p53 Antibodies as a Diagnostic Marker for Cancer: A Meta-Analysis

Importance: The protein p53 is an unequivocal tumor suppressor that is altered in half of all cancers. The immune system produces systemic p53 autoantibodies (p53 Abs) in many cancer patients. Objective: This systemic review and meta-analysis focuses on the prognostic value of p53 Abs expressed in the serum of patients with solid tumors. Data Sources: All the clinical investigations were searched on PubMed from the first study dated 1993 until May 2021 (date of submission of the manuscript). Study Selection: Studies were included that met the following criteria: (1) participants with cancer; (2) outcome results expressed in relation to the presence of a p53 antibody; (3) a primary outcome (disease-free survival, overall survival or progression-free survival) expressed as hazard ratio (HR). The following exclusion criteria were used: (1) insufficient data available to evaluate outcomes; (2) animal studies; (3) studies with less than 10 participants. As a result, 12 studies were included in the analysis. Data Extraction and Synthesis: PRISMA guidelines were used for abstracting and assessing data quality and validity by three independent observers. The summary estimates were generated using a fixed-effect model (Mantel–Haenszel method) or a random-effect model (DerSimonian–Laird method), depending on the absence or presence of heterogeneity (I2). Main Outcome(s) and Measure(s): The primary study outcome was to determine the prognostic value of p53 Abs from a large population of patients with solid tumors, as determined before data collection. Results: In total, 12 clinical studies involving 2094 patients were included in the meta-analysis, and it was determined that p53 Abs expression in the serum significantly correlated with poorer survival outcomes of cancer patients (95% CI 1.48 [1.24, 1.77]; p < 0.00001). Conclusions and Relevance: This is the first meta-analysis proving the diagnostic utility of p53-Abs for cancer patients in predicting poorer outcomes. The serum-p53 value (s-p53-value) may be useful for future theranostics.

Mice lacking MDM2 show embryonic lethality, while the dual presence of p53 and MDM2 can rescue lethality [7]. The p53 mutation in cancer (p53-mut) does not activate the expression of the E3 ligase. Consequently, degradation of p53 protein is not downmodulated [8]. High expression of p53 by cells recapitulates in T-cells the production of antibodies against mutant or wild type p53 [8]. On the other hand, in many cancer patients the p53-wt region is exposed and serum antibodies are generated against p53-wt. These can be detected by ELISA method. The roles of these antibodies are not yet clearly understood.
Prognostic biomarkers have a crucial role in measuring the progression of diseases from samples of patients, such as metastasis in cancer, and they can aid clinicians in intervening with more precise medical interventions. In addition to the common theory that in humans the loss of p53 increases genomic instability, this loss has been linked to the proliferation of the stem-cell characteristic that ultimately leads to highly aggressive cancers with invasive and metastatic properties. p53 antibodies (s-p53-Abs) are stably expressed in the sera of cancer patients, and could have an important prognostic application. Many clinical studies have assessed in cancer patients the correlation between the expression of s-p53-Abs with tumor invasiveness grades, metastasis and prognosis [9].
In our review, we performed a meta-analysis of the current literature, investigating the prognostic role of serum p53-Abs in cancer patients.

months (at least)
High levels of p53-Abs correlated with worse survival prospects compared to patients with lower levels of the antibodies (p = 0.02).   The funnel plot (Figure 3) of the included studies showed a symmetric funnel plot and no significant publication bias was identified.

Discussion
The meta-analysis showed that high levels of p53 antibodies significantly correlated with worse clinical outcomes. However, our study had some limitations. First, the retrospective nature of the study was intrinsically susceptible to biases. Second, different forms of solid tumors were included pre-or post-treatment with various types of therapies, as the typology requirements were at different stages. Consequently, in our analysis patients were observed independently of treatment and tumor type because of the relatively low number of randomized studies at our disposal. Third, there was a lack of follow-up with patients from different clinical trials. Thus, differences in survival probability may have been influenced by the durations of the studies. This may have given rise to different age populations, which could ultimately have affected the data. All these variables may ultimately have influenced the results.
As medicine advances, studies involving greater numbers of patients could help to evaluate the impact of our findings and treatment response.
In summary, p53 is a well-established tumor suppressor, and its absence is commonly found in patients diagnosed with cancer. The p53 protein has been demonstrated to be absent or mutated in approximately one out of two malignancies. It is known that p53wt cancers have a better prognosis compared to p53-mut cancers. Our data are not in contradiction with this notion. Although both mutated and wild type p53 antibodies can be detected in cancer patients, their role is still controversial and a matter for debate. Recently, a few studies have reported that these antibodies are statistically associated with the survival of patients diagnosed with different malignancies. To the best of our knowledge, our meta-analysis is original and is the first study gathering p53 (wild type/mutated) antibody data generated from 1993 thus far. Overall, the investigation includes 12 studies and a total of 2094 patients.

Materials and Methods
The studies were identified according to the following inclusion criteria: (1) participants with cancer; (2) outcome results expressed in relation to the presence of a p53 antibody; (3) a primary outcome (disease-free survival, overall survival or progression-free survival) expressed as hazard ratio (HR). The following exclusion criteria were used: (1) insufficient data available to evaluate outcomes; (2) animal studies; (3) studies with less than 10 participants.
Two independent researchers revised the included studies, and all potential disputes that could have arisen were evaluated with the corresponding author.
The summary estimates were generated using a fixed-effect model (Mantel-Haenszel method) [27] or a random-effect model (DerSimonian-Laird method) [28] depending on the absence or presence of heterogeneity (I 2 ). A subgroup analysis was performed to highlight any differences between studies in terms of Overall Survival (OS), Disease-Free Survival (DFS), Progression-Free Survival (PFS), as summarized in Table 1.
When we used the keywords "p53 antibodies in early cancer", p53 antibodies in metastatic cancer", "p53 antibodies impact on cancer progression", the PubMed search yielded 1375 potentially relevant articles. Studies as duplicates, animal studies, cellular studies, or letters to the editor or reviews were excluded. After viewing the titles and abstracts, the full texts of 34 studies were retrieved and 12 studies [10][11][12][13][14][15][16][17][18][19][20][21] were included in the analysis because they had the hazard ratio available for survivals (Tables 1 and 2) as summarized in the flow chart of Figure 1.

Conclusions
We observed that serum antibodies generated in the blood of cancer patients against p53 (and mostly p53-wt) were deleterious. Given the straightforward detection in blood of p53 antibodies as a biomarker for cancer survival, as summarized in a simple workflow in Figure 4, these antibodies, together with other biomarkers, potentially constitute a valid method for prediction of cancer patients' survival outcomes. The correlation could also play an important role for targeted therapies involving a cancer-suppressing p53 pathway.

Competing Interests
The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in, or financial conflict with, the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
Author Contributions: G.R. and N.S. conceived, designed and planned the study. G.R. and N.S. acquired data and produced original drafts and figures. G.R. conducted statistical analysis of the data. G.R. and N.S. drafted the manuscript. P.K.N., R.R., D.G. and A.D. revised and improved the manuscript's content and visualization. All authors helped interpret the results and draft the manuscript. All authors revised and reviewed this article. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable for studies directly not involving humans or animals. Also as to ethics approval, the article does not contain any direct studies with human participants or animals performed by any of the authors.

Informed Consent Statement:
Not applicable for studies directly not involving humans.
Data Availability Statement: All relevant data are within the paper.