The lymphatic vasculature transports extravasated tissue fluid, macromolecules and cells back into the blood circulation. Recent reports have focused on the molecular mechanisms regulating the lymphatic vessels. Vascular endothelial growth factor (VEGF)-C and VEGF-D have been shown to stimulate lymphangiogenesis and their receptor, VEGFR-3, has been linked to human hereditary lymphedema. Here we show that a soluble form of VEGFR-3 is a potent inhibitor of VEGF-C/VEGF-D signaling, and when expressed in the skin of transgenic mice, it inhibits fetal lymphangiogenesis and induces a regression of already formed lymphatic vessels, though the blood vasculature remains normal. Transgenic mice develop a lymphedema-like phenotype characterized by swelling of feet, edema and dermal fibrosis. They survive the neonatal period in spite of a virtually complete lack of lymphatic vessels in several tissues, and later show regeneration of the lymphatic vasculature, indicating that induction of lymphatic regeneration may also be possible in humans.

Glioblastoma is an aggressive brain tumor with a dismal prognosis. Gene therapy may offer a new option for the treatment of these patients. Several gene therapy approaches have shown anti-tumor efficiency in experimental studies, and the first clinical trials for the treatment of malignant glioma were conducted in the 1990s. HSV-tk gene therapy has been the pioneering and most commonly used approach, but oncolytic conditionally replicating adenoviruses and herpes simplex virus mutant vectors, p53, interleukins, interferons, and antisense oligonucleotides have also been used. During the past few years, adenoviruses have become the most popular gene transfer vectors, and some recent randomized, controlled trials have shown significant anti-tumor efficacy in clinical use. However, efficient gene delivery into the brain still presents a major problem, and there is a lack of definitive phase III trials, which would avoid potential problems associated with a small number of patients, inadvertent patient selection, and overinterpretation of results based on a few long-time survivors. For clinical efficacy, median survival is one of the most rigorous endpoints. It is used here to evaluate the usefulness of various treatment approaches and current clinical status of gene therapy for malignant glioma.

We have treated Caki -2 human renal cell carcinoma in vivo using herpes simplex virus thymidine kinase ( HSV -tk ) gene therapy. Both stably transduced Caki -2 tumors, generated using retrovirus -mediated ex vivo HSV -tk gene transfer and direct intratumoral adenovirus -mediated HSV -tk gene transfer of wild type tumors, were tested. Similar treatments with LacZ containing retro -and adenoviruses were used as controls. The outcome was evaluated by imaging the tumors before and after the treatment with magnetic resonance imaging, and using histology, immunocytochemistry, and survival analysis. When implanted orthotopically into nude mouse kidneys, Caki -2 cells formed reproducible cystic papillary kidney carcinomas. In vivo magnetic resonance imaging provided an important tool for the evaluation of tumor growth. Transduction efficiency of wild -type tumors in vivo with adeno -LacZ was 22 14%. Significant tumor regression was achieved with direct intratumoral adeno -HSV -tk transduction followed by intraperitoneal ganciclovir ( GCV ) ( P < .001 ). Also, the treatment of stably transduced Caki -2 tumors with intraperitoneal GCV resulted in a significant treatment response in the HSV -tk group as compared to the LacZ group ( P < .009 ). Increased apoptosis and macrophage infiltrations, reduced proliferation, and degenerative changes were observed in the tumors treated with HSV -tk and GCV. Also, significant prolongation in survival was achieved with adeno -HSV -tk ± and GCV -treated mice as compared to the controls. It is concluded that adeno -HSV -tk gene therapy may be useful for the treatment of renal cell carcinoma in vivo. Cancer Gene Therapy ( 2001 ) 8, 529 ± 536

Background and methods: Gene therapy may offer a new tool for the treatment of renal cell carcinoma ( RCC ). We have tested a combination of cytotoxic and antiangiogenic gene therapy for wild -type orthotopic human RCC xenografts in nude mice using intratumoral adenovirus -mediated herpes simplex virus thymidine kinase ( HSV -tk ) and endostatin ( ES ) gene therapy. In vivo magnetic resonance imaging, morphometry, immunocytochemistry, and survival were used to evaluate the treatment effect. Adenovirus -mediated marker gene transfers ( GFP ) were used as controls. Results: In vivo transduction efficiency, measured using GFP gene transfer, was 27 ± 7%. The combination gene therapy with HSV -tk and ES adenoviruses resulted in a significant antitumor effect ( P < .01 ) compared to single HSV -tk ( n.s. ) or ES ( n.s. ). In the survival study, all tumors with single gene therapy using HSV -tk, ES, and marker gene adenoviruses showed progression in magnetic resonance imaging. In contrast, the majority of the tumors in the combination treatment group remained dormant or were eradicated ( 57% ). Survival of these mice equaled healthy nude mice, and was significantly prolonged ( P < .0001 ) compared to HSV -tk ( P < .028 ) and ES ( n.s. ) groups. Conclusions: It is concluded that the inhibition of angiogenesis using ES gene transfer together with the cytotoxic HSV -tk gene therapy results in a significantly improved treatment effect in RCC compared to the single gene treatments.

The very high binding affinity of avidin to biotin is one of the highest to occur in nature. We constructed a fusion protein composed of avidin and the endocytotic LDL receptor in order to target biotinylated molecules to cells of the desired tissues. In addition to the native avidin, charge-mutated and nonglycosylated avidins were utilized as part of the fusion proteins, in order to modify its properties. All of the fusion protein versions retained the biotin-binding capacity. Although the specificity was not increased, however, fusion proteins composed of natural avidin and nonglycosylated avidin bound most efficiently to the biotinylated ligands. Fluorescence microscopy and atomic force microscopy studies revealed the expression of the fusion protein on cell membranes, and demonstrated specific and high-affinity binding of biotin to the low-density lipoprotein receptor (LDLR)-avidin fusion protein in vitro. Additionally, systemically administered biotinylated ligand targeted with high specificity the intracerebral tumors of rats that were expressing fusion protein after the virus-mediated gene transfer. These results suggest that local gene transfer of the fusion protein to target tissues may offer a novel tool for the delivery of biotinylated molecules in vitro and in vivo for therapeutic and imaging purposes.

BackgroundSix radium-223 injections at 4-week intervals is indicated for patients with castration-resistant prostate cancer and symptomatic bone metastases. However, patients usually develop disease progression after initial treatment. This prospective phase I/II study assessed re-treatment safety and efficacy of up to six additional radium-223 injections.Patients and methodsPatients had castration-resistant prostate cancer and bone metastases and six initial radium-223 injections with no on-treatment bone progression; all had subsequent radiologic or clinical progression. Concomitant agents were allowed at investigator discretion, excluding chemotherapy and initiation of new abiraterone or enzalutamide. The primary endpoint was safety; additional exploratory endpoints included time to radiographic bone progression, time to total alkaline phosphatase and prostate-specific antigen progression, radiographic progression-free survival, overall survival, time to first symptomatic skeletal event (SSE), SSE-free survival, and time to pain progression.ResultsAmong 44 patients, 29 (66%) received all six re-treatment injections. Median time from end of initial radium-223 treatment was 6 months. Forty-one (93%) reported ≥1 treatment-emergent adverse event. No grade 4–5 hematologic treatment-emergent adverse events occurred. Only one (2%) patient had radiographic bone progression; eight (18%) had radiographic soft tissue tumor progression (three lymph node and five visceral metastases). Median times to total alkaline phosphatase and prostate-specific antigen progression were not reached and 2.2 months, respectively. Median radiographic progression-free survival was 9.9 months (12.8-month maximum follow-up). Five (11%) patients died and eight (18%) experienced first SSEs. Median overall survival, time to first SSE, and SSE-free survival were not reached. Five (14%) of 36 evaluable patients (baseline worst pain score ≤7) had pain progression. After 2 years of follow-up, 28 (64%) patients died, and the median overall survival was 24.4 months.ConclusionsRe-treatment with a second course of six radium-223 injections after disease progression is well tolerated, with minimal hematologic toxicity and low radiographic bone progression rates in this small study with limited follow-up. Favorable safety and early effects on disease progression indicate that radium-223 re-treatment is feasible and warrants further evaluation in larger prospective trials.

In a few cases, gemcitabine (GCB) has been shown to result in systemic capillary leak syndrome (SCLS) in the treatment of non-small cell lung, pancreatic, and ovarian carcinomas. SCLS is a life-threatening condition characterized by increased capillary permeability resulting in manifest pulmonary and peripheral edema. Usually SCLS responds successfully to corticosteroid therapy and diuretics. We present a case where GCB treatment likely resulted in SCLS in a male patient with metastatic renal cell carcinoma (RCC) in the absence of predisposing cardiac, pleural, or pulmonary disease.

197 Background: Ra-223 tx for up to 6 injections (inj) is indicated for pts with symptomatic bone metastases. Ra-223 tx beyond 6 inj has not been previously reported. Herein we report the first safety and efficacy findings of Ra-223 re-tx from an international prospective trial in mCRPC pts. Methods: Pts with CRPC with ≥ 2 bone mets who completed 6 initial Ra-223 inj with no disease progression in bone and progressed after initial tx were potentially eligible for Ra-223 re-tx, provided that hematologic (heme) parameters were adequate. No concomitant cytotoxic agents were allowed; other agents were allowed at investigator discretion. Primary objective was safety. Exploratory objectives were time to radiographic bone progression, time to ALP progression, and radiographic progression-free survival (rPFS) based on MRI/CT and bone scans performed q 3 mo. Results: 44 pts had Ra-223 re-tx, 29 (66%) completed tx with all 6 inj; median (med) number inj = 6. Med time from initial Ra-223 tx = 6 mo. Besides prior Ra-223, all pts had ≥ 2 hormonal regimens; 45% had ≥ 1 chemotherapy regimen. 32 (73%) failed novel hormonal agents, eg, abiraterone and enzalutamide. Baseline characteristics were comparable to ALSYMPCA (Table). No new safety concerns were noted; incidence of tx-emergent adverse events (TEAEs) in re-tx pts was comparable to or lower than ALSYMPCA (Table). Only 2 re-tx pts had grade 3 heme TEAEs. Only 1 pt had radiographic bone progression; med time to ALP progression was not reached. Med rPFS = 9.9 mo. Conclusions: Ra-223 re-tx was well tolerated in this highly selected population, with minimal heme toxicity, and provided continued control of disease progression in bone. Clinical trial information: NCT01934790. [Table: see text] [Table: see text]