Tumor structure and extracellular matrix as a possible barrier for therapeutic approaches using immune cells or adenoviruses in colorectal cancer.

In this article we report about the role that tumor structure and extracellular matrix (ECM) may play in immunotherapy and in gene therapy using adenoviruses. We performed studies in a rat model for colorectal cancer, CC531, and in specimens of human colorectal cancer. The tumors were composed of two compartments, tumor cell nests surrounded by stromal cells. ECM proteins were expressed in the stromal part, where the blood vessels were also located. Furthermore, in several tumors, the tumor cell nests were surrounded by basal membrane-like structures. Therefore, in vascular approaches to treat cancer, therapeutic agents on their route to tumor cells may be hampered by ECM to reach tumor cells. We found that immune cells were abundantly present in tumors from colorectal origin. These cells were, however, not found in direct contact with tumor cells, but mainly in the stromal part of the tumor. Adenoviruses, when intravascularly injected, did not reach tumor cells in the CC531 rat model. Tumor cells were only infected, and even then in limited numbers, in cases of intratumoral injection. We hypothesize that ECM in a tumor is a barrier both for immune cells and for adenoviruses to make direct contact with these tumor cells, and thus limits colorectal tumor therapy.

Recombinant adenoviral vectors have adjuvant activity and stimulate T cell responses against tumor cells.

The host-immune response against adenoviruses forms a major obstacle for their use as gene therapy vectors for treatment of genetic defects. None the less, they are the preferred vectors for in vivo gene transfer in experimental gene therapy protocols for cancer. In this article we demonstrate the antitumor efficacy of adenovirus-mediated transfer of human interleukin-2 cDNA in the rat-CC531 model for hepatic metastases of colorectal cancer: intratumoral administration of 10 plaque-forming units of the hlL-2-expressing adenoviral vector, AdCAIL-2, resulted in a cessation of tumor growth in 80% of the injected tumors. In control groups receiving AdCnull, a vector with the same viral backbone, but lacking transgene expression, none of the tumors responded. However, intratumoral treatment with this vector significantly enhanced tumor regression induced by systemic IL-2 protein treatment, which was used as a positive control. In addition we show, by performing delayed-type of hypersensitivity assays, that AdCnull when injected intratumorally enhances recognition of tumor antigens by T lymphocytes to the same extent as intratumoral treatment with the IL-2-expressing vector. The replication-deficient adenoviruses appear to have a therapeutic advantage in cytokine-mediated immunotherapy: even adenovirus vectors that do not express a transgene, show adjuvant activity and stimulate an antitumor immune response.

Specific tumor-cell killing with adenovirus vectors containing the apoptin gene.

Specificity is an essential prerequisite for cancer gene therapy. Recently we described that apoptin, a protein of 121 amino acids which is derived from the chicken anemia virus, induces programmed cell death or apoptosis in transformed and malignant cells, but not in normal, diploid cells (Danen-van Oorschot AAAM et al, Proc Natl Acad Sci USA 1997; 94: 5843-5847). This protein has an intrinsic specificity that allows it to selectively kill tumor cells, irrespective of the p53 or Bcl-2 status of these cells. Hence, it is attractive to explore the use of the apoptin gene for therapeutic applications, viz cancer gene therapy. In this paper, we describe the generation and characterization of an adenovirus vector, AdMLPvp3, for the expression of apoptin. Despite the fact that apoptin ultimately induces apoptosis in the helper cells, which are transformed by the adenovirus type 5 early region 1 (E1), the propagation kinetics and yields of AdMLPvp3 are similar to those of control vectors. Infection with AdMLPvp3 of normal rat hepatocytes in cell culture did not increase the frequency of apoptosis. In contrast, in the hepatoma cell lines HepG2 and Hep3b, infection with AdMLPvp3, but not with control vectors, led to a rapid induction of programmed cell death. Experiments in rats demonstrated that AdMLPvp3 could be safely administered by intraperitoneal, subcutaneous or intravenous injection. Repeated intravenous doses of AdMLPvp3 were also well tolerated, indicating that the apoptin-expressing virus can be administered without severe adverse effects. In a preliminary experiment, a single intratumoral injection of AdMLPvp3 into a xenogeneic tumor (HepG2 cells in Balb/Cnu/nu mice) resulted in a significant reduction of tumor growth. Taken together, our data demonstrate that adenovirus vectors for the expression of the apoptin gene may constitute a powerful tool for the treatment of solid tumors.

Towards gene therapy for colorectal liver metastases.

Hepatic gene therapy may provide a new approach to treating hepatic malignancies. A promising strategy involves the infection of tumor and liver cells with replication-defective adenoviral vectors carrying suicide genes. The products of these genes convert prodrugs into cytotoxic derivates. In the transduced cells the subsequently administered prodrugs are thus activated and destroy replicating tumor cells, whereas the infected liver cells are thought to be unaffected because of their minimal proliferative activity. A major concern about this approach is that the suicide genes can theoretically enter the germline and other organs with high mitotic activity and as a consequence, cause their destruction. In vivo gene delivery should therefore be tissue-specific, and methods for targeted gene delivery are required. Targeting the colorectal liver metastases is pursued by combining the suicide gene principle and the surgical technique of isolated perfusion of the liver.

[Gene therapy for hemophilia: feasible in principle but not yet clinically applicable]. / Gentherapie voor hemofilie: in principe uitvoerbaar, maar nog niet klinisch toepasbaar.

The current treatment of haemophiliacs consists of injection of concentrates of blood clotting factors VIII (haemophilia A) and IX (haemophilia B). The inconvenience of the multiple injections needed, and the risk of transmission of infectious agents (HIV, hepatitis) prompted the development of alternative therapies. Gene therapy aims at introducing functional factor VIII and IX genes into the body cells of patients in order to make these cells produce the desired clotting factors. There are two strategies for gene therapy: (a) in the laboratory cells of the patient may be provided with the desired gene, followed by reintroduction of the cells that now produce factor VIII, into the patient (ex vivo strategy); (b) vectors with the desired genes may be injected into the patient in order to induce the modification (in vivo strategy) For both routes, the formal proof-of-principle has been acquired recently in animal experiments: cells modified by factor VIII or IX genes will produce adequate concentrations of the clotting products in plasma and will correct the bleeding tendency. Before the clinical evaluation and widespread application of the technology can be considered many technical problems have to be solved.

Severe hepatic dysfunction after adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene and ganciclovir administration.

The use of so-called 'suicide' genes to activate prodrugs has been effective in animal models for several solid tumor types and is now in phase I and II clinical trials. We have exploited adenovirus vectors (Ad) for transfer and expression of the herpes simplex virus thymidine kinase (HSVtk) gene to render rat colorectal liver metastases sensitive to the anti-herpetic agent ganciclovir (GCV). The efficacy and toxicity of this enzyme-prodrug combination were tested after in situ transduction of rat colorectal tumor cells and after intraportal administration of the vector Ad.CMV.TK. Our results demonstrate the validity of the approach but reveal that hepatic expression of HSVtk, both in tumor bearing and in tumor-free rats, provokes severe liver dysfunction and mortality upon GCV administration. These data show, that in contrast to the common assumption, normally non-mitotic tissues too, can be affected by adenovirus-mediated HSVtk transfer and subsequent GCV treatment. Given the hepatotropic nature of systemically administered adenovirus type 2- and 5-derived vectors, it will be essential to monitor liver functions of patients included in all gene therapy trials involving adenoviral vectors with the HSVtk gene.

Delivery of anticancer drugs via isolated hepatic perfusion: a promising strategy in the treatment of irresectable liver metastases?

The prognosis of patients with irresectable liver metastases derived from colorectal cancer is invariably poor; unfortunately, these tumours show only minor responses to conventional anticancer agents. The best responses have been obtained by fluoropyrimidines delivered as continuous infusion into the hepatic artery (HAI): their rapid uptake and detoxification by liver cells results in relatively low systemic drugs levels. This approach increases mean survival duration from 17 to 26 months and, in few patients, causes "down-staging" that may result in resectability. To improve opportunities for chemotherapy, the technique of 1-hour recirculating perfusion of the vascularly isolated liver (isolated hepatic perfusion, IHP) was developed. If leakage to the systemic circulation is negligible-and the compounds used do not readily cause hepatotoxicity-IHP allows usage of drug doses that would be fatal if delivered systemically. Because alkylating agents generally have steep dose-response curves, mitomycin C (MMC) and melphalan (L-PAM) entered phase I/II studies on IHP. Using these drugs, IHP was performed in principle as a single procedure in 60 otherwise untreated patients at our institution. However, despite preliminary data that indicate impressive clinical responses are obtained, improvement over HAI will probably be minor. Because IHP is a complicated way of drug delivery, one could argue that its use is justified only when it has the potential to kill all tumour cells in the liver. We critically discuss the possibilities of IHP and/or the use of gene therapy in an IHP setting.

Isolated-organ perfusion for local gene delivery: efficient adenovirus-mediated gene transfer into the liver.

Targeted gene delivery is essential for gene therapy involving in vivo gene transfer. In the present study we analyzed the efficiency and tissue-specificity of gene transfer into the liver with recombinant adenoviruses. Adenovirus vectors carrying the E. coli lacZ gene (Ad.RSV.beta-gal) and the firefly luciferase gene (AdCMV-luc) as reporters were administered to the liver of adult Wistar rats, either via infusion into the portal vein (intraportal infusion; IPI) or via perfusion of the vascularity isolated liver (isolated liver perfusion; ILP). Ex vivo liver perfusion experiments with Ad.RSV. beta-gal were used to optimize the conditions for hepatic gene transfer. Ex vivo perfusion of rat livers with 2 x 10(9) plaque forming units (p.f.u) Ad RSV.beta-gal was sufficient to infect about 20% of the liver parenchymal cells. Perfusion with chelating agents (1 mM EGTA, or 2 mM EDTA) prior to the administration of the vector increased the efficiency to at least 40%. Similar efficiencies were obtained in experiments with liver lobes of Rhesus monkeys. In vivo administration of AdCMV-luc via ILP resulted in a significantly more efficient (P = 0.028) and also more reproducible gene transfer when compared to IPI. Although detectable in both groups, extrahepatic luciferase expression was considerably reduced in the ILP group. Our data demonstrate that IPL can be used for efficient and reproducible liver-specific gene delivery. Therefore, we think that the perfusion of vascularly isolated organs is useful as a modality for the tissue-specific administration of recombinant adenovirus vectors.

Factors impeding efficient expression of factor VIII complementary DNA minigenes.

Haemophilia A is a bleeding disorder that affects approximately 1 in 10,000 males. It is caused by a deficiency of functional blood-clotting factor VIII. Protein-replacement therapy has been effective as treatment, resulting in a vast improvement in the quality of life and dramatically increasing the life expectancy of patients. However, therapy with plasma-derived factor VIII has allowed the transmission of several human viruses, such as hepatitis viruses, human immunodeficiency virus and parvovirus B19. To date, the safety of the therapeutic agent is one of the key issues in haemophilia A treatment. The use of recombinant factor VIII in haemophilia therapy can avoid the dependence on blood-derived products and prevent the occurrence of transfusion-associated infections with blood-borne pathogens. Gene therapy could go further, and offers the prospect of one-time treatment which may, optimally, achieve a total cure of the disease. Therefore, haemophilia is an appealing and challenging target for somatic-cell gene therapy. On the basis of the phenotypic correction that is achieved upon infusion of factor VIII protein, it is expected that an increase in the factor VIII plasma level to 10% of the level found in healthy individuals would suffice to prevent the manifestation of the bleeding tendency. In this paper, we review the progress and the problems of gene therapy for haemophilia, with special focus on the problems specifically associated with the transfer and expression of human factor VIII complementary DNA.

Reverse smoking as a risk factor for palatal cancer: a cross-sectional study in rural Andhra Pradesh, India.

A cross-sectional study of reverse smoking and its association with pre-malignant and malignant lesions of the palate was conducted in the north coastal areas of Andhra Pradesh, India. A total of 480 randomly selected persons were interviewed. Information about smoking status, diet and access to mass media was obtained in each case and an examination of the oral cavity was performed. Reverse smoking of chutta was practised by 33% of the total rural population. The prevalence rate of all palatal lesions was 55%. The prevalence rates of the separate lesions: leukoplakia palatii, palatal keratosis and palatal cancer, were 9.8%, 18.1% and 1.9%, respectively. The presence of these (pre-)malignant lesions was strongly associated with reverse smoking and also associated with conventional chutta smoking. Reverse smoking induced significantly more lesions than conventional chutta smoking, and was a major determinant of subsequent palatal cancer: all 9 newly diagnosed palatal cancers were observed within the group of reverse smokers. There was an inverse relationship between the incidence of palatal lesions and vitamin A intake. The study of access to mass media indicated that the most favourable medium for promoting a prevention campaign would be the cinema.