Recombinant Human Adenovirus-p53 Therapy for the Treatment of Cervical Cancer: A Meta-Analysis

Objectives To evaluate the clinical curative effects and toxicity of recombinant human adenovirus-p53 injection (rAd-p53) plus chemotherapy (CT), radiotherapy (RT), or concurrent chemoradiotherapy (CRT) for the treatment of cervical cancer. Methods We identified 14 eligible studies in the PubMed, Web of Science, Cochrane Library, Embase, CNKI, Wangfangdate, CBM, and VIP databases from their inception to May 2021 and performed meta-analyses using RevMan version 5.3. Results This analysis included 14 studies involving 737 patients. The results of the meta-analysis results showed significantly improved complete remission (odds ratio [OR] = 2.54, 95% confidence interval [CI]: 1.74–3.70, p < 0.00001), partial remission (OR = 1.56, 95% CI: 1.14–2.14, p = 0.006), and object response (OR = 4.47, 95% CI: 3.02–6.60, p < 0.00001) rates in the rAd-p53 combination therapy group compared to those in the CT/RT/CRT group. The results of subgroup analyses of CT/RT/CRT were consistent with the overall results. Regarding the incidence of adverse reactions, only the occurrence rate of fever (OR = 18.21, 95% CI: 10.54–31.47, p < 0.00001) in the rAd-p53 combination group was higher than that in the CT/RT/CRT group. No other significant differences were observed in other adverse reactions. Conclusion RAd-p53 combined with CT/RT/CRT for the treatment of cervical cancer showed significant advantages in efficacy and safety compared to those in the CT/RT/CRT group. Therefore, rAd-p53 has great potential as an effective therapy for cervical cancer. Systematic Review Registration https://inplasy.com/inplasy-2021-5-0058/.


INTRODUCTION
Cervical cancer is the fourth most common malignancy in women worldwide and represents a major global health challenge. The Global Cancer Observatory reported approximately 570,000 cases of cervical cancer and 311,000 deaths from the disease in 2018, with approximately 290,000 (51%) of new cases worldwide occurring in women living in lowand middle-income countries (500,000 [88%], including uppermiddle-income countries) (1)(2)(3).
Radical hysterectomy with pelvic lymphadenectomy remains the standard recommendation for patients with early-stage cervical cancer. The standard treatment for locally advanced cervical cancer is definitive chemoradiotherapy with pelvic radiotherapy (RT) and concurrent cisplatin-based chemotherapy (CT) (4,5). However, many patients present at an advanced stage owing to the low cervical cancer screening rate (6). While concurrent chemoradiotherapy (CRT) is the standard treatment mode recommended by the National Comprehensive Cancer Network (NCCN) guidelines, 29%-38% of the failure modes are uncontrolled or recurrent disease, with a 5-year survival rate of relapsed patients of only 3.8%-13% (7). Chemotherapy and RT resistance are the major causes of cervical cancer recurrence and mortality (8,9). Therefore, improving the sensitivity of RT and chemotherapy before treatment is particularly important to guide follow-up treatment (10).
The tumor suppressor gene p53 is widely regarded as the gene guardian of cells and plays a key role in cell cycle control, apoptosis, and the inhibition of tumor cell proliferation. The p53 gene is one of the most frequently mutated genes in human cancers, with over 50% of all cancers harboring p53 mutations (11). Inactivation of p53 function often correlates with increased malignancy, poor patient survival, and resistance to chemotherapy or RT (12)(13)(14). Accumulating evidence has demonstrated that the restoration of p53 activity can induce cell cycle arrest and apoptosis, eliminate RT and chemotherapy resistance, and inhibit tumor growth in cervical cancer cells (15). As such, the reactivation of the p53 protein has become an attractive approach for the effective treatment of cervical cancer (16). Recombinant human adenovirus p53 (rAd-p53) can transfer the p53 gene into tumor cells through recombinant human adenovirus-p53, rebuild p53 gene function in tumor cells, and cause tumor cells to undergo programmed death or develop a severe hibernation state, thereby increasing the sensitivity of tumor cells to RT and chemotherapy (17).
This meta-analysis aimed to systematically evaluate the efficacy and safety of rAd-p53 combined with CT/RT/CRT in the treatment of cervical cancer and to provide evidence-based medical data for the treatment of cervical cancer.

MATERIALS AND METHODS
This systematic review and meta-analysis was based on a preplanned protocol constructed according to the standard Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) and was prospectively registered on i n p l a s y . c o m ( I N P L A S Y p r o t o c o l 2 0 2 1 5 0 0 5 8 . d o i : 10.37766/inplasy2021.5.0058).

Study Inclusion Criteria
The inclusion criteria were as follows: (i) Randomized controlled clinical studies.
(ii) Diagnosis of cervical cancer by cytological and histopathological examinations and without serious cardiac, pulmonary, hepatic, or renal disease. (iii) Receipt of CT/RT/CRT combined with rAd-p53 for those in the experimental group was treated with and CT/RT/CRT alone for those in the control group. (iv) The primary efficacy outcomes were complete remission (CR), partial remission (PR), and objective tumor response rate (ORR) according to World Health Organization (WHO) criteria. The ORR was calculated as follows: (CR + PR)/total number of cases×100%. The secondary outcome measure was the number of adverse reactions, including fever, myelosuppression, gastrointestinal reaction, radio rectitis, radio cystitis, and liver damage.

Search Strategy and Study Selection
We identified RCTs of CT/RT/CRT plus rAd-p53 versus the control group without rAd-p53 in the treatment of cervical cancer through searches of the

Statistical Analysis
We used Cochrane RevMan version 5.3 to analyze the data. The results were reported as pooled odds ratios (ORs) with respective 95% confidence intervals (95% CIs). Heterogeneity was assessed using Cochran's Q test and I 2 statistics. If the heterogeneity was not significant (p > 0.1, I 2 < 50.0%), a fixed-effects model was used; otherwise, a random-effects model was used. The results of this meta-analysis were presented as forest plots. To detect potential publication bias, we generated a Begg's funnel plot and performed a sensitivity analysis. All p-values were two-sided, and p < 0.05 was considered statistically significant.

Study Characteristics
We identified 201 studies through the database search, 132 of which were duplicate studies, 88 were irrelevant research studies, 19 were reviews, and 9 were basic research based on title and abstract review. Among the remaining 16 articles selected for full-text review, we excluded one non-RCT and one article with an inconsistent outcome. Finally, the meta-analysis included 14 studies. A flow chart of the literature screening is shown in Figure 1. A total of 737 patients were enrolled, including 379 (51.4%) and 358 (48.6%) patients in the experimental and control groups, respectively. In the included literature, four RCTs (18-21) combined rAd-p53 with CT with platinumbased CT regimens; six RCTs (22)(23)(24)(25)(26)(27) administered rAd-p53 with RT and four RCTs (28-31) combined rAd-p53 with CRT; among these 10 articles, six studies were rAd-p53 combined with

Quality Assessment
The results of the quality evaluations of the included studies are shown in Figures 2 and 3. All the included studies were RCTs. Four studies assigned random numbers (18,19,24,31), while none of the remaining studies described any particular randomization method. Most of the included studies did not provide sufficient information to assess whether the allocation concealment was adequate. A total of 488 participants in nine studies (18, 20-25, 28, 31) signed informed consent forms, while the remaining five studies (19,26,27,29,30)  did not have sufficient information to assess the blinding method. All included articles had complete data, no data fall-off, no selective reports, and no other deviations.

CR
All the included studies reported CR. None of the trials showed significant heterogeneity (p = 0.79, I 2 = 0%); thus, a fixed-effects model was used for the meta-analysis. The results of the metaanalysis showed a significantly higher CR rate in the rAd-p53 combination group compared to that in the control group (OR = 2.54, 95% CI:1.74-3.70, p < 0.00001) ( Figure 4A).

PR
All 14 studies reported PR. We observed no statistically significant heterogeneity among the trials (p = 0.67, I 2 = 0%); thus, a fixed-effects model was used to perform the metaanalysis. The results of the meta-analysis showed a significantly higher PR rate in the rAd-p53 combination group compared to that in the control group (OR = 1.56, 95% CI: 1.14-2.14, p = 0.006) ( Figure 4B).

ORR
All 14 studies reported the ORR. We observed no statistically significant heterogeneity among the trials (p = 0.82, I 2 = 0%); thus, a fixed-effects model was used for the meta-analysis. The results of the meta-analysis showed a significantly higher CR rate in the rAd-p53 combination group compared to that in the control group (OR = 4.47, 95% CI: 3.02-6.60, p < 0.00001) ( Figure 4C).

Subgroup Analysis
The subgroup analysis of CT/RT/CRT revealed no heterogeneity in any subgroup. The RT subgroup included four studies involving 250 patients; the RT subgroup included six studies involving 329 patients; and the CRT subgroup included four studies, involving 158 patients. All subgroups demonstrated improved CR, PR, and ORR ( Figures 5-7). We also performed a subgroup analysis on IMRT/non-IMRT and observed no heterogeneity in all subgroups. Moreover, all subgroups     Figure S4).

Evaluation of the Sensitivity and Publication Bias
Sensitivity analysis was performed by omitting one study at a time to assess its influence on the overall estimates. The results showed that the deletion of any single study had no significant effect on the results (Figures 9B-11B), indicating that the results of this meta-analysis were relatively stable. Analysis of publication bias among the included articles showed no obvious bias in the CR, PR, and ORR. Begg's funnel plot and Egger's test indicated no significant publication bias ( Figures 9A-11A, Supplementary Figures S1-S3).

DISCUSSION
RAd-p53 was approved by the State Food and Drug Administration (SFDA) of China in 2004 as a gene therapy medicinal product for tumors. RAd−p53 is a replication-defective living adenovirus carrying the p53 gene. An advantage of the adenovirus delivery  system is that it does not result in the integration of the vector DNA into the host cells (32). Once cells have been infected, virus particles cannot replicate but can import the p53 gene, which exerts biological functions. This method has the advantages of high infection efficiency, no genetic toxicity to the human body, and safe clinical application. The basic mechanisms of rhAd−p53 reagents are as follows: (i) the inhibition of tumor growth by cell cycle arrest and induced programmed cell death; (ii) enhancement of chemotherapy−induced cell cycle arrest and apoptosis; (iii) stimulation of the body to produce an anti−tumor immune such that a large number of immune cells gather at the local injection site of tumors; (iv) inhibition of tumor vascular endothelial growth factor (VEGF) to suppress angiogenesis and tumor growth through the "bystander effect"; thus, the injection site of the local tumor tissue will block blood supply and induce tumor necrosis (33). RAd-p53 is currently used as a treatment for various cancers and has shown remarkable clinical efficacy in head and neck (34), lung (35), liver (36), colorectal (37), and ovarian (38) cancers. Meta-analysis studies have evaluated the safety and efficacy of rAd-p53 for the treatment of nasopharyngeal cancer (39) and malignant pleural effusion (40). To our knowledge, this is the first meta-analysis to systematically assess the effects and safety of rAd-p53 in the treatment of cervical cancer. This systematic review included 14 studies involving 737 patients. The results of the meta-analysis showed that CR, PR, and ORR in the rAd-p53 combined therapy group were significantly improved compared with the CT/RT/CRT alone group. Similar results were observed in the subgroup analysis. Two of our included 14 studies investigated the long-term survival of rAd-p53 treating cervical cancer. Zhang Dan et al. used the Kaplan-Meier method to estimate the progression-free survival (PFS) of patients administered rAd-p53 combined with chemotherapy. They reported that the PFS of the experimental group (6.38 ± 0.14) was higher than that of the control group (4.48 ± 0.14). Su et al. showed that the 5-year overall survival rate (OS) for rAd-p53 combined with radiotherapy was 17.5% higher than that for RT alone (hazard ratio [HR] = 0.551, 95% CI: 0.278-1.095, p = 0.084); the 5-year PFS for rAd-p53 combined with radiotherapy was 17.1% higher than that for RT alone (HR = 0.485,95% CI: 0.234-1.006, p = 0.0470). DNA aneuploidy is thought to reflect the biological behavior of malignancy, in which the higher the DNA aneuploidy of tumor tissue, the higher the degree of malignancy and the worse the prognosis (41). Two of the 14 included articles reported differences in DNA ploidy index before and after rAd-p53 combined with RT for cervical cancer. The results showed no significant difference in DNA aneuploidy between the two groups before treatment. After treatment, the positivity rate of DNA aneuploidy in the rAd-p53 combined group was lower than that in the RT group. The intratumoral injection of rhAd-p53 inhibited VEGF expression and angiogenesis and promoted tumor necrosis and shrinkage in advanced cancer (42). One of the included 14 studies was a related experiment. Xiao et al. determined the expression levels of vascular endothelial VEGF in three sets of cervical cancer specimens (blank control; CT alone; and rAd-p53+CT) before and after treatment by immunohistochemistry. The results showed significantly decreased VEGF expression in the CT alone and rAd-p53+CT groups compared to that in the control. In addition, the rhAd-p53 + CT group showed a more significant decrease in VEGF levels than that in the CT group. Based on the results of the above analyses, rad-p53 may be beneficial in patients with cervical cancer in terms of both shortand long-term effects.
Regarding the incidence of adverse reactions, only the occurrence rate of fever in the rAd-p53 combination group was higher than that in the control group. No significant differences were observed between the groups in myelosuppression, gastrointestinal reaction, radio rectitis, radio cystitis, and liver damage rate. All fevers were transient and self-limited and appeared within 24 h after injection. The grade II fevers resolved spontaneously, while the grade III fevers returned to normal levels after the administration of antipyretics. It is well  known that adenovirus vectors can induce a strong immune response in patients, which manifests as a self-limiting fever. Although fever is considered a side effect in clinical use, it also reflects the effectiveness and benefits of Adp53 in mobilizing the immune system. Thus, rAd-p53 was a safe and biologically active treatment for improving the curative effect in patients with cervical cancer.

Limitations
Some questions remain in our study. The issues requiring further study include the identification of the best chemotherapy regimen to be used in combination with rAd-p53, the recommended dose for radiotherapy, and whether the combination of rAd-p53 as a radiotherapy and chemotherapy sensitizer can produce long-term improvement in distant control. Moreover, most of the included studies did not perform long-term follow-up. Additional studies are needed to evaluate the relationship between rAd-p53 expression and longterm survival.

CONCLUSION
In conclusion, our results demonstrated that rAd-p53 combined with CT/RT/CRT was more effective and safer for the treatment of cervical cancer. Therefore, rAd-p53 showed great potential as an effective therapy for cervical cancer.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.

AUTHOR CONTRIBUTIONS
YG and JC proposed the study concept and design. YG and XZ collected the literature. YG, MF, MX, and YX analyzed the data.