivo conditions. Clinically, SLNs were identified in all patients (n = 24) using the ex vivo strategy within 5 min after injection of fluorescent tracer. Also, 9 patients showed lymph node involvement (N1 disease). In 1 patient, a 3-mm mesenteric metastasis was found adjacent to a tumor-negative SLN. CONCLUSIONS: The current pilot study shows proof of principle that ex vivo NIR fluorescence-guided SLN mapping can provide high-sensitivity, rapid, and accurate identification of SLNs in colon and rectum. This creates an experimental platform to test optimized, non-FDA-approved NIR fluorescent lymphatic tracers in a clinical setting.Imaging- and therapeutic targets in neoplastic and musculoskeletal inflammatory diseas

Preclinical evaluation of EpCAM-binding designed ankyrin repeat proteins (DARPins) as targeting moieties for bimodal near-infrared fluorescence and photoacoustic imaging of cancer

PURPOSE 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 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. METHODS 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. RESULTS 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. CONCLUSION 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

Toward Optimization of Imaging System and Lymphatic Tracer for Near-Infrared Fluorescent Sentinel Lymph Node Mapping in Breast Cancer

Near-infrared (NIR) fluorescent sentinel lymph node (SLN) mapping in breast cancer requires optimized imaging systems and lymphatic tracers. A small, portable version of the FLARE imaging system, termed Mini-FLARE, was developed for capturing color video and two semi-independent channels of NIR fluorescence (700 and 800 nm) in real time. Initial optimization of lymphatic tracer dose was performed using 35-kg Yorkshire pigs and a 6-patient pilot clinical trial. More refined optimization was performed in 24 consecutive breast cancer patients. All patients received the standard of care using (99m)Technetium-nanocolloid and patent blue. In addition, 1.6 ml of indocyanine green adsorbed to human serum albumin (ICG:HSA) was injected directly after patent blue at the same location. Patients were allocated to 1 of 8 escalating ICG:HSA concentration groups from 50 to 1000 mu M. The Mini-FLARE system was positioned easily in the operating room and could be used up to 13 in. from the patient. Mini-FLARE enabled visualization of lymphatic channels and SLNs in all patients. A total of 35 SLNs (mean = 1.45, range 1-3) were detected: 35 radioactive (100%), 30 blue (86%), and 35 NIR fluorescent (100%). Contrast agent quenching at the injection site and dilution within lymphatic channels were major contributors to signal strength of the SLN. Optimal injection dose of ICG:HSA ranged between 400 and 800 mu M. No adverse reactions were observed. We describe the clinical translation of a new NIR fluorescence imaging system and define the optimal ICG:HSA dose range for SLN mapping in breast cancer.EndocrinologyOV5Oncologic ImagingImaging- and therapeutic targets in neoplastic and musculoskeletal inflammatory diseas

Low Level of Microsatellite Instability Correlates with Poor Clinical Prognosis in Stage II Colorectal Cancer Patients

The influence of microsatellite instability (MSI) on the prognosis of colorectal cancer (CRC) requires more investigation. We assessed the role of MSI status in survival of individuals diagnosed with primary colorectal cancer. In this retrospective crosssectional study the MSI status was determined in 158 formalin-fixed paraffin-embedded tumors and their matched normal tissues from patients who underwent curative surgery. Cox proportional hazard modeling was performed to assess the clinical prognostic significance. In this study we found that MSI-H tumors were predominantly located in the colon versus rectum ( = 0.03), associated with poorer differentiation ( = 0.003) and TNM stage II/III of tumors ( = 0.02). In CRC patients with stage II, MSI-L cases showed significantly poorer survival compared with patients who had MSI-H or MSS tumors ( = 0.04). This study indicates that MSI-L tumors correlate with poorer clinical outcome in patients with stage II tumors ( = 0.04) or in tumors located in the colon ( = 0.02). MSI-L characterizes a distinct subgroup of CRC patients who have a poorer outcome. This study suggests that MSI status in CRC, as a clinical prognostic marker, is dependent on other factors, such as tumor stage and location

Nuclear Localization of CXCR4 Determines Prognosis for Colorectal Cancer Patients

Chemokines and their receptors are implicated in formation of colorectal cancer metastases. Especially CXCR4 is an important factor, determining migration, invasiveness, metastasis and proliferation of colorectal cancer cells. Object of this study was to determine expression of CXCR4 in tumor tissue of colorectal cancer patients and associate CXCR4 expression levels to clinicopathological parameters. Levels of CXCR4 expression of a random cohort of patients, who underwent primary curative resection of a colorectal carcinoma, were retrospectively determined by quantitative real-time RT-PCR and semi-quantitative analyses of immunohistochemical stained paraffin sections. Expression levels were associated to clinicopathological parameters. Using RT-PCR we found that a high expression of CXCR4 in the primary tumor was an independent prognostic factor for a poor disease free survival (p = 0.03, HR: 2.0, CI = 1.1–3.7). Immunohistochemical staining showed that nuclear distribution of CXCR4 in the tumor cells was inversely associated with disease free and overall survival (p = 0.04, HR: 2.6, CI = 1.0–6.2), while expression in the cytoplasm was not associated with prognosis. In conclusion, our study showed that a high expression of nuclear localized CXCR4 in tumor cells is an independent predictor for poor survival for colorectal cancer patients

Induction of lymphokine-activated killer activity in rat splenocyte cultures: The importance of 2-mercaptoethanol and indomethacin

The role of 2-mercaptoethanol and indomethacin in the induction of lymphokine-activated killer (LAK) activity by interleukin-2 (IL-2) in rat splenocyte cultures was investigated. Spleens from 4-month-old male rats of five different strains were tested. Splenocytes were cultured for 3-5 days in the presence of IL-2 (1000 U/ml) and LAK activity was assessed by 4-h51Cr release assays with P815 and YAC-1 cells as targets. LAK activity could be induced by IL-2 in splenocytes from all rat strains, but only when 2-mercaptoethanol was present in the culture medium. Optimal LAK activity was induced when the 2-mercaptoethanol concentration in splenocyte cultures was at least 5 μM. Different rat strains showed differences in levels of in vitro induction of LAK activity. In the presence of 2-mercaptoethanol the level of LAK activity induced by IL-2 was high in BN and Lewis rats, intermediate in Wistar and Wag rats, and low in DZB rats. In the absence of 2-mercaptoethanol no or minimal LAK activity was induced. Furthermore we observed that addition of 50 μm indomethacin to the culture medium in the presence of 2-mercaptoethanol augmented the induction of LAK activity to some extent. In the absence of 2-mercaptoethanol, addition of indomethacin resulted only in low levels or no induction of LAK activity. We conclude that for optimal induction of LAK activity by IL-2 in rat splenocyte cultures 2-mercaptoethanol is essential, while indomethacin can only marginally further improve this induction

Sialyl Lewis X Expression and Lymphatic Microvessel Density in Primary Tumors of Node-negative Colorectal Cancer Patients Predict Disease Recurrence

Up to 30% of curatively resected colorectal cancer patients with tumor-negative lymph nodes, show disease recurrence. We assessed whether these high-risk patients can be identified by examining primary tumors for the following blood and lymphatic vasculature markers: A) sialyl Lewis X (sLeX), vascular endothelial growth factor (VEGF)-C and VEGF-D expression; B) blood and lymphatic microvessel density (BMVD/LMVD); and C) the presence of blood and lymphatic vessel invasion. Thirty-six cases (disease recurrence within 5 years) and 72 controls (no disease recurrence for at least 5 years) were selected in a case-control design. Tumor sections were stained by antibodies CSLEX1 (sLeX), anti-VEGF-C, anti-VEGF-D, anti-CD31 (BMVD) or D2–40 (LMVD) to determine the parameters as mentioned above. A multivariate analysis showed sLeX expression and high LMVD (odds ratio 5.1, 95% confidence interval 1.3–20.0 and odds ratio 3.1, 95% confidence interval 1.0–10.0, respectively) to be independent factors predicting disease recurrence. Expression of sLeX correlated with liver metastases (P = 0.015). A high LMVD was related to regional intra-abdominal or intrapelvic metastases in lymph nodes and distant metastases other than in the liver and lungs such as peritoneum, bones, brain and adrenal glands (P = 0.004). A high BMVD in the invasive front correlated with lung metastases (P = 0.018). We show that high-risk node-negative colorectal cancer patients can be identified by primary tumor assessment for sLeX expression and LMVD. Our results are consistent with the notion that both lymphatic and hematogenous metastasis play a role in colorectal cancer

Clinical impact of HLA class I expression in rectal cancer

Contains fulltext : 69499.pdf (publisher's version ) (Open Access)PURPOSE: To determine the clinical impact of human leukocyte antigen (HLA) class I expression in irradiated and non-irradiated rectal carcinomas. EXPERIMENTAL DESIGN: Tumor samples in tissue micro array format were collected from 1,135 patients. HLA class I expression was assessed after immunohistochemical staining with two antibodies (HCA2 and HC10). RESULTS: Tumors were split into two groups: (1) tumors with >50% of tumor cells expressing HLA class I (high) and (2) tumors with < or =50% of tumor cells expressing HLA class I (low). No difference in distribution or prognosis of HLA class I expression was found between irradiated and non-irradiated patients. Patients with low expression of HLA class I (15% of all patients) showed an independent significantly worse prognosis with regard to overall survival and disease-free survival. HLA class I expression had no effect on cancer-specific survival or recurrence-free survival. CONCLUSIONS: Down-regulation of HLA class I in rectal cancer is associated with poor prognosis. In contrast to our results, previous reports on HLA class I expression in colorectal cancer described a large population of patients with HLA class I negative tumors, having a good prognosis. This difference might be explained by the fact that a large proportion of HLA negative colon tumors are microsatellite instable (MSI). MSI tumors are associated with a better prognosis than microsatellite stable (MSS). As rectal tumors are mainly MSS, our results suggest that it is both, oncogenic pathway and HLA class I expression, that dictates patient's prognosis in colorectal cancer. Therefore, to prevent confounding in future prognostic analysis on the impact of HLA expression in colorectal tumors, separate analysis of MSI and MSS tumors should be performed