This single-center, randomized, placebo-controlled, double-blind, phase III clinical study showed that intra-arterial infusion of rAd-p53 and chemotherapy is associated with significantly better outcomes than either treatment alone, with a three-fold advantage in CR rate of the primary tumor mass. The survival rate was significantly higher in group I patients (intra-arterial rAd-p53 plus chemotherapy) with UICC III oral cancer. Patients selected for this study did not undergo prior surgery or radiotherapy, and treatment outcomes were recorded before any post-treatment local management by surgery. For complete disclosure, we state that after recording of all outcomes, the percentage of patients who underwent surgery was similar in group I (n = 10, 30.3%), group II (n = 7, 23.3%) and group III (n = 7, 24.1%) (P = 0.787), and two patients received both surgery and radiotherapy. Thus, survival rates and other outcomes were related to the study treatments, and were not influenced by other interventions. Immunohistochemical analysis of biopsy specimens showed increased levels of staining for wt-p53 protein in those patients who received infusion of rAd-p53. Exogenous p53 protein produced by rAd-p53 uptake, and expression in the primary lesion appeared to be functionally active in the approximately 80% of patients who had higher Bax and lower Bcl-2 expression in the post-treatment primary tumor samples. Our results emphasize that rAd-p53 activity is intimately related to outcome. These findings are similar to those of Li et al., who observed an increase in p21 in 14 of 22 rAd-p53-treated tumor samples, a decline in bcl-2 staining in 8 of 22 samples, and detectable apoptosis (by terminal dUTP nick end labeling (TUNEL) staining) in 18 of 22 samples , suggesting that rAd-p53 intra-tumoral administration and p53 expression increased tumor cell death by apoptosis in most samples (81.8%). As an increase in Bax protein level was observed in all our patients achieving CR in groups I and II, we infer that p53 was expressed after rAd-p53 administration, and led to tumor cell death in these patients. The higher Bax expression is consistent with the rAd-p53-induced apoptosis observed by Li et al. . By contrast, Bax immunoreactivity remained unchanged in approximately half of the patients in groups I and II who did not show CR to rAd-p53 treatment. Thus, these data suggest that the increase in Bax expression in the primary lesion was associated with the functional activity of the exogenous wt-p53 protein.
The p53 protein is a key element in the apoptotic signaling cascade, and a mutation in the p53 gene reduces the susceptibility of a cell to apoptosis. Alterations in p53 can occur early in carcinogenesis, and are maintained during progression to overt malignancy. Flooding this mutant gene with wt p53 via adenovirally mediated p53 gene therapy has provided a modest therapeutic benefit. Sensitivity of head and neck SCC (HNSCC) to rAd-p53 therapy was associated with p53 status . High expression of mutated p53 in patients with recurrent HNSCC significantly decreased efficacy and tumor response of Ad-p53 gene therapy . Conversely, low levels of p53 protein were favorable for p53 gene therapy in patients with recurrent HNSCC . As expected, the p53 profiles predictive of efficacy of p53 gene therapy did not predict methotrexate response . In the current study, we detected wt-p53 in the patients (Figure 4A), but we did not examine the mutated p53 expression profile.
Compared with previous studies using intratumoral or intravenous infusion of rAd-p53 [10, 11, 36], this study achieved higher overall clinical response rates with intra-arterial administration. A comparison with our Kaplan-Meier plot shows that group II (rAd-p53 alone) was associated with longer median survival time than in the study of Clayman et al. [10, 11]. Direct comparison of outcomes is constrained because of differences in study populations (for example, our study excluded patients with refractory disease). However, the following factors may have contributed to our positive results. First, we delivered rAd-p53 and/or chemotherapy through selective intra-arterial infusion in a retrograde manner, which, compared with intravenous infusion, more accurately targets the tumor, increases the local effective therapeutic dosage, may distribute the rAd-p53 more evenly throughout the tumor, and is associated with fewer side effects. Second, the blood vessels feeding oral tumors are typically more anatomically apparent than in healthy tissue, which facilitates accurate delivery of drug to the target area. Indocyanine green fluorescence improve identification of the blood supply to the tumor . Third, because greater rAd-p53 doses yielded better responses , improved dose delivery via intra-arterial infusion may have contributed to the higher survival rate in patients with stage III oral carcinoma. Fourth, the physical status of our patients may have also contributed to survival, as our population was treatment-naive and did not have any morbidities resulting from toxicity or serious side effects associated with previous chemoradiotherapy or surgery.
The combination of intra-arterial rAd-p53 and chemotherapy showed a significantly greater treatment response and survival benefit compared with either treatment alone, consistent with a synergistic interaction. Combination of rAd-p53 with other forms of treatment has also significantly improved CR and patient survival. Weinrib et al.  suggested that rAd-p53 and cisplatin interact in an additive manner to kill C666-1 and CNE-1 cells. Somatic cell DNA damage inflicted by chemotherapy does not increase the risk of adenovirus DNA integration into genome. On the contrary, the non-specific induced DNA damage helps to activate the wt-p53 protein introduced by rAd-p53, with consequent synergistic effect . If wt-p53 function is lost by down-regulation or mutation, chemotherapy is less effective , which may explain the increased effectiveness of combination therapy. In this study, we did not see CR for cervical (neck) lesions in any of the groups, which might be related to the small number of patients. Despite the lack of statistical significance, we observed that for neck metastases, 15/33 group I patients (45.5%) achieved PR and 9/33 (27.3%) SD whereas 10/30 group II patients (33.3%) achieved PR and 8/30 (26.7%) SD, and (10/29 group III patients (34.5%) had PR and 5/29 (17.2%) SD. The higher percentage of PR in group I compared with groups II or III did not reach statistical significance. We propose that the following three criteria are necessary to enhance the efficacy of the treatment: 1) regular and sufficient administration of rAd-p53; 2) adequate local concentration of rAd-p53; and 3) combination of gene therapy with chemotherapy.
Results from other trials of rAd-p53 therapy have shown that rAd-p53 treatment appears safe and well-tolerated [10, 11, 15, 36, 48]. The limited dataset and statistical power in our small pilot study did not detect any novel safety concerns. Approximately 82% of patients who received intra-arterial infusion of rAd-p53 experienced flu-like symptoms and/or bone marrow suppression. The adverse events such as flu-like symptoms appeared to be associated with the adenovirus delivery vehicle itself rather than with the encoded exogenous wt-p53 gene, similar to previous studies . A noteworthy finding of our study was that bone marrow suppression was significantly less common with rAd-p53 plus chemotherapy compared with chemotherapy alone. We propose that the mechanisms by which wt-p53 protein may reduce the chemotherapy-induced toxicity involves at least one of the following three pathways: 1) interaction of p53 protein with DNA helicase; 2) up-regulation of ribonucleotide reductase (p53R2) by p53; or 3) the 3′→ 5′ exonuclease activity of the p53 protein. However, further studies are warranted.
This study had several limitations. Although larger than previous investigations [10, 11], this small clinical study (fewer than 40 patients were randomized to each group) of gene therapy for SCC was conducted at a single center. The low incidence of advanced (stage III/IV) oral carcinoma precluded recruitment of a larger study population. Nevertheless, the findings and protocol used in this study can be used to aid in designing a future larger multi-center clinical trial. Second, our study design did not contain a control group that received dosing through intratumoral or intravenous administration for direct comparison. Third, because the chemotherapeutic agent is a prodrug that must be metabolized in the liver into the active drug, intra-arterial delivery may also have reduced the effectiveness of chemotherapy. Fourth, the patients had a range of tumor characteristics (location, classification, stage). However, these potential sources of heterogeneity were minimized because the distribution of SCC was similar in the three groups. Disease heterogeneity did not appear to account for differences in outcomes among the three groups. Fifth, only patients with advanced disease (stage III or IV) who refused or were not suitable for surgery were enrolled. No patients with stage I or II disease were included, as most would have received surgical treatment. A treatment-naive population with advanced SCC does not reflect current, routine presentation in advanced industrialized countries. However, our study does provide proof of principle that intra-arterial infusion of rAd-p53 with chemotherapy has clinical benefit in advanced oral cancer. We are currently performing a clinical trial in which patients with advanced oral carcinoma receive chemotherapy and gene therapy after surgery.
In summary, the combination of gene therapy (rAD-p53) and chemotherapy produced significantly higher CR rates than either gene therapy alone or chemotherapy alone. These findings are consistent with a synergistic interaction, which has also been described in publications with other types of tumors. Interestingly, no significant adverse effects were found, and bone marrow suppression was significantly less common in group I (gene therapy plus chemotherapy) than in group III (chemotherapy alone). This study also demonstrated that the p53 protein delivered by gene therapy is functionally active in 80% of cases (based on analysis of Bax and Bcl-2 expression in primary tumor). This pilot study provides the proof of benefits of intra-arterial infusion for cancer gene therapy. These results are noteworthy, and suggest that intra-arterial infusion of combined rAd-p53 and chemotherapy is a viable strategy for the management of oral SCC, and may represent an alternative to chemoradiotherapy and surgery, which is the current standard of care.