PURPOSE:A feasibility study was performed to investigate the presence of VEGF in melanoma lesions by VEGF-SPECT with (111)In-bevacizumab. In addition the effect of a single therapeutic bevacizumab dose on (111)In-bevacizumab uptake was compared with VEGF levels in resected melanoma lesions. PATIENTS AND METHODS:Eligible were patients with stage III/IV melanoma who presented with nodal recurrent disease. VEGF-SPECT was performed after administration of 100 Mbq (111)In-bevacizumab (8 mg) at days 0, 2, 4 and 7 post injection. Tumour visualisation and quantification were compared with CT and FDG-PET. On day 7 a single dose of 7.5mg/kg bevacizumab was administered intravenously. On day 21, a second tracer dose (111)In-bevacizumab was administered and scans were obtained on days 21, 25 and 28. Metastases were surgically resected within 2 weeks after the last VEGF-SPECT scan and immunohistological (IHC) VEGF tumour expression was compared with (111)In-bevacizumab tumour uptake. RESULTS:Nine patients were included. FDG-PET and CT detected both in total 12 nodal lesions which were all visualised by VEGF-SPECT. At baseline, (111)In-bevacizumab tumour uptake varied 3-fold between and 1.6 ± 0.1-fold within patients. After a therapeutic dose of bevacizumab there was a 21 ± 4% reduction in (111)In-bevacizumab uptake. The (111)In-bevacizumab tumour uptake in the second series positively correlated with the VEGF-A expression in the resected tumour lesions. CONCLUSION:VEGF-SPECT could visualise all known melanoma lesions. A single dose of bevacizumab slightly lowered (111)In-bevacizumab uptake. Future studies should elucidate the role of VEGF-SPECT in the selection of patients and the individual dosing of bevacizumab treatment.

Background: Isolated groups, such as first generation non-Western immigrants, are at risk for suboptimal utilisation of the health care system resulting in a worse outcome. Methods: From 1989 to 2007, all patients with stomach cancer were selected from the Comprehensive Cancer Centre North-East cancer registry. Associations between country of birth and patient, tumour and treatment characteristics were determined using χ 2 analysis. Relative survival analysis was used to estimate relative excess risk of dying according to country of birth (non-Western vs Western). Results: After adjusting for confounding factors (patient, tumour and treatment related), the risk of dying was lower for first generation non-Western immigrants (relative excess risk 0.55, 95% confidence interval 0.43–0.70) compared with Western patients. Conclusion: Although the better survival of first generation non-Western immigrants with stomach cancer remains unexplained, it argues against accessibility problems within the Dutch health care system.

Background: Surgical resection is an important factor in the curative treatment of gastric cancer. However a variety of aspects of surgical treatment that potentially influence outcome are still not well defined. This study aims to assess the influence of hospital type, referral pattern and proximal or distal location of the tumour on the ultimate survival.Methods: From January 1994 to January 2007, a total of 5245 patients were diagnosed with gastric adenocarcinoma in the region of the Comprehensive Cancer Centre North-East Netherlands. Hospitals in this region were categorized into three types: teaching university (TU), teaching non-university (TNU), and non-teaching hospitals (NT). The influence of hospital type, referral for surgery and location of the tumour on the relative survival of operated patients was studied.Results: Of the 5245 patients, 2334 patients underwent surgery. For operated patients, the 5-year relative survival was 42.5% for the TU versus 34.0% and 35.5% for respectively TNU and NT hospitals (p = 0.064), with no difference (p = 0.38) in relative survival (25.6-31.9%) in the proximal tumours. A significant difference was found between the hospitals in the 5-year relative survival in the distal tumours; 59.7% in the TU versus 36.4% in the TNU and 36% in the NT (p = 0.03 univariate), however this was not confirmed in the multivariate analysis (p = 0.184). High referral centres did not perform better as far as survival is concerned than low referral hospitals.In conclusion the hospital type in our region did not significantly influence outcome of surgery for gastric cancer. (C) 2010 Elsevier Ltd. All rights reserved.

Aim: Tumor marker based recurrences of previously treated testicular cancer are generally detected with CT scan. They sometimes cannot be visualized with conventional morphologic imaging. FDG-PET has the ability to detect these recurrences. PET probe-guided surgery, may facilitate the extent of surgery and optimize the surgical resection.Methods: Three patients with resectable 2nd or 3rd recurrent testicular cancer based on elevated tumor markers after previous various chemotherapy schedules and resections of residual retroperitoneal tumor masses were included in this study. A diagnostic FDG-PET was performed and a hotspot in previously operated area of the retroperitoneal space in all three patients was visualized. PET probe-guided surgery was performed using a high-energy gamma probe 3 h post-injection of 500 MBq FDG.Results: All patients showed extended adhesions and scar tissue in the retroperitoneal area due to the previous surgeries. Pre-operative PET/CT scan showed a good correlation with intra-operative PET probe-guided detection of recurrent lesions. There was a high target to background ratio (TGB) of 5:1 during the procedure. In one patient, a 2 cm large lesion, which did not show on pre-operative FDG-PET scan, was detected with the PET probe. Histopathologic tissue evaluation demonstrated recurrent vital tumor in all PET probe positive lesions.Conclusions: PET probe-guided surgery seems to be a promising tool to localize FDG-PET positive lesion in recurrent testicular cancer in hardly accessible surgical locations. PET probe-guided surgery might be a useful technique in surgical oncology for recurrent testicular cancer and has the potential to be applied in surgery of other malignant diseases. (C) 2010 Elsevier Ltd. All rights reserved.

e15564 Background: To optimize treatment results of patients with stomach cancer some institutions prompt referral to high volume hospitals with experienced surgeon's analogue to esophageal cancer. We analyzed if evidence can be found for superior treatment outcome depending on hospital type, referral pattern or hospital volume. Methods: Data from all patients diagnosed with primary invasive stomach cancer from 1994–2006 were obtained from the nationwide Netherlands Cancer Registry of the north-east part of the Netherlands. This area is served by one university hospital (U), 4 teaching hospitals (T), and 18 non-teaching hospitals (N-T). Results: In the study period, 5243 patients were diagnosed with stomach cancer (65.6% men, 34.4% female), 44.5% underwent surgery. Older age, advanced stage and proximal tumor location resulted in a lower probability of tumor resection. The 5 year relative survival (5yrsurv) was similar in the three hospital types. However, patients with proximal tumors were more often referred for surgery to U (18%), especially by N-T hospitals. In the non-proximal tumors the 5yrsurv was significantly better in U versus T and N-T hospitals, also after multivariate analysis. The significantly better 5yrsurv of low referral centers disappeared after excluding hospital U. No volume effect could be observed. Postoperative mortality registered in the period from 2004–2006 showed no differences between the hospitals. Conclusions: We found highly superior survival for non-proximal tumors but not for proximal cancer in an university hospital. In this study no evidence was found for a referral or volume effect. [Table: see text] No significant financial relationships to disclose.

There was evidence of impaired cellular immunity in 10 patients with myelofibrosis. In-vitro lymphocyte transformation with phytohaemagglutin, concanavalin A, and dinitrochlorobenzene skin reaction were diminished. Signs of impaired humoral activity were also found, the primary response to alpha-Helix pomatia haemocyanin being impaired, particularly in the immunoglobulin-A class. Moreover three patients had a benign paraproteinaemia. Immunecomplexes (IC) could be demonstrated with various test systems. The indirect granulocyte phagocytosis test was positive in 50%, the C1q-binding in 70% and the polyethylene glycol precipitation test in 50%. In most patients complement levels were normal, although the patient with the most advanced disease had low C3A, C3 and C4 levels accompanied by high levels of IC. No correlation could be shown between impaired immune response or levels of IC when they were related to spleen diameter or degree of anaemia. Some relation however existed between disturbed immune response and IC when they were related to time elapsed since diagnosis. It is suggested that the impaired immune response is the result of primary bone marrow disease and that the presence of IC may reflect the extent of fibrosis.

3547 Background: VEGF, released by tumor cells, is an important growth factor in tumor angiogenesis as it induces tumor neovascularization. VEGF121 is freely soluble whereas splice variants such as VEGF165, 189, 206 are mainly located in the extra cellular matrix, resulting in high concentrations in the tumor micro-environment. Non-invasive assessment of VEGF-levels in the micro-environment of the tumor could guide in the development and follow up of anti-VEGF targeted therapy. For this aim, we radiolabeled the anti-VEGF monoclonal antibody bevacizumab and the Fab-fragment ranibizumab, both binding to all VEGF-A isoforms, with SPECT (111In) and PET (89Zr) isotopes. Methods: Labeling, stability and in-vitro immunoreactivity studies were performed. Thereafter, nude mice were inoculated with VEGF producing human SKOV-3 ovarian tumor cells. After tumor size reached 6–8 mm in diameter, microPET and biodistribution studies were performed at multiple time points corresponding with the different elimination half-lives of radiolabeled bevacizumab and ranibizumab. A feasibility study in patients with recurrent melanoma and metastatic colon cancer has been started. Results: Uptake was significantly higher for both SPECT- and PET-tracers compared to radiolabeled control (Fab)-IgG, namely 7.74 ± 1.99 %ID/g and 4.66 ± 1.91 %ID/g for respectively 111In-bevacizumab at 168 h and 111In- ranibizumab at 24 h post injection. MicroPET imaging with 89Zr-bevacizumab and 89Zr-ranibizumab showed clear tumor localization respectively 72 and 3 h post injection. Furthermore, uptake of 89Zr-bevacizumab was predominantly seen in liver and spleen whereas 89Zr-ranibizumab was mainly cleared by kidney. Preliminary results of SPECT imaging using 111In- bevacizumab revealed tumor lesions in both recurrent melanoma and metastatic colon cancer patients. Conclusions: Radiolabeled bevacizumab and ranibizumab showed to be specific tracers for non-invasive (pre)clinical VEGF imaging, with distinctive pharmacokinetics. Bevacizumab shows the highest tumor uptake whereas ranibizumab allows imaging shorter after injection of the tracer. Both tracers could potentially guide anti-angiogenic VEGF targeted therapy. Supported by a grant from the Dutch Cancer Society. No significant financial relationships to disclose.

Neutropenia following high-dose chemotherapy leads to a high incidence of infectious complications, of which central venous catheter-related infections predominate. Catheter-related infections and associated risk factors in 392 patients participating in a randomized adjuvant breast cancer trial and assigned to receive high-dose chemotherapy and peripheral stem-cell reinfusion were evaluated. Median catheter dwell time was 25 days (range 1–141). Catheter-related infections were seen in 28.3% of patients (11 infections per 1000 catheter-days). Coagulase-negative staphylococci were found in 104 of 186 positive blood cultures (56%). No systemic fungal infections occurred. Cox regression analysis showed that duration of neutropenia >10 days ( P =0.04), using the catheter for both stem-cell apheresis and high-dose chemotherapy ( P =<0.01), and use of total parenteral nutrition (TPN, P =0.04) were predictive for catheter-related infections. In conclusion, a high incidence of catheter-related infections after high-dose chemotherapy was seen related to duration of neutropenia, use of the catheter for both stem-cell apheresis and high-dose chemotherapy, and use of TPN. Selective use and choice of catheters could lead to a substantial reduction of catheter-related infectious complications.

AIMS:To measure capillary permeability, assessed by skin capillary sodium fluorescein (NaF) leakage, in patients with diabetes mellitus with critical limb ischaemia (DM-CLI) and to compare the effects of vascular endothelial growth factor (VEGF) with those of placebo.METHODS:NaF leakage was assessed in 17 patients with DM-CLI, in 24 diabetes mellitus (DM) patients without clinical signs of macrovascular disease or neuropathy (DM-C) and in 22 healthy control subjects. The 17 DM-CLI patients were randomized to receive phVEGF165 gene product (n = 11) or placebo (n = 6). Measurements were repeated after 28 days.RESULTS:DM-CLI patients had a longer dye arrival time (DAT), but NaF leakage was similar to control subjects, while capillary permeability was increased in DM-C compared with control subjects. Leakage curve rose in patients receiving VEGF and fell in those receiving placebo, 28 days after administration. The decrease in DAT in the VEGF group was not significant, whilst DAT rose in the placebo group. Perfusion pressures were similar in the two groups.CONCLUSION:No increase in capillary leakage in DM-CLI was found, probably because an increased capillary filtration coefficient is counterbalanced by a marked fall in perfusion pressures. Increased capillary leakage may be one explanation for oedema formation after VEGF treatment.

To detect low-level DNA platination, a sensitive immunocytoand histochemical technique was developed using a polyclonal antibody. The antibody GPt, derived after immunization of rabbits with highly platinated DNA and purified with affinity chromatography, detected the main platinum (Pt)-containing intrastrand and interstrand adducts. Double-fluorescence microscopy image analysis was used to quantify Pt-DNA adducts with Hoechst 33258 fluorescence to locate the nuclei and with fluorescein isothiocyanate fluorescence to measure the immunosignal. A twoto five-fold dose-dependent difference in the level of cisplatin (CDDP)-induced Pt-DNA adducts between a CDDPsensitive and -resistant human tumour cell line was detected. Large differences in Pt-DNA adduct levels after in vitro CDDP incubation between human buccal cells, lymphocytes and biopsies of different tumour types were observed. Pt-DNA adduct levels were fivefold higher in human testicular tumours than in colon tumours, representing CDDP-sensitive and -resistant tumours, respectively, in the clinic. These data suggest the possibility of predictive testing by measuring Pt-DNA adduct levels. Pt-DNA adducts in patients after treatment with CDDP were shown in normal buccal cells and in imprints of fresh tumour biopsies as well as in paraffin-embedded tumour cells. The analysis of Pt-DNA adducts at a single-cell level in small samples of normal and tumour cells during and/or after treatment is feasible with GPt and will hopefully enable more selective treatment of patients.

Despite advances in revascularization techniques, limb salvage and relief of pain cannot be achieved in many diabetic patients with diffuse peripheral vascular disease. Our objective was to determine the effect of intramuscular administration of phVEGF(165) (vascular endothelial growth factor gene-carrying plasmid) on critical limb ischemia (CLI) compared with placebo (0.9% NaCl). A double-blind, placebo-controlled study was performed in 54 adult diabetic patients with CLI. The primary end point was the amputation rate at 100 days. Secondary end points were a 15% increase in pressure indices (ankle-to-brachial index and toe-to-brachial index), clinical improvement (skin, pain, and Quality of Life score), and safety. In patients (n = 27) treated with placebo versus phVEGF(165)-treated patients (n=27) the following results were found: 6 amputations versus 3 (p = not significant [NS]); hemodynamic improvement in 1 versus 7 (p = 0.05); improvement in skin ulcers, 0 versus 7 (p = 0.01); decrease in pain, 2 versus 5 (p = NS); and overall, 3 versus 14 responding patients (p = 0.003). No grade 3 or 4 adverse effects were seen in these patients. We conclude that this small, randomized gene therapy study failed to meet the primary objective of significant amputation reduction. However, significant and meaningful improvement was found in patients treated with a VEGF(165)-containing plasmid. There were no substantial adverse events.

Objective The herpes simplex virus thymidine kinase (HSVtk) gene has frequently been applied as a reporter gene for monitoring transgene expression in animal models. In clinical gene therapy protocols, however, extremely low expression levels of the transferred gene are generally observed. Consequently, sensitive and selective radiotracers for imaging are required. This study describes the in-vitro evaluation of 2'-[F-18]fluoro-5-ethyl-1-beta-D-arabi-nofuranosyluracil (F-18-FEAU) as a candidate tracer for HSVtk imaging with positron emission tomography (PET).Methods In cellular accumulation experiments, the potential of F-18-FEAU as a PET tracer for HSVtk was compared to the known acyclic guanosine derivatives 9-[(3-[F-18]fluoro-1-hydroxy-2-propoxy)methyl]guanine (F-18-FHPG) and 9-[4-[F-18]fluoro-3-(hydroxymethyl)butyl]-guanine (F-18-FHBG), and the thymidine derivatives 3'-deoxy-3'-[F-18]fluorothymidine (F-18-FLT), 2-deoxy-2'-[F-18]fluoro-5-methyl-1-beta-D-arabi-nofuranosyluracil (F-18-FMAU) and 2'-deoxy-2'-[F-18]fluoro-5-iodo-1-beta-D-arabi-nofuranosyluracil (F-18-FIAU). For this purpose, C6 control cells and HSVtk-expressing C6tk cells were incubated with the different tracers for various periods of time and cellular uptake and initial uptake rates were analysed. The initial rate of tracer uptake was determined from the slope of the plot of tracer uptake versus incubation time.Results After 2 h of tracer incubation, the C6tk/C6 accumulation ratio was 1.6 for F-18-FLT, 2.4 for F-18-FMAU, 5.5 for F-18-FHPG, 10.3 for F-18-FIAU, 40.8 for F-18-FHBG and 84.5 for F-18-FEAU. The initial tracer uptake rate in C6tk cells was in the order FLT > FMAU > FEAU > FIAU > FHBG > FHPG, whereas the initial tracer uptake rate in C6 control cells was FLT > FMAU > FIAU > FEAU congruent to FHBG congruent to FHPG. The highest HSVtk specific uptake was observed for FEAU.Conclusion This study indicates that the high uptake rate of FEAU together with its high selectivity make this tracer an excellent candidate as a PET tracer for HSVtk gene expression.

The p53 protein is mutated in 50% of all human tumors and plays a key role in apoptosis, cell cycle, and the expression of several genes. We investigated if p53 mutations influence the herpes simplex virus thymidine kinase (HSV-tk)/ganciclovir (GCV)-induced bystander effect (BE). Additionally, we studied some of the underlying mechanisms. GCV sensitivity and BE were studied in a human ovarian carcinoma cell line transfected with an empty vector (A2780/cmv) or a vector containing a p53 hotspot mutation at codon 175 (A2780/m175) or 248 (A2780/m248). In addition, expression levels of two nucleoside analogue transporters, multidrug resistance-related protein 4 and 5 (MRP4/MRP5), were determined. Finally, differences in gap junctional intercellular communication (GJIC) were studied by determining connexin 43 (Cx43) expression and by modulating GJIC with 18-alpha-glycyrrhetinic acid. Our results showed that compared to A2780/cmv, GCV sensitivity was significantly decreased in A2780/m175 and A2780/m248. Additionally, a significant BE (relative increase in cell kill) was found in A2780/cmv and A2780/m248. In contrast, an increased survival was observed in A2780/m175. No MRP4 or MRP5 expression was detected. However, all A2780 cell lines expressed Cx43. Modulating the GJIC significantly decreased the BE in A2780/cmv and A2780/ m248. In conclusion, HSV-tk/GCV-induced BE is influenced by p53 mutations. Differences in GJIC could be one of the underlying mechanisms.

Drug resistance is a major problem in the treatment of solid tumours. Based on a steep dose–response relationship for especially alkylating agents on tumour cell survival, high-dose chemotherapy was considered of interest for the treatment of solid tumours. Results of phase 1 and 2 studies with high-dose chemotherapy in a variety of tumour types showed good response rates. Nowadays, several phase 3 studies are available especially in metastatic and high-risk breast cancer patients. The high expectations of high-dose chemotherapy did not come true. This review analyses results of randomised studies and comments on the discrepancy between findings in patients versus those in tissue culture. Potential factors involved are the presence of tumour stem cells with different characteristics from more mature tumour cells, limitations in drug escalation in the clinic, transplant mortality, trial design and tumour cell contamination of the haematopoietic stem cell transplant. Maturation of the results from recent studies indicating a more modest benefit in, e.g., adjuvant breast cancer balanced versus long-term side effects will ultimately determine the role of high-dose chemotherapy in certain solid tumours. In case of well-defined indications for high-dose chemotherapy, further selection of patients based on patient and tumour characteristics as well as the introduction of new agents will most likely play a role.

Our study focused on the influence of herpes simplex virus thymidine kinase (HSV-tk) expression and ganciclovir (GCV) treatment on the sensitivity of C6 glioma cells to frequently used chemotherapeutic drugs, i.e. adriamycin (ADR), cisplatin (CDDP), 5-fluorouracil (5-FU), and methotrexate (MTX). Transfection with HSV-tk revealed an increased sensitivity to GCV and CDDP and a decreased sensitivity to ADR and MTX. No significant differences were found in sensitivity to 5-FU. Combined treatment in a HSV-tk negative cell line revealed an additive effect when GCV was combined with ADR, whereas an antagonistic effect was found when GCV was combined with CDDP, 5-FU, or MTX. Comparable results were obtained in an HSV-tk positive cell line, apart from CDDP, which showed an additive effect. In conclusion, both HSV-tk transfection and subsequent GCV treatment can influence the sensitivity of tumor cells to various chemotherapeutic drugs in an antagonistic manner. Therefore, combining HSV-tk/GCV gene therapy with chemotherapy might not always be beneficial.