Louis A. Zumstein

Adenovirus-mediated p53 gene transfer in patients with advanced recurrent head and neck squamous cell carcinoma.

PURPOSE Standard therapies of head and neck squamous cell carcinoma (HNSCC) often cause profound morbidity and have not significantly improved survival over the last 30 years. Preclinical studies showed that adenoviral vector delivery of the wild-type p53 gene reduced tumor growth in mouse xenograft models. Our purpose was to ascertain the safety and therapeutic potential of adenoviral (Ad)-p53 in advanced HNSCC. PATIENTS AND METHODS Patients with incurable recurrent local or regionally metastatic HNSCC received multiple intratumoral injections of Ad-p53, either with or without tumor resection. Patients were monitored for adverse events and antiadenoviral antibodies, tumors were monitored for response and p53 expression, and body fluids were analyzed for Ad-p53. RESULTS Tumors of 33 patients were injected with doses of up to 1 x 10(11) plaque-forming units (pfu). No dose-limiting toxicity or serious adverse events were noted. p53 expression was detected in tumor biopsies despite antibody responses after Ad-p53 injections. Clinical efficacy could be evaluated in 17 patients with nonresectable tumors: two patients showed objective tumor regressions of greater than 50%, six patients showed stable disease for up to 3.5 months, and nine patients showed progressive disease. One resectable patient was considered a complete pathologic response. Ad-p53 was detected in blood and urine in a dose-dependent fashion, and in sputum. CONCLUSION Patients were safely injected intratumorally with Ad-p53. Objective antitumor activity was detected in several patients. The infectious Ad-p53 in body fluids was asymptomatic, and suggests that systemic or regional treatment may be tolerable. These results suggest the further investigation of Ad-p53 as a therapeutic agent for patients with HNSCC.

Inhibition of human lung cancer growth following adenovirus-mediated mda-7 gene expression in vivo.

Overexpression of the melanoma differentiation associated gene-7 (mda-7) in vitro results in suppression of lung cancer cell proliferation. However, the ability of MDA-7 to suppress lung cancer in vivo has not been previously demonstrated. In this study, we investigated the possibility of inducing overexpression of the mda-7 gene in human non-small cell lung carcinoma cells in vivo and its effects on tumor growth. Adenovirus-mediated overexpression of MDA-7 in p53-wild-type A549 and p53-null H1299 subcutaneous tumors resulted in significant tumor growth inhibition through induction of apoptosis. In addition, decreased CD31/PECAM expression and upregulation of APO2/TRAIL were observed in tumors expressing MDA-7. In vivo studies correlated well with in vitro inhibition of lung tumor cell proliferation and endothelial cell differentiation mediated by Ad-mda7. These data demonstrate that Ad-mda7 functions as a multi-modality anti-cancer agent, possessing both, pro-apoptotic and anti-angiogenic properties. We demonstrate for the first time the potential therapeutic effects of Ad-mda7 in human lung cancer.

INGN 007, an oncolytic adenovirus vector, replicates in Syrian hamsters but not mice: comparison of biodistribution studies

Preclinical biodistribution studies with INGN 007, an oncolytic adenovirus (Ad) vector, supporting an early stage clinical trial were conducted in Syrian hamsters, which are permissive for Ad replication, and mice, which are a standard model for assessing toxicity and biodistribution of replication-defective (RD) Ad vectors. Vector dissemination and pharmacokinetics following intravenous administration were examined by real-time PCR in nine tissues and blood at five time points spanning 1 year. Select organs were also examined for the presence of infectious vector/virus. INGN 007 (VRX-007), wild-type Ad5 and AdCMVpA (an RD vector) were compared in the hamster model, whereas only INGN 007 was examined in mice. DNA of all vectors was widely disseminated early after injection, but decayed rapidly in most organs. In the hamster model, DNA of INGN 007 and Ad5 was more abundant than that of the RD vector AdCMVpA at early times after injection, but similar levels were seen later. An increased level of INGN 007 and Ad5 DNA but not AdCMVpA DNA in certain organs early after injection, and the presence of infectious INGN 007 and Ad5 in lung and liver samples at early times after injection, strongly suggests that replication of INGN 007 and Ad5 occurred in several Syrian hamster organs. There was no evidence of INGN 007 replication in mice. In addition to providing important information about INGN 007, the results underscore the utility of the Syrian hamster as a permissive immunocompetent model for Ad5 pathogenesis and oncolytic Ad vectors.

Adenovirus-mediated wild-type p53 gene expression radiosensitizes non-small cell lung cancer cells but not normal lung fibroblasts

Purpose : We compared the ability of adenoviral-mediated wildtype p53 RPR/INGN201(Ad5/CMV/p53) to radiosensitize nonsmall cell lung carcinoma (NSCLC) and normal lung fibroblast cells. Materials and methods : NSCLC cell lines (A549 and H322) and human lung fibroblast cells (MRC-9 and CCD-16) were used in this study. Radiosensitivity was determined by clonogenic assay and tumor growth delay. Expression of p53, Bax, and p21 WAF1 protein were evaluated by immunoblot. A FITC conjugate of annexin V was used for flow cytometric detection of apoptosis. Results : Clonogenic and apoptotic assays indicated that Ad5/CMV/p53 enhanced the radiosensitivity of both NSCLC cell lines. On the other hand, the two normal human fibroblast cell lines appeared to be resistant to the cytotoxic effects of Ad5/CMV/p53 and were not radiosensitized compared to the NSCLC cells. According to immunoblot analysis, Bax expression was increased in the NSCLC cells treated with the combination therapy; Bax expression, however, was unchanged in normal cells. In in vivo studies, tumor growth suppression was enhanced by this combination strategy in xenograft tumors growing in nude mice compared to Ad5/CMV/p53 or radiation therapy when used alone. Conclusions : Our data indicate that therapy using Ad5/CMV/p53 and irradiation in combination is more effective than either treatment when used alone on NSCLC cells, is not limited to cells with defective endogenous p53, and does not enhance the radiosensitivity of normal cells.

p53 therapy in a patient with Li-Fraumeni syndrome

Li-Fraumeni syndrome is an autosomal dominant disorder that greatly increases the risk of developing multiple types of cancer. The majority of Li-Fraumeni syndrome families contain germ-line mutations in the p53 tumor suppressor gene. We describe treatment of a refractory, progressive Li-Fraumeni syndrome embryonal carcinoma with a p53 therapy (Advexin) targeted to the underlying molecular defect of this syndrome. p53 treatment resulted in complete and durable remission of the injected lesion by fluorodeoxyglucose-positron emission tomography scans with improvement of tumor-related symptoms. With respect to molecular markers, the patients tumor had abnormal p53 and expressed coxsackie adenovirus receptors with a low HDM2 and bcl-2 profile conducive for adenoviral p53 activity. p53 treatment resulted in the induction of cell cycle arrest and apoptosis documented by p21 and cleaved caspase-3 detection. Increased adenoviral antibody titers after repeated therapy did not inhibit adenoviral p53 activity or result in pathologic sequelae. Relationships between these clinical, radiographic, and molecular markers may prove useful in guiding future application of p53 tumor suppressor therapy. [Mol Cancer Ther 2007;6(5):1478–1482

An acute toxicology study with INGN 007, an oncolytic adenovirus vector, in mice and permissive Syrian hamsters; comparisons with wild-type Ad5 and a replication-defective adenovirus vector

Oncolytic (replication-competent) adenoviruses as anticancer agents provide new, promising tools to fight cancer. In support of a Phase I clinical trial, here we report safety data with INGN 007 (VRX-007), an oncolytic adenovirus with increased anti-tumor efficacy due to overexpression of the adenovirus-encoded ADP protein. Wild-type adenovirus type 5 (Ad5) and a replication-defective version of Ad5 were also studied as controls. A parallel study investigating the biodistribution of these viruses is described elsewhere in this issue. The toxicology experiments were conducted in two species, the Syrian hamster, which is permissive for INGN 007 and Ad5 replication and the poorly permissive mouse. The studies demonstrated that the safety profile of INGN 007 is similar to Ad5. Both viruses caused transient liver damage upon intravenous injection that resolved by 28 days post-infection. The No-Observable-Adverse-Effect-Level (NOAEL) for INGN 007 in hamsters was 3 × 1010 viral particles per kg. In hamsters, the replication-defective vector caused less toxicity, indicating that replication of Ad vectors in the host is an important factor in pathogenesis. With mice, INGN 007 and Ad5 caused toxicity comparable to the replication-defective adenovirus vector. Partially based on these results, the FDA granted permission to enter into a Phase I clinical trial with INGN 007.

Adenoviral-mediated PTEN expression radiosensitizes non-small cell lung cancer cells by suppressing DNA repair capacity

Expression of the PTEN tumor suppressor gene is abnormal in many human cancers. Loss of PTEN expression leads to the activation of downstream signaling pathways that have been associated with resistance to radiation. In non-small cell lung carcinoma (NSCLC), suppressed expression of PTEN is frequently due to methylation of its promoter region. In this study, we tested whether gene transfer of wild-type PTEN into an NSCLC cell line with a known methylated PTEN promoter, H1299, would increase its sensitivity to ionizing radiation. Pretreating H1299 cells with an adenoviral-mediated PTEN (Ad-PTEN)-expressing vector sensitized H1299 cells to radiation. To determine the mechanism responsible for radiosensitization, we first examined radiation-induced apoptosis, which was enhanced but did not correlate with radiosensitizing effect of Ad-PTEN. Therefore, we next examined the ability of Ad-PTEN to modulate the repair of radiation-induced DNA double-strand breaks (DSBs) using the detection of repair foci positive for γ-H2AX, a protein that becomes evident at the sites of each DSB and that can be visualized by immunofluorescent staining. Compared with controls, the repair of radiation-induced DSBs was retarded in H1299 cells pretreated with Ad-PTEN, consistent with the radiosensitizing effect of the vector. We conclude that signal transduction pathways residing primarily in the cytoplasm may intersect with DNA damage and repair pathways in the nucleus to modulate cellular responses to radiation. Elucidating the mechanisms responsible for this intersection may lead to novel strategies for improving therapy for cancers with defective PTEN.

Adenovirus-mediated PTEN treatment combined with caffeine produces a synergistic therapeutic effect in colorectal cancer cells.

The tumor suppressor phosphatase and tensin homologue deleted from chromosome 10 (PTEN) gene is a negative regulator of the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt/PKB) signaling pathway. Overexpression of PTEN in cancer cells results in cell-cycle arrest and cell death through inhibition of PI3K. Caffeine, a xanthine analogue, is well known to enhance the cytocidal and growth-inhibitory effects of DNA-damaging agents such as radiation, UV light, and anticancer agents on tumor cells by abrogating DNA-damage checkpoints through inhibition of ataxia-telangiectasia-mutated (ATM), and ATM and Rad3-related (ATR) kinase activity. In this study, we demonstrate that treatment with a combination of adenovirus-mediated transfer of PTEN (Ad-PTEN) and caffeine synergistically suppressed cell growth and induced apoptosis in colorectal cancer cells but not in normal colorectal fibroblast cells. This synergistic effect was induced through abrogation of G2/M arrest, downregulation of the Akt pathway, and modulation of the p44/42MAPK pathway. Thus, combined treatment with Ad-PTEN and caffeine is a potential therapy for colorectal cancer.

Bivariate analysis of the p53 pathway to evaluate Ad-p53 gene therapy efficacy

BACKGROUND Gene therapy of human tumors with adenovirus vectors presents a clinical research challenge and a potential opportunity in cancer therapy. One of the research challenges is that endpoints like tumor reduction, time to recurrence, and survival do not provide information about whether a potential therapeutic infects the targeted cells or whether the transferred gene functions or induces a cellular response. Therefore, a flow cytometric approach was developed for a wildtype, p53 encoding adenoviral vector (Ad-p53) that provides (1) the relative level of p53 transferred by p53 immunoreactivity, (2) mdm2 immunoreactivity as an assay of p53 activity, and (3) estimates of the percentage of infected cells by dual parameter analysis (p53 versus mdm2). METHODS Three prostate cancer cell lines (PC-3, LNCaP, DU 145) that are null, wild-type, and mutant for p53, respectively, and two ovarian cancer cell lines (PA1, MDAH 2774) that are wild-type and mutant for p53, respectively, were tested for immunoreactivity and lack of cross-reactivity with the monoclonal antibodies, DO-7 (anti-p53) and IF2 (anti-mdm2). Optimal dual staining conditions for a flow cytometric assay employing saturating levels of antibody were developed and tested by infection of PC-3, PA1, and MDAH 2774 with Ad-p53 or a control virus, Ad-luc. Dual staining with DO-7 and propidium iodide was used to determine any biological effect of the transferred gene. RESULTS Neither DO-7 nor IF2 showed appreciable cross-reactions by Western blot analysis of representative prostate or ovarian cell lines. By flow cytometric titration, DO-7 appears to be a high avidity antibody (saturation staining of 10(6) DU 145 cells with 0.5ug) whereas IF2 appears less so (optimum signal to noise ratio at 1ug/10(6) cells). Infection with Ad-p53 was detected at 6 to 48 hours post infection as a uniform relative increase in p53 levels over background p53 levels. Coincident increases in mdm2 immunoreactivity were also detected. DNA content measurements of PA1 and MDAH 2774 cells indicated that G1 arrest and/or apoptosis occurred subsequent to Ad-p53 infection. p53 and mdm2 levels and DNA content distributions for Ad-luc infected cells were equivalent to uninfected cells. CONCLUSIONS A flow cytometric approach to measure the efficacy of an Ad-p53 gene therapy vector was developed that detects not only the gene transferred but also the activity of the transferred gene product.