Abstract
Purpose
The aim of this study is to determine the ability of intratumorally delivered docetaxel to enhance the antitumor activity of adenovirus-mediated delivery of p53 (Ad-p53) in murine head and neck cancer xenograft model.
Materials and methods
A xenograft head and neck squamous cell carcinoma mouse model was used. Mice were randomized into 4 groups of 6 mice receiving 6 weeks of biweekly intratumoral injection of ( a ) diluent, ( b ) Ad-p53 (1 × 10 10 viral particles per injection), ( c ) docetaxel (1 mg/kg per injection), and ( d ) combination of Ad-p53 (1 × 10 10 viral particles per injection) and docetaxel (1 mg/kg per injection). Tumor size, weight, toxicity, and overall and disease-free survival rates were determined.
Results
Intratumoral treatments with either docetaxel alone or Ad-p53 alone resulted in statistically significant antitumor activity and improved survival compared with control group. Furthermore, combined delivery of Ad-p53 and docetaxel resulted in a statistically significant reduction in tumor weight when compared to treatment with either Ad-p53 or docetaxel alone.
Conclusion
Intratumoral delivery of docetaxel enhanced the antitumor effect of Ad-p53 in murine head and neck cancer xenograft model. The result of this preclinical in vivo study is promising and supports further clinical testing to evaluate efficacy of combined intratumoral docetaxel and Ad-p53 in treatment of head and neck cancer.
1
Introduction
The American Cancer Society estimates that approximately 45 660 new cases of head and neck squamous cell carcinoma (HNSCC) will be diagnosed in the United States and 11 210 Americans will die of this disease in the year 2007 . The treatment of advanced head and neck cancer includes surgery, radiotherapy, chemotherapy, or more commonly a combined treatment regimen. Platinum-based chemoradiotherapy compared with radiotherapy alone has been shown to improve survival for nasopharyngeal , unresectable , and oropharyngeal squamous carcinomas. When cisplatin was used together with 5-fluorouracil, organ preservation of the larynx and hypopharynx was achieved. Chemoradiation using cisplatin as compared with radiation alone after surgical resection has demonstrated a benefit in overall survival in the European Organization for Research and Treatment of Cancer trial (53% vs 40%, P = .02) . However, results from the US Intergroup phase III trial demonstrated only an improvement in disease-free survival ( P = .04) and did not confirm benefit in overall survival ( P = .19). Although systemic cisplatin-based chemotherapy has significantly reduced the incidence of distant metastasis , local-regional failure frequently occurs. Thus, efforts toward the development and investigation of more efficacious therapeutic regiments and delivery strategy are ongoing.
Intratumoral (IT) injection of chemotherapeutic agents have been described as a novel approach of drug delivery, which allows delivery of high therapeutic dose of chemotherapeutic agent locally while limiting systemic toxicity . Because chemotherapy-induced cytotoxicity in tumor cells is proportional to drug concentration, intratumoral injection of supratherapeutic dose of chemotherapeutic agent may lead to increased dose-related cancer cell killing and reduced systemic toxicity.
Previous studies by other investigators using cisplatin-based IT therapy failed to demonstrate improvement in overall survival . Taxanes are anti-microtubule agents that have antiproliferative and suppressive effects on cancer cells. Paclitaxel and its alternative taxane, docetaxel, have been shown to improve survival in breast , lung , and ovarian cancers. The use of taxane in the treatment of HNSCC also appeared promising . Taxane, in combination with other agents, have demonstrated significant clinical response rates (75%–100%) in newly diagnosed HNSCC, but the response rates in recurrent disease are not as high (13%–77%) . Recently, 2 prospective multicenter phase III clinical trials showed the addition of docetaxel to the standard induction regimen of cisplatin and fluorouracil followed by chemoradiotherapy or radiation alone improved survivals in patients with HNSCC.
The tumor suppressor gene p53 is mutated in about 50% of human cancers and approximately 33%–45% of HNSCC . The p53 gene is known to be involved in DNA repair, cell cycle, apoptosis, senescence, and genomic stability along with many other cellular functions. Studies have shown suppression of tumor growth when exogenous p53 is expressed through gene transfer in cancer cells including HNSCC . Tumor suppression has been shown to occur in both mutated and wild-type p53 human HNSCC cell lines, which suggests that the newly introduced gene may act in a dominant fashion and selects against proliferation. Ad-p53 is a constructed adenoviral vector that contains the wild-type p53 gene driven by a cytomegalovirus promoter. In a phase I trial, patients with recurrent HNSCC received multiple IT injections of Ad-p53 . No dose-limiting toxicity or related serious adverse events were noted. In a phase II multicenter trial, IT delivery of Ad-p53 was used in patients with recurrent or unresectable HNSCC, which have been heavily treated previously . Of the 112 patients, 7 (6%) achieved either partial or complete response with median survival of 40.8 months. Eighteen patients (16%) had stabilization of disease with median survival of 10.2 months .
We have previously demonstrated in HNSCC cell line models that docetaxel enhanced Ad-p53 cytotoxic effect via increased expression of exogeneous p53 gene transfer, apoptosis, and antitumor mechanisms . Given the promising in vitro result, we want to assess the clinical efficacy of the combined delivery of docetaxel and Ad-p53 in murine head and neck cancer xenograft model. Although a clinically relevant concentration of docetaxel can be delivered to the tumor site via an intravenous route, a number of studies have shown that supratherapeutic dose of docetaxel can significantly increase tumor cytotoxicity in cell line models . This supratherapeutic dose, although impossible to attain via an intravenous route due to systemic toxicity, can be achieved via the IT route. In this study, we set out to determine whether the addition of docetaxel, delivered via IT route, enhance the antitumor effect of Ad-p53 in murine head and neck cancer xenograft model.
2
Materials and methods
2.1
Ad-p53 and docetaxel
The Ad-p53 (Introgen Therapeutics, Houston, TX) viral particles (VP) were stored at −80°C. Just before the injections, the virus vectors were thawed on ice and mixed with lactated ringer to obtain the desired concentrations (VP per milliliter). Docetaxel (sanofi-aventis, Vitry sur Seine, France) in concentrated form (20 mg/0.5 mL) was initially reconstituted using 1.5 mL of 13% ethanol solution, and the reconstituted docetaxel solution (10 mg/mL) was stored at −20°C. Immediately before use, the reconstituted docetaxel solution was diluted using 5% dextrose to make final desired concentrations. The final ethanol concentration was less than 0.05% by volume.
2.2
Head and neck squamous cell carcinoma xenograft model
The HNSCC cell line, HN30, which has a wild-type p53 gene, was grown in Dulbecco modified Eagle medium with 10% fetal calf serum. After being washed twice in phosphate-buffered saline, the cells (15 × 10 6 ) were suspended in 0.1 mL of phosphate-buffered saline and 0.1 mL MATRIGEL (Becton Dickinson, Bedford, MA) to reach a total volume of 0.2 mL per injection per site. Two tumor sites per mouse were injected. All mice studies were done in compliance with and approval from the Wayne State University Institutional Animal Care and Use Committee-Division of Laboratory Animal Research. Female CB17 Severe Combined Immunodeficiency Disorder (SCID) mice, 6–11 weeks of age, were obtained from Harlan (Frederick, MD). Tumor nodule growth was monitored biweekly.
2.3
Intratumoral therapy using Ad-p53 alone in the mouse model
We have previously shown that IT injection of docetaxel, at a dose of 7.5 mg/kg per injection, was extremely effective at inhibiting the growth of tumor in the HN30 xenograft SCID mice model . This high degree of antitumoral activity would potentially mask any enhanced antitumoral effect when IT docetaxel is combined with another antitumor agent. To determine whether docetaxel can potentiate the antitumor effect of Ad-p53, a low IT docetaxel dose of 1 mg/kg per injection was used. Furthermore, to establish the IT activity of Ad-p53 in the HN30 xenograft model, 2 different doses of Ad-p53 were tested. Twelve mice were randomized into 3 groups and received IT injection of Ad- β GAL (1 × 10 10 VP per injection), Ad-p53 (1 × 10 9 VP per injection), or Ad-p53 (1 × 10 10 VP per injection).
2.4
Combined intratumoral therapy using both docetaxel and Ad-p53 in the mouse model
Intratumoral therapy was started when the tumor nodules reached a diameter of 7–10 mm. A total of 24 mice were randomized into 4 groups of 6 and underwent biweekly IT injection of ( a ) diluent, ( b ) Ad-p53 (1 × 10 10 VP per injection), ( c ) docetaxel (1 mg/kg per injection), and ( d ) combination of Ad-p53 (1 × 10 10 VP per injection) and docetaxel (1 mg/kg per injection) for a total of 6 weeks. For the combination group, the tumors were injected with docetaxel on alternate days (ie, docetaxel injected on Monday and Wednesday of each week and Ad-p53 injected on Tuesday and Thursday of each week). The total volume of injections did not exceed 0.2 mL, and the delivery of the dose into the tumor nodule was performed via multiple injections using a 27-gauge needle. After this 6 weeks of IT therapy course, mice were observed for 6 more weeks until day 85 if a complete or partial regression of the tumor was observed or if there was no change in tumor size. Mice were sacrificed if the tumors reached 1500 mg or 10% of the animal’s body weight. The surviving mice were sacrificed at day 95.
2.5
Tumor measurements in experimental vs control groups
The experimental and control groups were compared with each other with regard to tumor size, weight, and overall and disease-free survival rates, during and after IT therapy. At the time the mice were sacrificed, the tumor nodules were weighed, or the tumor weights were estimated with the following formula: tumor weight = length (cm) × width (cm) × 0.5 × 1 g/cm 3 . Calipers were used to measure the tumor length and width. Tumor size was used to estimate tumor weight. This relies on the assumption that the density of the tissue was 1 g/cm 3 . This is similar to human trials, in which using 2 measurements is standard. Statistical analysis using paired t test on tumor weight was calculated.
2.6
Clinical evaluation of IT docetaxel injections
Although the tumors were measured twice a week by calipers, the mice were also observed for changes in appearance. These changes included weight loss, scruffy appearance, listlessness, uncoordinated movements, inability to grasp a pencil, and walking splay-footed. Any dead mice were necropsied (spleen size, gastrointestinal toxicity, liver, kidneys). Mice were sacrificed by carbon dioxide narcosis and cervical dislocation when tumors reach 1500 mg or 10% of the animal’s body weight, animals displayed any open lesion, or weight loss more than 20% of body weight.
2
Materials and methods
2.1
Ad-p53 and docetaxel
The Ad-p53 (Introgen Therapeutics, Houston, TX) viral particles (VP) were stored at −80°C. Just before the injections, the virus vectors were thawed on ice and mixed with lactated ringer to obtain the desired concentrations (VP per milliliter). Docetaxel (sanofi-aventis, Vitry sur Seine, France) in concentrated form (20 mg/0.5 mL) was initially reconstituted using 1.5 mL of 13% ethanol solution, and the reconstituted docetaxel solution (10 mg/mL) was stored at −20°C. Immediately before use, the reconstituted docetaxel solution was diluted using 5% dextrose to make final desired concentrations. The final ethanol concentration was less than 0.05% by volume.
2.2
Head and neck squamous cell carcinoma xenograft model
The HNSCC cell line, HN30, which has a wild-type p53 gene, was grown in Dulbecco modified Eagle medium with 10% fetal calf serum. After being washed twice in phosphate-buffered saline, the cells (15 × 10 6 ) were suspended in 0.1 mL of phosphate-buffered saline and 0.1 mL MATRIGEL (Becton Dickinson, Bedford, MA) to reach a total volume of 0.2 mL per injection per site. Two tumor sites per mouse were injected. All mice studies were done in compliance with and approval from the Wayne State University Institutional Animal Care and Use Committee-Division of Laboratory Animal Research. Female CB17 Severe Combined Immunodeficiency Disorder (SCID) mice, 6–11 weeks of age, were obtained from Harlan (Frederick, MD). Tumor nodule growth was monitored biweekly.
2.3
Intratumoral therapy using Ad-p53 alone in the mouse model
We have previously shown that IT injection of docetaxel, at a dose of 7.5 mg/kg per injection, was extremely effective at inhibiting the growth of tumor in the HN30 xenograft SCID mice model . This high degree of antitumoral activity would potentially mask any enhanced antitumoral effect when IT docetaxel is combined with another antitumor agent. To determine whether docetaxel can potentiate the antitumor effect of Ad-p53, a low IT docetaxel dose of 1 mg/kg per injection was used. Furthermore, to establish the IT activity of Ad-p53 in the HN30 xenograft model, 2 different doses of Ad-p53 were tested. Twelve mice were randomized into 3 groups and received IT injection of Ad- β GAL (1 × 10 10 VP per injection), Ad-p53 (1 × 10 9 VP per injection), or Ad-p53 (1 × 10 10 VP per injection).
2.4
Combined intratumoral therapy using both docetaxel and Ad-p53 in the mouse model
Intratumoral therapy was started when the tumor nodules reached a diameter of 7–10 mm. A total of 24 mice were randomized into 4 groups of 6 and underwent biweekly IT injection of ( a ) diluent, ( b ) Ad-p53 (1 × 10 10 VP per injection), ( c ) docetaxel (1 mg/kg per injection), and ( d ) combination of Ad-p53 (1 × 10 10 VP per injection) and docetaxel (1 mg/kg per injection) for a total of 6 weeks. For the combination group, the tumors were injected with docetaxel on alternate days (ie, docetaxel injected on Monday and Wednesday of each week and Ad-p53 injected on Tuesday and Thursday of each week). The total volume of injections did not exceed 0.2 mL, and the delivery of the dose into the tumor nodule was performed via multiple injections using a 27-gauge needle. After this 6 weeks of IT therapy course, mice were observed for 6 more weeks until day 85 if a complete or partial regression of the tumor was observed or if there was no change in tumor size. Mice were sacrificed if the tumors reached 1500 mg or 10% of the animal’s body weight. The surviving mice were sacrificed at day 95.
2.5
Tumor measurements in experimental vs control groups
The experimental and control groups were compared with each other with regard to tumor size, weight, and overall and disease-free survival rates, during and after IT therapy. At the time the mice were sacrificed, the tumor nodules were weighed, or the tumor weights were estimated with the following formula: tumor weight = length (cm) × width (cm) × 0.5 × 1 g/cm 3 . Calipers were used to measure the tumor length and width. Tumor size was used to estimate tumor weight. This relies on the assumption that the density of the tissue was 1 g/cm 3 . This is similar to human trials, in which using 2 measurements is standard. Statistical analysis using paired t test on tumor weight was calculated.
2.6
Clinical evaluation of IT docetaxel injections
Although the tumors were measured twice a week by calipers, the mice were also observed for changes in appearance. These changes included weight loss, scruffy appearance, listlessness, uncoordinated movements, inability to grasp a pencil, and walking splay-footed. Any dead mice were necropsied (spleen size, gastrointestinal toxicity, liver, kidneys). Mice were sacrificed by carbon dioxide narcosis and cervical dislocation when tumors reach 1500 mg or 10% of the animal’s body weight, animals displayed any open lesion, or weight loss more than 20% of body weight.
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