Johannes S. de Jong

Intraoperative tumor-specific fluorescence imaging in ovarian cancer by folate receptor-α targeting: first in-human results

The prognosis in advanced-stage ovarian cancer remains poor. Tumor-specific intraoperative fluorescence imaging may improve staging and debulking efforts in cytoreductive surgery and thereby improve prognosis. The overexpression of folate receptor-α (FR-α) in 90–95% of epithelial ovarian cancers prompted the investigation of intraoperative tumor-specific fluorescence imaging in ovarian cancer surgery using an FR-α–targeted fluorescent agent. In patients with ovarian cancer, intraoperative tumor-specific fluorescence imaging with an FR-α–targeted fluorescent agent showcased the potential applications in patients with ovarian cancer for improved intraoperative staging and more radical cytoreductive surgery.

Expression of growth factors, growth-inhibiting factors, and their receptors in invasive breast cancer. II: Correlations with proliferation and angiogenesis

Growth factors may play an important role in tumour growth and angiogenesis by their influence on tumour cell proliferation or their effect on neovascularization. The aim of the present study was to determine which of the growth factors, growth‐inhibiting factors, and their receptors investigated in a previous study are correlated with proliferation and angiogenesis in invasive breast cancer, with emphasis on the impact of possible autocrine and paracrine loops. Five growth factors and their receptors: platelet‐derived growth factor A chain (PDGF‐AA) and PDGF alpha receptor (PDGFαR), PDGF‐BB and PDGF beta receptor (PDGFβR), transforming growth factor alpha (TGFα) and its receptor epidermal growth factor receptor (EGFR), and vascular endothelial growth factor (VEGF) and its receptors (Flt‐1 and Flk‐1/KDR; two growth‐inhibiting factors: transforming growth factor beta‐1 (TGFβ1) and TGFβ2 and their receptor couple TGFβR‐I and TGFβR‐II; and basic fibroblast growth factor (bFGF) were stained in 45 cases of invasive breast cancer by standard immunohistochemistry on frozen sections. Staining in tumour cells, stromal cells, and endothelial cells was scored as negative or positive. Proliferation was determined by assessment of the mitotic activity index (MAI) and the degree of angiogenesis was measure by counting the number of microvessels (microvessel density: MVD) in the most vascularized area of the tumour. bFGF and EGFR showed positive correlations with the MAI, while TGFβ2 showed a negative correlation. Expression of bFGF, TGFα, TGFβ2, and EGFR correlated positively with the MVD. Co‐expression of the TGFα/EGFR growth factor/receptor combination showed a stronger correlation with the MAI and the MVD than EGFR or TGFα alone, and the TGFβ2/TGFβR‐I/TGFβR‐II combination showed a positive correlation with the MVD. In conclusion, the expression of several growth factors, growth factor receptors, and growth‐inhibiting factors showed correlations with the rate of proliferation and the degree of angiogenesis in invasive breast cancer. Some growth factor/receptor combinations showed stronger correlations with proliferation and angiogenesis than the growth factor or receptor alone, pointing to the importance of possible auto‐ and paracrine loops for stimulation of proliferation and angiogenesis by growth factors and their receptors.

Obtaining Adequate Surgical Margins in Breast-Conserving Therapy for Patients with Early-Stage Breast Cancer: Current Modalities and Future Directions

Inadequate surgical margins represent a high risk for adverse clinical outcome in breast-conserving therapy (BCT) for early-stage breast cancer. The majority of studies report positive resection margins in 20% to 40% of the patients who underwent BCT. This may result in an increased local recurrence (LR) rate or additional surgery and, consequently, adverse affects on cosmesis, psychological distress, and health costs. In the literature, various risk factors are reported to be associated with positive margin status after lumpectomy, which may allow the surgeon to distinguish those patients with a higher a priori risk for re-excision. However, most risk factors are related to tumor biology and patient characteristics, which cannot be modified as such. Therefore, efforts to reduce the number of positive margins should focus on optimizing the surgical procedure itself, because the surgeon lacks real-time intraoperative information on the presence of positive resection margins during breast-conserving surgery. This review presents the status of pre- and intraoperative modalities currently used in BCT. Furthermore, innovative intraoperative approaches, such as positron emission tomography, radioguided occult lesion localization, and near-infrared fluorescence optical imaging, are addressed, which have to prove their potential value in improving surgical outcome and reducing the need for re-excision in BCT.

Expression of growth factors, growth inhibiting factors, and their receptors in invasive breast cancer. I: An inventory in search of autocrine and paracrine loops

The aim of the present study was to investigate which growth factors, receptors, and growth inhibiting factors are expressed in invasive breast cancer. Five (angiogenic) growth factors and their receptors: platelet‐derived growth factor A chain (PDGF‐AA) and PDGF receptor alpha (PDGFαR), PDGF‐BB and PDGF beta receptor, transforming growth factor alpha (TGFα) and its receptor epidermal growth factor receptor (EGFR), and vascular endothelial growth factor (VEGF) and its receptors vascular endothelial growth factor receptor I (Flt‐1) and vascular endothelial growth factor receptor II (Flk‐1/KDR); two growth inhibiting factors: transforming growth factor beta‐1 (TGFβ1) and TGFβ2) and their receptor couple transforming growth factor beta receptor I (TGFβR‐I) and TGFβR‐II; and basic fibroblast growth factor (bFGF) were stained by standard immunohistochemistry on frozen sections in 45 cases of invasive carcinoma of the breast. Staining was scored as negative or positive in tumour epithelium, stroma, and blood vessels. TGFβ1 and TGFβ2 were expressed in the tumour cells in 67 per cent and 76 per cent of cases, respectively, whereas PDGFβR and TGFβR‐II were expressed in 0 per cent and 2 per cent, respectively. The other factors showed variable expression in tumour cells. All factors were expressed in the stroma in most cases, except Flt‐1, Flk‐1/KDR, TGFβ2, and TGFβR‐II, which showed variable expression, and EGFR, which showed no expression. The endothelium was in most cases positive for bFGF, PDGF‐AA, PDGF‐BB, VEGF, PDGFαR, PDGFβR, and TGFβ1 but TGFβ2 was negative in most cases and TGFα, EGFR, Flt‐1, Flk‐1/KDR, TGFβR‐I, and TGFβR‐II were variably expressed. The most interesting possible auto/paracrine loops, as demonstrated on serial sections and by fluorescence double staining, were the TGFα/EGFR, TGFβs/TGFβR, VEGF/Flt‐1, and the VEGF/Flk‐1 combinations. In conclusion, growth factors, growth inhibiting factors, and their receptors are frequently expressed in invasive breast cancer. Indications for some possible auto‐and paracrine loops have been found, which should encourage further study on the role of these factors in breast cancer proliferation and angiogenesis.

Intraoperative assessment of biliary anatomy for prevention of bile duct injury: a review of current and future patient safety interventions

BackgroundBile duct injury (BDI) is a dreaded complication of cholecystectomy, often caused by misinterpretation of biliary anatomy. To prevent BDI, techniques have been developed for intraoperative assessment of bile duct anatomy. This article reviews the evidence for the different techniques and discusses their strengths and weaknesses in terms of efficacy, ease, and cost-effectiveness.MethodPubMed was searched from January 1980 through December 2009 for articles concerning bile duct visualization techniques for prevention of BDI during laparoscopic cholecystectomy.ResultsNine techniques were identified. The critical-view-of-safety approach, indirectly establishing biliary anatomy, is accepted by most guidelines and commentaries as the surgical technique of choice to minimize BDI risk. Intraoperative cholangiography is associated with lower BDI risk (OR 0.67, CI 0.61–0.75). However, it incurs extra costs, prolongs the operative procedure, and may be experienced as cumbersome. An established reliable alternative is laparoscopic ultrasound, but its longer learning curve limits widespread implementation. Easier to perform are cholecystocholangiography and dye cholangiography, but these yield poor-quality images. Light cholangiography, requiring retrograde insertion of an optical fiber into the common bile duct, is too unwieldy for routine use. Experimental techniques are passive infrared cholangiography, hyperspectral cholangiography, and near-infrared fluorescence cholangiography. The latter two are performed noninvasively and provide real-time images. Quantitative data in patients are necessary to further evaluate these techniques.ConclusionsThe critical-view-of-safety approach should be used during laparoscopic cholecystectomy. Intraoperative cholangiography or laparoscopic ultrasound is recommended to be performed routinely. Hyperspectral cholangiography and near-infrared fluorescence cholangiography are promising novel techniques to prevent BDI and thus increase patient safety.

Multispectral Near-Infrared Fluorescence Molecular Imaging of Matrix Metalloproteinases in a Human Carotid Plaque Using a Matrix-Degrading Metalloproteinase–Sensitive Activatable Fluorescent Probe

Formation of unstable atherosclerotic plaque in the internal carotid artery carries a high risk for emboli and subsequent cerebral ischemic events. The fibrous cap of such a plaque may become thin and rupture as a result of the depletion of matrix components through the activation of proteolytic enzymes such as matrix-degrading proteinases. Enhanced matrix breakdown has been attributed primarily to a family of matrix-degrading metalloproteinases (MMPs) that are highly concentrated in atherosclerotic plaques by inflammatory cells (eg, macrophages, foam cells), smooth muscle cells and endothelial cells.1 Elevated serum MMP-9 concentration is associated with carotid plaque instability and the presence of infiltrated macrophages.2 Furthermore, analysis of the presence of MMP-9 protein by ELISA within excised carotid plaques revealed high MMP-9 protein mass in calcified segments at or near the carotid bifurcation and in segments with intraplaque hemorrhage. Gelatin zymography showed an increased gelatinase activity of MMP-9 in these segments.3 These data favor the important role of MMP-9 in the pathogenesis of plaque instability. We analyzed the topographic distribution of MMPs within an excised human carotid plaque by applying multispectral near-infrared fluorescence (NIRF) imaging (IVIS Spectrum, Caliper Life Sciences, Hopkinton, Mass). A surgical endarterectomy was performed on a 74-year-old women with a left-sided, symptomatic, >70% carotid stenosis. Immediately after endarterectomy, the plaque was placed in PBS and transported to the NIRF system. The plaque was then stretched out and fixed on a silicon plate with 25G needles. A PBS NIRF image was generated from both the intraluminal and extraluminal side of the plaque to determine the level of autofluorescence (background) (Figure 1). After incubation with …

Counting of apoptotic cells: a methodological study in invasive breast cancer

Aims—To arrive at a reproducible sampling technique for counting apoptotic cells in tissue sections of invasive breast cancer that can serve as a protocol for further clinical studies. Methods—In 4 μm thick haematoxylin and eosin stained tissue sections of 12 breast cancers, apoptotic cells, recognised by strict morphological criteria, were counted in consecutive fields of vision at ×1000 magnification in a marked area in the most poorly differentiated region of tumour. These counts were regarded as the gold standard. Subsequently, in a systematic sampling experiment, the number of fields that had to be counted to derive an acceptable coefficient of variation (CV) was determined. To compare counts at different magnifications, all fields of vision were also counted at ×630 and ×400. The intra- and inter-observer reproducibility was tested by repeated measurements at these magnifications in 10 systematically selected fields of vision. Results—Apoptosis seemed to be a rare event, affecting, on average, about 1% of tumour cells. Noticeable clustering of apoptotic cells was observed. The systematic sampling experiment showed that at ×1000 magnification, the CV was improved by counting up to 20 fields. When comparing ×400 and ×630 magnifications with the ×1000 magnification, the correlation coefficients were 0.88 and 0.87, respectively. However, the lower magnifications yielded lower counts. With regard to reproducibility, the intra-observer correlation coefficient was 0.91 at ×630 and 0.76 at ×400. The inter-observer correlation coefficient was 0.77 at ×630 and 0.68 at ×400. Conclusions—Apoptotic cells can be counted readily in haematoxylin and eosin stained tissue sections. However, a systematic sampling protocol must be followed and cells should be counted at a relatively high magnification to obtain acceptable reproducibility. The suggested protocol will permit further correlative and prognostic studies and the monitoring of the effects of treatment.

Tumor-specific uptake of fluorescent bevacizumab-IRDye800CW microdosing in patients with primary breast cancer: a phase I feasibility study

Purpose: To provide proof of principle of safety, breast tumor–specific uptake, and positive tumor margin assessment of the systemically administered near-infrared fluorescent tracer bevacizumab–IRDye800CW targeting VEGF-A in patients with breast cancer. Experimental Design: Twenty patients with primary invasive breast cancer eligible for primary surgery received 4.5 mg bevacizumab–IRDye800CW as intravenous bolus injection. Safety aspects were assessed as well as tracer uptake and tumor delineation during surgery and ex vivo in surgical specimens using an optical imaging system. Ex vivo multiplexed histopathology analyses were performed for evaluation of biodistribution of tracer uptake and coregistration of tumor tissue and healthy tissue. Results: None of the patients experienced adverse events. Tracer levels in primary tumor tissue were higher compared with those in the tumor margin (P < 0.05) and healthy tissue (P < 0.0001). VEGF-A tumor levels also correlated with tracer levels (r = 0.63, P < 0.0002). All but one tumor showed specific tracer uptake. Two of 20 surgically excised lumps contained microscopic positive margins detected ex vivo by fluorescent macro- and microscopy and confirmed at the cellular level. Conclusions: Our study shows that systemic administration of the bevacizumab–IRDye800CW tracer is safe for breast cancer guidance and confirms tumor and tumor margin uptake as evaluated by a systematic validation methodology. The findings are a step toward a phase II dose-finding study aimed at in vivo margin assessment and point to a novel drug assessment tool that provides a detailed picture of drug distribution in the tumor tissue. Clin Cancer Res; 23(11); 2730–41. ©2016 AACR.

In Vivo Fluorescence Immunohistochemistry: Localization of Fluorescently Labeled Cetuximab in Squamous Cell Carcinomas

Anti-EGFR (epidermal growth factor receptor) antibody based treatment strategies have been successfully implemented in head and neck squamous cell carcinoma (HNSCC). Unfortunately, predicting an accurate and reliable therapeutic response remains a challenge on a per-patient basis. Although significant efforts have been invested in understanding EGFR-mediated changes in cell signaling related to treatment efficacy, the delivery and histological localization in (peri-)tumoral compartments of antibody-based therapeutics in human tumors is poorly understood nor ever made visible. In this first in-human study of a systemically administered near-infrared (NIR) fluorescently labeled therapeutic antibody, cetuximab-IRDye800CW (2.5 mg/m2, 25 mg/m2, and 62.5 mg/m2), we show that by optical molecular imaging (i.e. denominated as In vivo Fluorescence Immunohistochemistry) we were able to evaluate localization of fluorescently labeled cetuximab. Clearly, optical molecular imaging with fluorescently labeled antibodies correlating morphological (peri-)tumoral characteristics to levels of antibody delivery, may improve treatment paradigms based on understanding true tumoral antibody delivery.

Expression of growth factors, growth factor receptors and apoptosis related proteins in invasive breast cancer: relation to apoptotic rate

AbstractPurpose. To assess the relation between growth factors, growth-factor receptors, p53, bcl-2 and bax expression, and the rate of apoptosis in invasive breast cancer patients. Materials and methods. Tumors from 45 patients were assessed by immunohistochemistry for expression patterns of five growth factors and their receptor platelet-derived growth factor A chain (PDGF-AA) and PDGF-receptor alpha (PDGFαR), PDGF-BB and PDGFβR, transforming growth-factor alpha (TGFα) and its receptor-epidermal growth factor receptor (EGFR) and vascular-endothelial growth factor (VEGF) and its receptors vascular-endothelial growth factor receptor I (FLT-1) and vascular-endothelial growth factor receptor II (FLK-1/KDR), two growth-inhibiting factors; transforming-growth factor beta 1 (TGFβ1) and TGFβ2 and their receptor couple TGFβ receptor I (TGFβR-I) and TGFβR-II, and basic-fibroblast growth factor (bFGF). Besides, the expression patterns of the bcl-2, bax and p53 gene products were investigated. Expression patterns were correlated to the number of apoptotic cells assessed by light microscopy. Results. PDGF-BB and bFGF showed a positive correlation with the AI (p=0.006 and p=0.030, respectively). EGFR expression was associated with a high number of apoptotic cells but did not reach significance (p=0.10). None of the other individual growth factors, growth-inhibiting factors or receptors showed a significant relation with the AI. The presence of a possible auto- or paracrine loop of the TGFα/EGFR combination was associated with a high number of apoptotic cells but did not reach significance (p=0.20). PDGF-AA, bFGF and EGFR expression showed a significant relation to p53 overexpression. TGFβ2 expression showed an inverse correlation with p53 overexpression. Conclusion. We found an association between several growth factors and growth-factor receptors with number of apoptotic cells. This underlines the importance of growth factors and their receptors not just in proliferation, but also, directly and/or indirectly, in regulating the rate of apoptosis in invasive breast cancer. Growth factors and their receptors may therefore be useful as targets of anti-cancer therapy by inducing apoptosis or increasing the sensitivity of cells for chemo- or hormonal therapy induced apoptosis.