Geke A.P. Hospers

Endothelium in vitro: a review of human vascular endothelial cell lines for blood vessel-related research.

Endothelial cells (EC) are currently used as in vitro model systems for various physiological and pathological processes, especially in angiogenesis research. Primary EC have a limited lifespan and display characteristics that differ from batch to batch due to their multidonor origin. In recent years many groups have established EC lines. This Review gives an overview of the advantages and disadvantages of currently available vascular EC lines. Its aim is to help the investigator to decide which cell line matches his or her research goal best. Truly immortalized cell lines are generally better characterized and more stable in their endothelial traits than EC that were given an extended life span. Presently the best characterized macro- and micro-vascular EC lines are EA.hy926 and HMEC-1, respectively.

Platelets and granulocytes, in particular the neutrophils, form important compartments for circulating vascular endothelial growth factor.

The measurement of circulating vascular endothelial growth factor (VEGF) levels as a prognostic factor will gain increasing relevance in the diagnosis and evaluation of treatment in cancer patients. Angiogenesis is an absolute requirement in tumour growth and metastatic disease. In the present study data are presented which indicate that circulating VEGF mainly resides in peripheral blood cells. In 15 healthy volunteers we demonstrated that approximately 34% of the circulating VEGF resides in platelets and approximately 11% in patients with cancer (n= 4). An important part namely 58% in healthy volunteers and 69% in patients with cancer of the total circulating VEGF is contained in granulocytes, particular in the neutrophils, as confirmed by fluorescence-activated cell sorting (FACS). Also an increased VEGF level per granulocyte is found in patients with cancer (77 μg VEGF/l) compared with the healthy volunteers (164 μg VEGF/l). In contrast only 2% was present in plasma. The biological significance of platelet- or granulocyte-derived VEGF is not yet known. Liberation of VEGF from these compartments could well be of importance for tumour angiogenesis. Therefore, future studies on the clinical value of circulating VEGF as a prognostic factor in cancer patients should include measurements of VEGF in peripheral blood cells.

In Vivo VEGF Imaging with Radiolabeled Bevacizumab in a Human Ovarian Tumor Xenograft

Vascular endothelial growth factor (VEGF), released by tumor cells, is an important growth factor in tumor angiogenesis. The humanized monoclonal antibody bevacizumab blocks VEGF-induced tumor angiogenesis by binding, thereby neutralizing VEGF. Our aim was to develop radiolabeled bevacizumab for noninvasive in vivo VEGF visualization and quantification with the single γ-emitting isotope 111In and the PET isotope 89Zr. Methods: Labeling, stability, and binding studies were performed. Nude mice with a human SKOV-3 ovarian tumor xenograft were injected with 89Zr-bevacizumab, 111In-bevacizumab, or human 89Zr-IgG. Human 89Zr-IgG served as an aspecific control antibody. Small-animal PET and microCT studies were obtained at 24, 72, and 168 h after injection of 89Zr-bevacizumab and 89Zr-IgG (3.5 ± 0.5 MBq, 100 ± 6 μg, 0.2 mL [mean ± SD]). Small-animal PET and microCT images were fused to calculate tumor uptake and compared with ex vivo biodistribution at 168 h after injection. 89Zr- and 111In-bevacizumab ex vivo biodistribution was compared at 24, 72, and 168 h after injection (2.0 ± 0.5 MBq each, 100 ± 4 μg in total, 0.2 mL). Results: Labeling efficiencies, radiochemical purity, stability, and binding properties were optimal for the radioimmunoconjugates. Small-animal PET showed uptake in well-perfused organs at 24 h and clear tumor localization from 72 h onward. Tumor uptake determined by quantification of small-animal PET images was higher for 89Zr-bevacizumab—namely, 7.38 ± 2.06 %ID/g compared with 3.39 ± 1.16 %ID/g (percentage injected dose per gram) for human 89Zr-IgG (P = 0.011) at 168 h and equivalent to ex vivo biodistribution studies. Tracer uptake in other organs was seen primarily in liver and spleen. 89Zr- and 111In-bevacizumab biodistribution was comparable. Conclusion: Radiolabeled bevacizumab showed higher uptake compared with radiolabeled human IgG in a human SKOV-3 ovarian tumor xenograft. Noninvasive quantitative small-animal PET was similar to invasive ex vivo biodistribution. Radiolabeled bevacizumab is a new tracer for noninvasive in vivo imaging of VEGF in the tumor microenvironment.

Vemurafenib in patients with BRAFV600 mutated metastatic melanoma: an open-label, multicentre, safety study

BACKGROUND The orally available BRAF kinase inhibitor vemurafenib, compared with dacarbazine, shows improved response rates, progression-free survival (PFS), and overall survival in patients with metastatic melanoma that has a BRAF(V600) mutation. We assessed vemurafenib in patients with advanced metastatic melanoma with BRAF(V600) mutations who had few treatment options. METHODS In an open-label, multicentre study, patients with untreated or previously treated melanoma and a BRAF(V600) mutation received oral vemurafenib 960 mg twice a day. The primary endpoint was safety. All analyses were done on the safety population, which included all patients who received at least one dose of vemurafenib. This report is the third interim analysis of this study. This study is registered with ClinicalTrials.gov, number NCT01307397. FINDINGS Between March 1, 2011, and Jan 31, 2013, 3226 patients were enrolled in 44 countries. 3222 patients received at least one dose of vemurafenib (safety population). At data cutoff, 868 (27%) patients were on study treatment and 2354 (73%) had withdrawn, mainly because of disease progression. Common adverse events of all grades included rash (1592 [49%]), arthralgia (1259 [39%]), fatigue (1093 [34%]), photosensitivity reaction (994 [31%]), alopecia (826 [26%]), and nausea (628 [19%]). 1480 (46%) patients reported grade 3 or 4 adverse events, including cutaneous squamous cell carcinoma (389 [12%]), rash (155 [5%]), liver function abnormalities (165 [5%]), arthralgia (106 [3%]), and fatigue (93 [3%]). Grade 3 and 4 adverse events were reported more frequently in patients aged 75 years and older (n=257; 152 [59%, 95% CI 53-65] and ten [4%, 2-7], respectively) than in those younger than 75 years (n=2965; 1286 [43%, 42-45] and 82 [3%, 2-3], respectively). INTERPRETATION Vemurafenib safety in this diverse population of patients with BRAF(V600) mutated metastatic melanoma, who are more representative of routine clinical practice, was consistent with the safety profile shown in the pivotal trials of this drug. FUNDING F Hoffmann-La Roche.

Short-course radiotherapy followed by neo-adjuvant chemotherapy in locally advanced rectal cancer – the RAPIDO trial

BackgroundCurrent standard for most of the locally advanced rectal cancers is preoperative chemoradiotherapy, and, variably per institution, postoperative adjuvant chemotherapy. Short-course preoperative radiation with delayed surgery has been shown to induce tumour down-staging in both randomized and observational studies. The concept of neo-adjuvant chemotherapy has been proven successful in gastric cancer, hepatic metastases from colorectal cancer and is currently tested in primary colon cancer.Methods and designPatients with rectal cancer with high risk features for local or systemic failure on magnetic resonance imaging are randomized to either a standard arm or an experimental arm. The standard arm consists of chemoradiation (1.8 Gy x 25 or 2 Gy x 25 with capecitabine) preoperatively, followed by selective postoperative adjuvant chemotherapy. Postoperative chemotherapy is optional and may be omitted by participating institutions. The experimental arm includes short-course radiotherapy (5 Gy x 5) followed by full-dose chemotherapy (capecitabine and oxaliplatin) in 6 cycles before surgery. In the experimental arm, no postoperative chemotherapy is prescribed. Surgery is performed according to TME principles in both study arms. The hypothesis is that short-course radiotherapy with neo-adjuvant chemotherapy increases disease-free and overall survival without compromising local control. Primary end-point is disease-free survival at 3 years. Secondary endpoints include overall survival, local control, toxicity profile, and treatment completion rate, rate of pathological complete response and microscopically radical resection, and quality of life.DiscussionFollowing the advances in rectal cancer management, increased focus on survival rather than only on local control is now justified. In an experimental arm, short-course radiotherapy is combined with full-dose chemotherapy preoperatively, an alternative that offers advantages compared to concomitant chemoradiotherapy with or without postoperative chemotherapy. In a multi-centre setting this regimen is compared to current standard with the aim of improving survival for patients with locally advanced rectal cancer.Trial registrationClinicalTrials.gov NCT01558921

Baseline Biomarkers for Outcome of Melanoma Patients Treated with Pembrolizumab

Purpose: Biomarkers for outcome after immune-checkpoint blockade are strongly needed as these may influence individual treatment selection or sequence. We aimed to identify baseline factors associated with overall survival (OS) after pembrolizumab treatment in melanoma patients. Experimental Design: Serum lactate dehydrogenase (LDH), routine blood count parameters, and clinical characteristics were investigated in 616 patients. Endpoints were OS and best overall response following pembrolizumab treatment. Kaplan–Meier analysis and Cox regression were applied for survival analysis. Results: Relative eosinophil count (REC) ≥1.5%, relative lymphocyte count (RLC) ≥17.5%, ≤2.5-fold elevation of LDH, and the absence of metastasis other than soft-tissue/lung were associated with favorable OS in the discovery (n = 177) and the confirmation (n = 182) cohort and had independent positive impact (all P < 0.001). Their independent role was subsequently confirmed in the validation cohort (n = 257; all P < 0.01). The number of favorable factors was strongly associated with prognosis. One-year OS probabilities of 83.9% versus 14.7% and response rates of 58.3% versus 3.3% were observed in patients with four of four compared to those with none of four favorable baseline factors present, respectively. Conclusions: High REC and RLC, low LDH, and absence of metastasis other than soft-tissue/lung are independent baseline characteristics associated with favorable OS of patients with melanoma treated with pembrolizumab. Presence of four favorable factors in combination identifies a subgroup with excellent prognosis. In contrast, patients with no favorable factors present have a poor prognosis, despite pembrolizumab, and additional treatment advances are still needed. A potential predictive impact needs to be further investigated. Clin Cancer Res; 22(22); 5487–96. ©2016 AACR.

VEGF-PET Imaging Is a Noninvasive Biomarker Showing Differential Changes in the Tumor during Sunitinib Treatment

Non-invasive imaging of angiogenesis could ease the optimization of antiangiogenesis treatments for cancer. In this study, we evaluated the role of VEGF-PET as a biomarker of dynamic angiogenic changes in tumors following treatment with the kinase inhibitor sunitinib. The effects of sunitinib treatment and withdrawal on the tumor was investigated using the new VEGF-PET tracer (89)Zr-ranibizumab as well as (18)F-FDG PET, and (15)O-water PET in mouse xenograft models of human cancer. The obtained imaging results were compared with tumor growth, VEGF plasma levels and immunohistologic analyzes. In contrast to (18)F-FDG and (15)O-water PET, VEGF-PET demonstrated dynamic changes during sunitinib treatment within the tumor with a strong decline in signal in the tumor center and only minimal reduction in tumor rim, with a pronounced rebound after sunitinib discontinuation. VEGF-PET results corresponded with tumor growth and immunohistochemical vascular- and tumor- markers. Our findings highlight the strengths of VEGF-PET imaging to allow serial analysis of angiogenic changes in different areas within a tumor.

PET imaging of oestrogen receptors in patients with breast cancer

Oestrogen receptors are overexpressed in around 70% of all breast cancers, and are a target for endocrine therapy. These receptors can be visualised on PET with use of 16α-[(18)F]-fluoro-17β-oestradiol ((18)F-FES) as a tracer. Compared with biopsy, which enables assessment of individual sites, whole-body (18)F-FES-PET enables quantification of oestrogen-receptor expression in all metastases. In several studies, measurement of tumour protein expression in oestrogen receptors by (18)F-FES-PET, concurrent with biopsy, detected oestrogen-receptor-positive tumour lesions with a sensitivity of 84% and specificity of 98%. Roughly 45% of patients with metastatic breast cancer have discordant oestrogen-receptor expression across lesions (ie, (18)F-FES-positive and (18)F-FES-negative metastases). Low tumour (18)F-FES uptake in metastases can predict failure of hormonal therapy in patients with oestrogen-receptor-positive primary tumours. Finally, (18)F-FES-PET has shown that oestrogen-receptor binding capacity changes after intervention with hormonal drugs, but findings need to be confirmed. Factors other than oestrogen-receptor expression, including menopausal status and concomitant therapies, that can affect tumour (18)F-FES uptake must be taken into account.

PET Imaging of Estrogen Receptors as a Diagnostic Tool for Breast Cancer Patients Presenting with a Clinical Dilemma

16α-18F-fluoro-17β-estradiol (18F-FES) is an estrogen receptor (ER)–specific PET tracer with various potential interesting applications. The precise contribution of this technique in current clinical practice, however, has yet to be determined. Therefore, the aim of this study was to evaluate the value of 18F-FES PET in breast cancer patients presenting with a clinical dilemma. Methods: 18F-FES PET examination could be requested by referring physicians for patients with a history of ER-positive breast cancer and the presence of a clinical dilemma despite complete standard work-up. All requests for 18F-FES PET required a positive arbitration by a dedicated medical oncologist and nuclear medicine physician. The referring physician was asked to fill in validated questionnaires before, shortly after, and at more than 3 mo after 18F-FES PET to determine indication, diagnostic value, and therapeutic consequences of 18F-FES PET. To further validate 18F-FES PET findings, 18F-FES PET lesions were quantified and compared with centrally reviewed conventional imaging. Results: Thirty-three patients underwent 18F-FES PET between December 2008 and October 2010. 18F-FES PET was requested to evaluate equivocal lesions on conventional work-up (n = 21), ER status in metastatic patients (n = 10), and the origin of metastases (n = 2). 18F-FES–positive lesions were observed in 22 patients. 18F-FES PET was especially sensitive for bone metastases, detecting 341 bone lesions, compared with 246 by conventional imaging. The sensitivity for liver metastases was poor, and quantification of 18F-FES uptake in liver lesions was hampered by high physiologic background. 18F-FES uptake was highly variable between all metastases (range of standardized uptake value, 1.20–18.81), and 45% of the patients with a positive 18F-FES PET finding had both 18F-FES–positive and 18F-FES–negative metastases. 18F-FES PET improved diagnostic understanding in 88% of the patients and led to therapy change in 48% of the patients. Conclusion: With the exception of liver metastases, whole-body imaging of ER expression with 18F-FES PET can be a valuable additional diagnostic tool when standard work-up is inconclusive. 18F-FES PET supported therapy decisions by improving diagnostic understanding and providing information on ER status of tumor lesions.

Towards Sustained Silencing of HER2/neu in Cancer By Epigenetic Editing

The human epidermal growth factor receptor-2 (HER2/neu/ERBB2) is overexpressed in several cancer types. Although therapies targeting the HER2/neu protein result in inhibition of cell proliferation, the anticancer effect might be further optimized by limiting HER2/neu expression at the DNA level. Towards this aim, epigenetic editing was performed to suppress HER2/neu expression by inducing epigenetic silencing marks on the HER2/neu promoter.HER2/neu expression and HER2/neu promoter epigenetic modification status were determined in a panel of ovarian and breast cancer cell lines. HER2/neu-overexpressing cancer cells were transduced to express a zinc finger protein (ZFP), targeting the HER2/neugene, fused to histone methyltransferases (G9a, SUV39-H1)/super KRAB domain (SKD). Epigenetic assessment of the HER2/neu promoter showed that HER2/neu-ZFP fused to G9a efficiently induced the intended silencing histone methylation mark (H3K9me2). Importantly, H3K9me2 induction was associated with a dramatic downregulation of HER2/neu expression in HER2/neu- overexpressing cells. Downregulation by SKD, traditionally considered transient in nature, was associated with removal of the histone acetylation mark (H3ac). The downregulation of HER2/neu by induced H3K9 methylation and/or reduced H3 acetylation was sufficient to effectively inhibit cellular metabolic activity and clonogenicity. Furthermore, genome-wide analysis indicated preferential binding of the ZFP to its target sequence. These results not only show that H3K9 methylation can be induced but also that this epigenetic mark was instructive in promoting downregulation of HER2/neu expression. Implications: Epigenetic editing provides a novel (synergistic) approach to modulate expression of oncogenes. Mol Cancer Res; 11(9); 1029–39. ©2013 AACR.