Selective intra-arterial infusion of rAd-p53 with chemotherapy for advanced oral cancer: a randomized clinical trial

Oral cancer is a major global health problem with 300,000 new cases diagnosed each year. Alcohol and tobacco remain prominent etiologic factors in oral cancer [1], but high-risk human papillomavirus (HPV) has emerged as an important etiologic agent [2]. Several potentially curative surgical and non-surgical treatment options exist for patients with oropharyngeal cancer [3, 4], but treatment selection (chemoradiotherapy and primary surgery) is complex, reflecting the need for primary tumor and nodal disease control to be balanced against preservation of function and minimizing long-term toxicities. Despite advances in treatment with surgery, radiotherapy, and chemotherapy, this aggressive epithelial malignancy is associated with severe morbidity, and patients have a long-term survival rate of less than 50% [5, 6]. Treatment failures mainly involve development of second primary tumors in patients with early stage disease (stages I and II), and local recurrence and metastases in patients with locally advanced disease [7, 8]. New approaches are urgently needed to improve the outcomes of patients with oral cancer.

Gene therapy with wild-type human tumor suppressor p53 (wt-p53) is a promising treatment strategy for some malignant tumors [9‐12]. The wt-p53 protein is a primary mediator of cell cycle arrest, DNA repair, and apoptosis. The mutation and inactivation of p53 may be a crucial event in the origin and progression of head and neck carcinoma [13‐15], and potentially contributes to the development of drug resistance in tumors of epithelial origin [16]. The incidence of p53 mutation is approximately 31% in squamous cell carcinoma (SCC) [17, 18]. Introduction of the wt-p53 gene inside tumor cells may restore tumor suppressor functions and improve treatment outcomes in some patients [12, 15, 19]. Reintroduction of wt-p53 has been accomplished with recombinant adenoviral p53 (rAd-p53) [20]. Adenoviral delivery of wt-p53 results in strong wt-p53 protein expression in tumor cells, with minimal hematopoietic toxicity [10, 11, 13, 14, 17, 19‐27]. Clinical trials have demonstrated that rAd-p53 is effective against a variety of malignancies [10, 11, 13‐15, 17, 19‐27]. Routes of administration typically used for rAd-p53 are intratumoral injection, perfusion, and intravenous infusion [15, 28‐33]. Although intratumoral rAd-p53 combined with radiotherapy has led to higher rates of locoregional control [34], and rates of complete response (CR) were up to 75% of patients by 12 weeks in a phase II study [32, 33], rAd-p53 alone has led to CR in only a few cases [12, 28]. Although intratumoral injection delivers the recombinant adenovirus to its target, the spread of the recombinant adenoviral vectors from the injection site is minimal [35].

Several studies have examined the use of selective intra-arterial infusion of chemotherapy for head and neck cancers [36‐39], and suggest modestly higher responses or at least non-inferiority [39‐41]. Potential benefits of intra-arterial infusion include more precise delivery to the target organ, potentially wider dissemination in the tumor than occurs with intratumoral injection, and fewer systemic side effects [39]. Intra-arterial chemotherapeutic administration can also provide high response rates and acceptable palliative treatment without surgery or radiotherapy in elderly patients [39]. Because a high concentration of rAd-p53 can be delivered to an oral tumor through intra-arterial infusion with minimal systemic toxicity, the aims of this randomized, placebo-controlled, double-blind, phase III clinical trial were to compare the efficacy and safety outcomes of intra-arterial infusion of combined rAd-p53 and chemotherapeutic drugs with those of the single treatments individually for treatment of oral carcinoma. This study also assessed the expression of p53 and two transcriptional targets, Bax and Bcl-2, in the biopsy specimens, using immunohistochemical analysis.

In this randomized, placebo-controlled, double-blind, phase III clinical trial, 99 patients with stage III or IV oral SCC were randomly assigned to group I (n = 35; rAd-p53 plus chemotherapy), group II (n = 33; rAd-p53 plus placebo chemotherapy), or group III (n = 31; placebo rAd-p53 plus chemotherapy). Baseline characteristics of the three groups were similar (Table 1). All tumors were SCC (presence of adenocarcinoma was an exclusion criterion). Most tumors were classified as T3 or T4, and were well balanced across the three groups. Each group included approximately 60% of patients with stage IVa, approximately 11% with stage IVb and 30% with stage III disease, based on the UICC TNM cancer staging system. The treatments were administered by intra-arterial infusion into the primary lesion. Because of financial reasons, seven patients dropped out of the study after randomization. Dropouts occurred during the early years of the study, reflecting the financial or coverage environment in the geographic area at that time. As a result, 33, 30, and 29 patients in groups I, II, and III, respectively, completed the study.

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 [15], 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. [15]. 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 [12]. High expression of mutated p53 in patients with recurrent HNSCC significantly decreased efficacy and tumor response of Ad-p53 gene therapy [12]. Conversely, low levels of p53 protein were favorable for p53 gene therapy in patients with recurrent HNSCC [12]. As expected, the p53 profiles predictive of efficacy of p53 gene therapy did not predict methotrexate response [12]. 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 [46]. Third, because greater rAd-p53 doses yielded better responses [10], 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. [47] 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 [27]. If wt-p53 function is lost by down-regulation or mutation, chemotherapy is less effective [45], 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 [48]. 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.