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
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
A growing body of scientific literature documents a putative role of commensal and pathogenic bacteria in the initiation and progression of cancers. One such bacterium is nontyphoidal Salmonella ... Show moreA growing body of scientific literature documents a putative role of commensal and pathogenic bacteria in the initiation and progression of cancers. One such bacterium is nontyphoidal Salmonella (NTS), which has been associated with colon cancer in a few studies. Yet, a lot is still unknown about the magnitude and underlying mechanisms, including the necessary conditions or ‘prerequisites’, of the potential colon carcinogenesis promoting effects of Salmonella. In this thesis, we performed several complementary analyses based on both experimental and epidemiological study designs. Significant excess risk of NTS infection was observed among several occupational groups including those involving contact with live animals or animal manure and animal-derived food sale. The risk of colon cancer was not elevated in these groups. Also, no clear association between NTS and colon cancer was found in a Danish cohort. Two-fold infection of mouse embryonic fibroblasts (MEFs) with a low dose of NTS was more successful than a single high-dose infection (i.e. more and larger colonies). Substantial variation between NTS isolates was found in their capacity to infect MEFs and to induce cellular transformation, with a tendency towards higher transformation efficiency in isolates originating from people who were diagnosed with colon cancer later in life. Show less
This thesis describes the steps necessary for the addition of the tumor-stroma ratio (TSR) into clinical practice as high-risk factor besides the TNM classification. The route from laboratory... Show moreThis thesis describes the steps necessary for the addition of the tumor-stroma ratio (TSR) into clinical practice as high-risk factor besides the TNM classification. The route from laboratory biomarker development to clinical implementation is followed. During this process, the relationship of the TSR to other available biomarkers for prognostic information for breast and colon cancer patients is investigated. Additionally, the prognostic value of the TSR in lung cancer is studied. Show less
Major achievements in the field of immune oncology have demonstrated the ability of the immune system to induce a response against cancer. The prognostic impact of pre-existing immunity in several... Show moreMajor achievements in the field of immune oncology have demonstrated the ability of the immune system to induce a response against cancer. The prognostic impact of pre-existing immunity in several cancer types, including breast and colon cancer, demonstrates the influence of the immune system on disease progression. At the same time, immunotherapeutic approaches that aim to enhance antitumor immune reactions have significantly improved the clinical outcome for a subset of patients. However, a large proportion of patients (60-80%) do not respond to immunotherapeutic treatments. To extend the benefit of immunotherapeutic strategies to a larger number of patients, it is imperative to understand the mechanisms associated with immune responsiveness. Different variables have been described to influence the development of antitumor immunity in cancer patients, including the tumor’s genetic program, the genetic makeup of the patients, and environmental factors such as the microbiome. These factors likely act in concert to modulate antitumor immune responses. This thesis aimed to dissect the molecular determinants of cancer immune responsiveness in human tumors. A systems biology approach was used to define underlying factors that shape the tumor microenvironment and reveal potential mechanisms of immune escape. Show less
Purpose Integrin subunit beta 4 (beta 4) has been proposed to play an important role in colon cancer progression through its involvement in hemidesmosome disassembly processes and tumor cell... Show morePurpose Integrin subunit beta 4 (beta 4) has been proposed to play an important role in colon cancer progression through its involvement in hemidesmosome disassembly processes and tumor cell migration. However, the association between beta 4 expression and clinicopathological outcomes in colon cancer remains unclear.Methods Expression of beta 4 was assessed by immunohistochemistry in a large cohort of 651 colon cancer patients, the largest colon cancer cohort so far. Chi-squared tests were used to study the association between beta 4 expression and clinicopathological features. Overall and disease-free survival were assessed by Cox proportional hazard models.Results Loss of beta 4 expression was associated with local tumor invasion. Only 17.9% of the pT1 tumors displayed weak beta 4 expression level versus 28.1% of pT4 tumors, and 25.0% of the pT1 tumors had a high expression level versus 8.6% of the pT4 tumors (p = 0.012). No association between beta 4 expression and overall (p = 0.845) or disease-free survival (p = 0.767) was encountered, which disputes the role of beta 4 as a biomarker of malignant behavior in colon cancer.Conclusion Contradictory reports have suggested opposite roles for beta 4 expression in (colon) cancer progression. In the present large cohort of colon cancer patients, we found that beta 4 expression was not associated with worse clinical prognosis, but decreased with advanced pathological tumor stage. Future studies should establish whether loss of beta 4 expression promotes invasive characteristics of colon cancer cells. Show less
Purpose Integrin subunit beta 4 (beta 4) has been proposed to play an important role in colon cancer progression through its involvement in hemidesmosome disassembly processes and tumor cell... Show morePurpose Integrin subunit beta 4 (beta 4) has been proposed to play an important role in colon cancer progression through its involvement in hemidesmosome disassembly processes and tumor cell migration. However, the association between beta 4 expression and clinicopathological outcomes in colon cancer remains unclear.Methods Expression of beta 4 was assessed by immunohistochemistry in a large cohort of 651 colon cancer patients, the largest colon cancer cohort so far. Chi-squared tests were used to study the association between beta 4 expression and clinicopathological features. Overall and disease-free survival were assessed by Cox proportional hazard models.Results Loss of beta 4 expression was associated with local tumor invasion. Only 17.9% of the pT1 tumors displayed weak beta 4 expression level versus 28.1% of pT4 tumors, and 25.0% of the pT1 tumors had a high expression level versus 8.6% of the pT4 tumors (p = 0.012). No association between beta 4 expression and overall (p = 0.845) or disease-free survival (p = 0.767) was encountered, which disputes the role of beta 4 as a biomarker of malignant behavior in colon cancer.Conclusion Contradictory reports have suggested opposite roles for beta 4 expression in (colon) cancer progression. In the present large cohort of colon cancer patients, we found that beta 4 expression was not associated with worse clinical prognosis, but decreased with advanced pathological tumor stage. Future studies should establish whether loss of beta 4 expression promotes invasive characteristics of colon cancer cells. Show less
Strous, M.T.A.; Faes, T.K.E.; Gubbels, A.L.H.M.; Linden, R.L.A. van der; Mesker, W.E.; Bosscha, K.; ... ; Bruine, A.P. de 2022
Purpose: Despite known high-risk features, accurate identification of patients at high risk of cancer recurrence in colon cancer remains a challenge. As tumour stroma plays an important role in... Show morePurpose: Despite known high-risk features, accurate identification of patients at high risk of cancer recurrence in colon cancer remains a challenge. As tumour stroma plays an important role in tumour invasion and metastasis, the easy, low-cost and highly reproducible tumour-stroma ratio (TSR) could be a valuable prognostic marker, which is also believed to predict chemo resistance. Methods: Two independent series of patients with colon cancer were selected. TSR was estimated by microscopic analysis of 4 mu m haematoxylin and eosin (H&E) stained tissue sections of the primary tumour and the corresponding metastatic lymph nodes. Patients were categorized as TSR-low (<= 50%) or TSR-high (> 50%). Differences in overall survival and cancer-free survival were analysed by Kaplan-Meier curves and cox-regression analyses. Analyses were conducted for TNM-stage I-II, TNM-stage III and patients with an indication for chemotherapy separately. Results: We found that high TSR was associated with poor cancer-free survival in TNM-stage I-II colon cancer in two independent series, independent of other known high-risk features. This association was also found in TNM-stage III tumours, with an additional prognostic value of TSR in lymph node metastasis to TSR in the primary tumour alone. In addition, high TSR was found to predict chemo resistance in patients receiving adjuvant chemotherapy after surgical resection of a TNM-stage II-III colon tumour. Conclusion: In colon cancer, the TSR of both primary tumour and lymph node metastasis adds significant prognostic value to current pathologic and clinical features used for the identification of patients at high risk of cancer recurrence, and also predicts chemo resistance. Show less
Ozmen, I.; Grupa, V.E.M.; Bedrikovetski, S.; Dudi-Venkata, N.N.; Huisman, D.E.; Reudink, M.; ... ; LekCheck Study Grp 2022
Purpose Anastomotic leakage (AL) is a dreaded complication after colorectal surgery. Preoperatively identifying high-risk patients can help to reduce the incidence of this complication. For this... Show morePurpose Anastomotic leakage (AL) is a dreaded complication after colorectal surgery. Preoperatively identifying high-risk patients can help to reduce the incidence of this complication. For this reason, AL risk nomograms have been developed. The objective of this study was to test the AL risk nomogram developed by Frasson, et al. for validity and to identify risk-factors for AL. Methods From the international multi-center LekCheck study database, patients who underwent colonic surgery with the formation of an anastomosis were included. Data were prospectively collected between 2016 and 2019 at 14 hospitals. Univariate and multivariable regression analyses, and area under receiver operating characteristic curve analysis (AUROC) were performed. Results A total of 643 patients were included. The median age was 70 years and 51% were male. The majority underwent surgery for malignancies (80.7%). The overall AL rate was 9.2%. The risk nomogram was not predictive for AL in the population tested (AUROC 0.572). Low preoperative haemoglobin (p = 0.006), intraoperative hypothermia (p = 0.02), contamination of the operative field (p = 0.004), and use of epidural analgesia (p = 0.02) were independent risk-factors for AL. Conclusion The AL risk nomogram could not be validated using the international LekCheck study database. In the future, intraoperative predictive factors for AL, as identified in this study, should also be included in AL risk predictors. Show less
Survival of patients with colorectal cancer improved markedly over the past decades, as a result of advances in screening, staging procedures, treatment, and surveillance. However, still about 20%... Show moreSurvival of patients with colorectal cancer improved markedly over the past decades, as a result of advances in screening, staging procedures, treatment, and surveillance. However, still about 20% of patients with colorectal cancer develop metachronous metastases and 20% of all patients with colorectal cancer have metastatic disease at diagnosis. Several treatment modalities, such as total mesorectal excision (TME) and preoperative (chemo)radiotherapy for rectal cancer, as well as adjuvant chemotherapy for stage III colon cancer, have been studied extensively and showed to improve cancer-related outcomes. On the contrary, the effectiveness of other treatment modalities including adjuvant chemotherapy for rectal cancer and for stage II colon cancer, and surgery of the primary tumour in incurable metastatic colorectal cancer are still subject of debate. Moreover, there is considerable short-term and long-term morbidity after (chemo)radiotherapy or surgery which should be taken into account. Further defining optimal treatment strategies is therefore of great importance. This thesis focused on improving evidence for treatment modalities that are currently subject of debate for patients with colorectal cancer. This was done using data from randomised controlled trials as well as cancer registry data. 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
The tumour-stroma ratio (TSR) and tumour budding (TB) are two high-risk factors with potential to be implemented in the next TNM classification. The aim of the current study was to evaluate the... Show moreThe tumour-stroma ratio (TSR) and tumour budding (TB) are two high-risk factors with potential to be implemented in the next TNM classification. The aim of the current study was to evaluate the practical application of the two biomarkers based on reproducibility, independency and prognostic value. Patients diagnosed with stage II or III colon cancer who underwent surgery between 2005 and 2016 were included. Both TSR and TB were scored on haematoxylin and eosin-stained tissue sections. The TSR, based on the relative amount of stroma, was scored in increments of 10%. TB was scored following the consensus guidelines; a bud was defined as <= 4 tumour cells. For analysis, three categories were used. Cohen's kappa was used for reproducibility. The prognostic value was determined with survival analysis. In total, 246 patients were included. The TSR distribution was N = 137 (56%) stroma-low and N = 109 (44%) stroma-high. The TB distribution was TB-low N = 194 (79%), TB-intermediate N = 35 (14%) and TB-high N = 17 (7%). The reproducibility of the TSR was good (interobserver agreement kappa = 0.83 and intraobserver agreement kappa = 0.82), whereas the inter- and intraobserver agreement for scoring TB was moderate (kappa 0.47 and 0.45, respectively). The survival analysis showed an independent prognostic value for disease-free survival for TSR (HR 1.57; 95% CI 1.01-2.44; p = 0.048) and for TB-high (HR 2.01; 95% CI 1.02-3.96; p = 0.043). Based on current results, we suggest the TSR is a more reliable parameter in daily practice due to better reproducibility and independent prognostic value for disease-free survival. Show less
Duijster, J.W.; Hansen, J.V.; Franz, E.; Neefjes, J.J.C.; Frisch, M.; Mughini-Gras, L.; Ethelberg, S. 2021
Laboratory data increasingly suggest that Salmonella infection may contribute to colon cancer (CC) development. Here, we examined epidemiologically the potential risk of CC associated with... Show moreLaboratory data increasingly suggest that Salmonella infection may contribute to colon cancer (CC) development. Here, we examined epidemiologically the potential risk of CC associated with salmonellosis in the human population. We conducted a population-based cohort study using four health registries in Denmark. Person-level demographic data of all residents were linked to laboratory-confirmed non-typhoidal salmonellosis and to CC diagnoses in 1994-2016. Hazard ratios (HRs) for CC (overall/proximal/distal) associated with reported salmonellosis were estimated using Cox proportional hazard models. Potential effects of serovar, age, sex, inflammatory bowel disease and follow-up time post-infection were also assessed. We found no increased risk of CC >= 1 year post-infection (HR 0.99; 95% confidence interval (CI) 0.88-1.13). When stratifying by serovar, there was a significantly increased risk of proximal CC >= 1 year post-infection with serovars other than Enteritidis and Typhimurium (HR 1.40; 95% CI 1.03-1.90). CC risk was significantly increased in the first year post-infection (HR 2.08; 95% CI 1.48-2.93). The association between salmonellosis and CC in the first year post-infection can be explained by increased stool testing around the time of CC diagnosis. The association between proximal CC and non-Enteritidis/non-Typhimurium serovars is unclear and warrants further investigation. Overall, this study provides epidemiological evidence that notified Salmonella infections do not contribute significantly to CC risk in the studied population. Show less
Background: Treatment decisions concerning older patients can be very challenging and individualised treatment plans are often required in this very heterogeneous group. In 2015 we have implemented... Show moreBackground: Treatment decisions concerning older patients can be very challenging and individualised treatment plans are often required in this very heterogeneous group. In 2015 we have implemented a routine clinical care pathway for older patients in need of intensive treatment, including a comprehensive geriatric assessment (CGA) that was used to support clinical decision making. An ongoing prospective cohort study, the Triaging Elderly Needing Treatment (TENT) study, has also been initiated in 2016 for participants in this clinical care pathway, to study associations between geriatric characteristics and outcomes of treatment that are relevant to older patients. The aim of this paper is to describe the implementation and rationale of the routine clinical care pathway and design of the TENT study.Methods: A routine clinical care pathway has been designed and implemented in multiple hospitals in the Netherlands. Patients aged >= 70 years who are candidates for intensive treatments, such as chemotherapy, (chemo-)radiation therapy or major surgery, undergo frailty screening based on the Geriatric 8 (G-8) questionnaire and the Six-Item Cognitive Impairment Test (6CIT). If screening reveals potential frailty, a CGA is performed. All patients are invited to participate in the TENT study. Clinical data and blood samples for biomarker studies are collected at baseline. During follow-up, information about treatment complications, hospitalisations, functional decline, quality of life and mortality is collected. The primary outcome is the composite endpoint of functional decline or mortality at 1 year.Discussion: Implementation of a routine clinical care pathway for older patients in need of intensive treatment provides the opportunity to study associations between determinants of frailty and outcomes of treatment. Results of the TENT study will support individualised treatment for future patients.Trial registration: The study is retrospectively registered at the Netherlands Trial Register (NTR), trial number NL81 07. Date of registration: 22-10-2019. Show less
This thesis describes the detailed method of scoring the tumor-stroma ratio and the different possibilities to use it in routine clinical diagnostics, for different types of cancer. It can be used... Show moreThis thesis describes the detailed method of scoring the tumor-stroma ratio and the different possibilities to use it in routine clinical diagnostics, for different types of cancer. It can be used not only for prognostic purposes, but it might also be useful for predicting the response on neo-adjuvant therapy. As it is an easy and cheap method, based on routine hematoxylin-eosin stained tissue slides used for daily pathology routine, it can be implemented in clinical diagnostics with little effort. Show less
Patients presenting with ovarian tumors may pose diagnostic challenges. Primary ovarian tumors should be distinguished from metastases to the ovaries from other primary tumors. Treatment choices... Show morePatients presenting with ovarian tumors may pose diagnostic challenges. Primary ovarian tumors should be distinguished from metastases to the ovaries from other primary tumors. Treatment choices and statements about prognosis are different for localised or metastatic disease. Diagnostic workup including imaging and histologic examination of tumors is not always successful in deciphering the origin of tumors.Therefore, additional characterisation of tumors at a molecular level might be helpful. Show less
