Purpose For patients with epithelial ovarian cancer (EOC) cytoreduction, with a combination of taxane and platinum, is the standard of care. Despite this, approximately 50% of patients with advanced disease will relapse and moreover 15-20% of cases of EOC are resistant to platinum based chemotherapy. Vascular Endothelial Growth Factor (VEGF), an angiogenic factor, is associated with poor prognosis. This study was undertaken to examine whether there is an association between VEGF-A expression in the tumour of EOC patients and their response to platinum based chemotherapy.Methods The study cohort consisted of 66 patients with advanced stage EOC (FIGO III-IV). Ovarian cancer tissue was analysed for VEGF-A expression immunohistochemically. Protein expression was measured and correlated, with platinum sensitivity and overall patient survival.Results Median age of patients was 53 years, 45 patients had platinum sensitive disease (68%), the remaining patients being platinum resistant (32%). Of the platinum resistant group, 18 (86%) patients had high VEGF score compared to only 1 (2%) with high VEGF score in the platinum sensitive group. Median survival was 11 months in the patient group with high VEGF score versus 32 months in that cohort with low VEGF score. VEGF expression was significantly inversely correlated with overall survival (P < 0.0001).Conclusion We demonstrated that tumours of patients with platinum resistant EOC exhibit higher levels of VEGF expression compared to the platinum sensitive group. VEGF in EOC, may be of clinical and therapeutic relevance and suggests a role for first line anti-angiogenic therapy.

LB-200 Abstract LB-200 was submitted as a Late-Breaking Abstract and is embargoed until the time of presentation. The complete abstract will be posted to this site at or after the following date and time: 4/14/2008 2:30 PM.

PURPOSE:Antibody-directed enzyme prodrug therapy is a two-stage treatment whereby a tumor-targeted antibody-enzyme complex localizes in tumor for selective conversion of prodrug. The purpose of this study was to establish optimal variables for single administration of MFECP1, a recombinant antibody-enzyme fusion protein of an anti-carcinoembryonic antigen single-chain Fv antibody and the bacterial enzyme carboxypeptidase G2 followed by a bis-iodo phenol mustard prodrug. MFECP1 is manufactured in mannosylated form to facilitate normal tissue elimination.EXPERIMENTAL DESIGN:Pharmacokinetic, biodistribution, and tumor localization studies were used to test the hypothesis that MFECP1 localizes in tumor and clears from normal tissue via the liver. Firstly, safety of MFECP1 and a blood concentration of MFECP1 that would avoid systemic prodrug activation were tested. Secondly, dose escalation of prodrug was done. Thirdly, the dose of MFECP1 and timing of prodrug administration were optimized.RESULTS:MFECP1 was safe and well tolerated, cleared rapidly via the liver, and was less immunogenic than previously used products. Eighty-fold dose escalation from the starting dose of prodrug was carried out before dose-limiting toxicity occurred. Confirmation of the presence of enzyme in tumor and DNA interstrand cross-links indicating prodrug activation were obtained for the optimal dose and time point. A total of 28 of 31 patients was evaluable for response, the best response being a 10% reduction of tumor diameter, and 11 of 28 patients had stable disease.CONCLUSIONS:Optimal conditions for effective therapy were established. A study testing repeat treatment is currently being undertaken.

Purpose: Antibody-directed enzyme prodrug therapy (ADEPT) requires highly selective antibody-mediated delivery of enzyme to tumor. MFE-CP, a multifunctional genetic fusion protein of antibody and enzyme, was designed to achieve this by two mechanisms. First by using a high affinity and high specificity single chain Fv antibody directed to carcinoembryonic antigen. Second by rapid removal of antibody-enzyme from normal tissues by virtue of post-translational mannosylation. The purpose of this paper is to investigate these dual functions in an animal model of pharmacokinetics, pharmacodynamics, toxicity, and efficacy.Experimental Design: MFE-CP was expressed in the yeast Pichia pastoris and purified via an engineered hexahistidine tag. Biodistribution and therapeutic effect of a single ADEPT cycle (1,000 units/kg MFE-CP followed by 70 mg/kg ZD2767P prodrug at 6, 7, and 8 hours) and multiple ADEPT cycles (9-10 cycles within 21-24 days) was studied in established human colon carcinoma xenografts, LS174T, and SW1222.Results: Selective localization of functional enzyme in tumors and rapid clearance from plasma was observed within 6 hours, resulting in tumor to plasma ratios of 1,400:1 and 339:1, respectively for the LS174T and SW1222 models. A single ADEPT cycle produced reproducible tumor growth delay in both models. Multiple ADEPT cycles significantly enhanced the therapeutic effect of a single cycle in the LS174T xenografts (P = 0.001) and produced regressions in the SW1222 xenografts (P = 0.0001), with minimal toxicity.Conclusions: MFE-CP fusion protein, in combination with ZD2767P, provides a new and successful ADEPT system, which offers the potential for multiple cycles and antitumor efficacy. These results provide a basis for the next stage in clinical development of ADEPT.

This manuscript reports a carefully controlled study of patients with Dukes B colorectal cancer (Dukes stage A, n=12 and Dukes stage B, n=44). Immunohistochemistry has been used to demonstrate reactivity for vascular endothelial growth factor (VEGF), and to measure levels of microvessel density (MVD) in order to assess the relationship of tumor angiogenesis with clinical outcome. Immunohistochemistry was performed using antibodies to VEGF and CD34 (for intratumoral vessel identification) and counting was performed at the invasive margin of the tumor. Results showed that for Dukes stage A patients 4/12 died of their disease, none of whose tumor was VEGF positive. In contrast, 2 patients who survived were positive for VEGF cytoplasmically, but neither showed increased tumor MVD. In Dukes B patients 10/44 died, 5 of whose tumor demonstrated VEGF reactivity, both in malignant cells and in tumor vascular endothelium. MVD ranged from 11 to 53 (median 28) for Dukes A cases and from 9 to 69 (median 32.5) for the Dukes B group. Kaplan-Meier plots and log rank test statistics for Dukes B patients demonstrated that VEGF reactivity in cells, and in tumor vascular endothelium was correlated with survival (p=0.047 and p < or = 0.06, respectively). There was a significant relationship between the presence of VEGF reactivity on vascular endothelium and outcome by Fisher's exact test (p=0.018). Similarly, by the same test VEGF positivity was significantly correlated with patient mortality (p=0.032). The presence of endothelial VEGF reactivity correlated with VEGF in malignant cells (p=0.0001) by Mann-Whitney U test and a significant inverse relationship between vessel density and patient survival was demonstrated (p = 0.019). The finding that in Dukes B patients MVD was inversely correlated with mortality supports the hypothesis that a low microvascular count is predicted close to the invasive margin, where VEGF expression is upregulated in response to hypoxia, induced by a lack of a functional vasculature. These data will be used to identify cohorts of patients who have a high risk of relapse and can be selected for adjuvant therapies such as VEGF antibody or antitumor antibody-directed therapy.

Combretastatin A-4 phosphate (CA4-P) is an antivascular agent which inhibits tumour blood flow. The effects of CA4-P were studied at 1 and 24h in colorectal xenografts by the concomitant imaging of multiple physiological parameters (hypoxia, blood vessels and perfusion), selected to demonstrate changes related to vascular shut-down. Untreated tumours were viable, with perfused blood vessels throughout and only small areas of hypoxia. At 1h post-treatment, although blood vessels remained throughout the tumour, perfused vessels were mainly restricted to the rim. However, hypoxia was widespread in both peripheral and central parts of the tumour. Quantitative analysis also revealed a significant decrease in perfusion and a maximum increase in hypoxia at this time-point. Conversely, at 24h after treatment, when most of the tumour was necrotic, pathophysiological conditions in the surviving viable rim were already returning to normal: perfusion was increasing, and hypoxia was greatly reduced and restricted to regions bordering central necrosis. In conclusion, these data provide an insight into the actions by which CA4-P may exert its effects on solid tumours.

UNLABELLED:It has been shown in vitro that the cell uptake of (18)F-FDG, a tracer of glucose metabolism, increases under hypoxia. This is consistent with increased glycolytic metabolism. We have previously shown that in ischemic heart ex vivo the rates of uptake of (18)F-FDG and 2-(14)C-deoxy-D-glucose ((14)C-2DG) are both reduced. In this study, we investigated this effect in tumors by comparing the microdistribution of (18)F-FDG and (14)C-2DG in hypoxic and normoxic regions.METHODS:Mice (MF1) bearing LS174T human tumor xenografts were injected with premixed (18)F-FDG (100 MBq), (14)C-2DG (0.37 MBq), and pimonidazole hydrochloride (60 mg/kg). After 30, 60, and 120 min, tissues (n = 4) were taken and counted for whole-body biodistribution. Tumors were frozen, sectioned, and exposed to phosphor image plates to obtain a quantitative digital image of radionuclide distribution. Sections were then stained to reveal tumor pathophysiology: Hematoxylin and eosin staining demonstrated viable and necrotic regions, and immunohistochemical staining detected pimonidazole metabolism in hypoxic cells. The images of radionuclide microdistribution and histology were then coregistered and analyzed to assess radionuclide trapping throughout the tumor on a pixel-by-pixel basis. The Pearson correlation coefficients between the 2 radionuclides were calculated. The relative amounts of nuclide were then analyzed in viable and necrotic regions and in normoxic and hypoxic regions.RESULTS:Whole-body biodistributions for the 2 radiotracers were similar. A high Pearson correlation coefficient was obtained for the 2 radionuclides throughout the tumors (r = 0.85 +/- 0.10, P < 0.0001), indicating a highly similar microdistribution. When the tumors were divided into viable and necrotic regions, the ratio of mean counts per pixel was 1.96 (P < 0.0001), whereas for hypoxic versus normoxic regions it was 1.26 (P < 0.0001). There was no significant difference in selectivity for hypoxia between the 2 radiotracers (P = 0.86).CONCLUSION:The tumor microdistribution of deoxyglucose in viable, hypoxic, and necrotic regions show that there was little change in the microdistribution of deoxyglucose throughout this time course. This study extends previous in vitro work and confirms the selectivity of deoxyglucose for viable cells over necrotic regions and for hypoxic cells over normoxic regions in vivo.

The therapeutic efficacy of radiolabeled antibody fragments can be limited by nephrotoxicity, particularly when the kidney is the major route of extraction from the circulation. Conventional dose estimates in kidney assume uniform dose deposition, but we have shown increased antibody localization in the cortex after glomerular filtration. The purpose of this study was to measure the radioactivity in cortex relative to medulla for a range of antibodies and to assess the validity of the assumption of uniformity of dose deposition in the whole kidney and in the cortex for these antibodies with a range of radionuclides. Storage phosphor plate technology (radioluminography) was used to acquire images of the distributions of a range of antibodies of various sizes, labeled with 125I, in kidney sections. This allowed the calculation of the antibody concentration in the cortex relative to the medulla. Beta-particle point dose kernels were then used to generate the dose-rate distributions from 14C, 131I, 186Re, 32P and 90Y. The correlation between the actual dose-rate distribution and the corresponding distribution calculated assuming uniform antibody distribution throughout the kidney was used to test the validity of estimating dose by assuming uniformity in the kidney and in the cortex. There was a strong inverse relationship between the ratio of the radioactivity in the cortex relative to that in the medulla and the antibody size. The nonuniformity of dose deposition was greatest with the smallest antibody fragments but became more uniform as the range of the emissions from the radionuclide increased. Furthermore, there was a strong correlation between the actual dose-rate distribution and the distribution when assuming a uniform source in the kidney for intact antibodies along with medium- to long-range radionuclides, but there was no correlation for small antibody fragments with any radioisotope or for short-range radionuclides with any antibody. However, when the cortex was separated from the whole kidney, the correlation between the actual dose-rate distribution and the assumed dose-rate distribution, if the source was uniform, increased significantly. During radioimmunotherapy, the extent of nonuniformity of dose deposition in the kidney depends on the properties of the antibody and radionuclide. For dosimetry estimates, the cortex should be taken as a separate source region when the radiopharmaceutical is small enough to be filtered by the glomerulus.