Value of TP53 Status for Predicting Response to Neoadjuvant Chemotherapy in Breast Cancer: A Meta-Analysis

Background Numerous studies have yielded inconclusive results regarding the relationship between tumor suppressor protein TP53 overexpression and/or TP53 gene mutations and the response to neoadjuvant chemotherapy in patients with breast cancer. The purpose of the current study was therefore to evaluate the relationship between TP53 status and response to chemotherapy in breast cancer. Methods and Findings A total of 26 previously published eligible studies including 3,476 cases were identified and included in this meta-analysis. TP53 status (over expression of TP53 protein and/or TP53 gene mutations) was associated with good response in breast cancer patients who received neoadjuvant chemotherapy (total objective response: risk ratio [RR]  = 1.20, 95% confidence interval [CI]  = 1.09–1.33, p<0.001; pathological objective response: RR = 1.37, 95% CI = 1.20–1.57, p<0.01; total complete response: RR = 1.33, 95% CI = 1.15–1.53, p<0.001; pathological complete response: RR = 1.45, 95% CI = 1.25–1.68, p<0.001). In further stratified analyses, this association also existed among the studies using anthracycline-based neoadjuvant chemotherapy, and the association between response and the presence of gene alterations was stronger than that between response and immunohistochemistry positivity. Conclusion The results of the present meta-analysis suggest that TP53 status is a predictive factor for response in breast cancer patients undergoing neoadjuvant chemotherapy. Further larger and well-designed prospective studies are required to evaluate the predictive role of TP53 status in clinical practice.


Introduction
Neoadjuvant chemotherapy, also known as primary or induction chemotherapy, refers to chemotherapy administered before locoregional treatment, such as surgery and/or irradiation. Neoadjuvant chemotherapy has become the standard treatment for the management of locally advanced breast cancer, primarily because of its ability to downsize large tumors. Neoadjuvant chemotherapy is increasingly used for the treatment of early-stage breast cancer. However, despite generally high response rates, a small proportion of patients fail to respond to neoadjuvant chemotherapy, or even progress during therapy. Recent evidence suggests that biological markers may be useful for identifying those patients who would benefit from neoadjuvant chemotherapy [1].
The TP53 gene is a prime candidate for predicting the response of tumors to classic chemotherapy [2]. It is a master gene in the stress response that plays a critical role in cancer development. TP53 is the most frequently mutated gene in human cancer, with mutations occurring in at least 50% of human cancers [1]. It mediates checkpoint or stress responses to several insults and suppresses tumor formation through several mechanisms, including apoptosis, senescence, and autophagy [3]. Experimental evidence suggests a key role for TP53 in apoptosis in response to genotoxic agents [4,5].
The use of TP53 status as a biological marker to predict the response of breast cancer to neoadjuvant chemotherapy, however, is disappointing, and the findings to date have shown conficting results [6][7][8][9][10]. Several studies [6,[9][10][11] found that patients with TP53 mutations often had better responses to therapy than those with normal TP53 status. Other studies [7,8,12,13], however, evaluated TP53 status in breast cancer patients and drew different conclusions. The relevance of this gene to clinical therapy thus remains unknown. We therefore performed a meta-analysis of the value of TP53 status for predicting response to neoadjuvant chemotherapy in breast cancer.

Publication Search
PubMed, Embase, and Web of Science databases were searched (up to December 20, 2011) using the search terms: 'TP53', 'p53', 'p53 protein', 'p53 mutation', '17p13 gene', 'chemotherapy' and 'breast cancer'. All potentially eligible studies were retrieved and their bibliographies were carefully scanned to identify other eligible studies. Additional studies were identified by a hand search of the references cited in the original studies. When multiple studies of the same patient population were identified, we included the published report with the largest sample size. Only studies published in English were included in this meta-analysis.

Inclusion and Exclusion Criteria
Studies included in this meta-analysis had to meet all of the following criteria: (a) evaluation of TP53 status for predicting the response to neoadjuvant chemotherapy in early-stage breast cancer, locally-advanced breast cancer, (b) described therapeutic response, (c) retrospective or prospective cohort study, (d) inclusion of sufficient data to allow the estimation of a risk ratio (RR) with 95% confidence intervals (95% CI), and (e) studies published in English. Letters to the editor, reviews, and articles published in books, or papers published in a language other than English were excluded.

Data Extraction and Definitions
According to the inclusion criteria listed above, the following data were extracted for each study: the first author's surname, publication year, country of origin, number of patients analyzed, types of measurement, and treatment. Data on the main outcomes were entered in tables showing the clinical and pathological responses to chemotherapy with respect to TP53 status. Information was carefully and independently extracted from all eligible publications by two of the authors (Chen and Zhu). Any disagreement between the researchers was resolved by discussions until a consensus was reached. If they failed to reach a consensus, a third investigator (Lu) was consulted to resolve the dispute.
We used the definitions and standardizations for 'TP53' and 'response to chemotherapy' as reported by Pakos et al. [14]. For consistency, we used 'TP53' to denote the gene, 'TP53' for the expressed protein, and 'TP53 status' to refer to both the gene and protein markers. The correlation between protein and gene detection is not straightforward [9,15]. TP53 alterations increase the half-life of the TP53 protein, leading to nuclear accumulation of mutant TP53, which can be detected by immunohistochemistry (IHC). However TP53 protein accumulation measured by IHC does not necessarily correspond to TP53 mutations. Thus, the overall analysis considered all studies, regardless of whether protein expression or gene mutation was being evaluated. Separate analyses for TP53 protein expression and TP53 gene alterations were also performed. For studies using both protein and gene detection, we used the protein data but also examined the gene detection data, and found similar results (data not shown). TP53 status positive means patients with over expression of TP53 protein and/or TP53 gene mutations. Response was defined as complete response (CR), partial response (PR), or objective response (OR) (OR = CR +PR). Non-response was defined as stable disease (SD) or progressive disease (PD), according to WHO criteria [16] or RECIST (Response Evaluation Criteria in Solid Tumors) criteria [17].

Statistical Analysis
RR with 95% CIs was used to estimate the association between TP53 status and response to neoadjuvant chemotherapy in breast cancer patients. Subgroup analyses were performed to evaluate the effects of treatment regimens (anthracycline-based) and different methods of TP53 gene determination (protein and gene). Heterogeneity assumption was checked using the Q test, and a p value .0.10 indicated a lack of heterogeneity among studies. The pooled RR was calculated using a fixed-effects model (the Mantel-Haenszel method) or a random-effects model (the DerSimonian and Laird method), according to the heterogeneity. Funnel plots and the Egger's test were employed to estimate the possible publication bias. We also performed sensitivity analysis by omitting each study or specific studies to find potential outliers. Statistical analyses were conducted using Stata (version SE/10; StataCorp, College Station, TX). p values for all comparisons were two-tailed and statistical significance was defined as p,0.05 for all tests, except those for heterogeneity.

Correlation of TP53 Status with Response to Neoadjuvant Chemotherapy in Breast Cancer Patients
Among the studies of breast cancer patients who received neoadjuvant therapy, 26 studies involving 3,476 patients contributed data on total OR (clinical OR + pathological OR). TP53 status-positivity was significantly associated with improved total OR among patients treated with neoadjuvant therapy (RR = 1.20; 95% CI = 1.09-1.33; p,0.001, Figure S2). Thirteen studies involving 2,761 patients contributed data on pathological OR. TP53 status-positivity was significantly associated with improved pathological response (RR = 1.37; 95% CI = 1.20-1.57; p,0.001). Fifteen studies involving 2,736 patients contributed data on total CR. TP53 status-positivity was significantly associated with improved total CR (RR = 1.33; 95% CI = 1.15-1.53; p,0.001). Finally, 12 studies involving 2,434 patients provided information on pathological CR. TP53 status-positivity was significantly associated with significant improvements in pathological CR (RR = 1.45; 95% CI = 1.25-1.68; p,0.001, Figure. S3). For studies using both clinical and pathological responses, we used the pathological-response data, but also examined the clinicalresponse data and found similar results (data not shown).
Different measurements of TP53 status (either by protein or gene detection) have been used to evaluate associations with favorable responses to neoadjuvant chemotherapy. We therefore calculated the associations using both protein and gene statuses of TP53. The results of subgroup analysis are presented in Table 2. For gene detection, TP53 status-positivity was significantly associated with increased total OR (RR = 1.41, 95% CI = 1.20-1.65, p,0.001, Figure S5

Publication Bias
Begg's funnel plot and Egger's test were used to estimate the publication bias of the included literature. The shapes of the funnel plots showed no evidence of obvious asymmetry( Figure S6), and Egger's test indicated the absence of publication bias (p.0.05). Moreover, sensitivity analysis was carried out to assess the influence of individual studies on the summary effect. Trastuzumab would likely have increased the chances of response when combined with a taxane in two of the studies which included patients with HER-2 positive disease, there may be some discordance in response rates in the newer studies compared to the older studies prior to the advent of trastuzumab, this may have falsely credited the anthracycline for the benefit seen and introduced confounding. However, the corresponding pooled RRs were not substantially altered whether or not these studies were included. No individual study dominated this meta-analysis, and the removal of any single study had no significant effect on the overall results (total OR: RR ranged from 1.

Discussion
TP53 status had been shown to play a pivotal role in the response to a large panel of anticancer drugs. Previous studies suggested that breast cancers with TP53 mutations might be either resistant or sensitive to anticancer drugs. However, the issue could not be resolved, because most of the available clinical reports involved small sample sizes, and the results were therefore unable to determine the value of TP53 status for predicting the response to chemotherapy. Additionally, IHC, which lacks sensitivity and specificity, or various DNA sequencing techniques, some of which also lack sensitivity, were the main techniques used in these studies. We therefore concluded that a meta-analysis was the best way of evaluating the association between TP53 status and response to neoadjuvant chemotherapy in a large population.
The current meta-analysis of 26 studies systematically evaluated the association between TP53 status and response to neoadjuvant chemotherapy in a large population. The results indicate that altered TP53 status may predict good response rates to neoadjuvant chemotherapy in patients with breast cancer. TP53 status was associated with total and pathologically relevant increases in OR and CR. Stratification according to different treatments showed that altered TP53 status was significantly associated with increased OR and CR in patients who received anthracycline-based neoadjuvant chemotherapy. Further stratification by gene detection revealed imprecise results, but amplification of the TP53 gene was also associated with relevant increases in OR and CR (both total and pathological); however, although overexpression of TP53 was associated with relevant increases in pathological OR and CR, it was not associated with total OR and CR. Gene detection was associated with advantages regarding response rates to neoadjuvant chemotherapy in patients with breast cancer. Gene detection may thus be a useful approach in future prospective studies.
Despite our attempts to perform a comprehensive analysis, there were some limitations associated with this meta-analysis. First, the meta-analysis may have been influenced by publication bias, we limited the search to studies performed in English, and we did not search conference proceedings and abstract books, which may have introduced publication bias to meta-analysis. We tried to identify all relevant data and retrieve additional unpublished information, some missing data were unavoidable. Second, the studies used different measurements of TP53 status (either protein or gene detection), and the cut-off values for TP53 for overexpression by IHC and for gene amplification differed between studies. Standardization is therefore of great importance for obtaining an accurate assessment of the clinical significance of TP53 status. Although we made considerable efforts to standardize definitions, some variability in definitions of methods, measurements, and outcomes among studies was inevitable. Third, our analysis was observational in nature, and we therefore cannot exclude confounding as a potential explanation of the observed results. Despite these limitations, this meta-analysis had several strengths. First, a substantial number of cases were pooled from different studies, and 3,476 subjects represent a sizeable number, significantly increasing the statistical power of the analysis. Secondly, no publication biases were detected, indicating that the pooled results may be unbiased.
This study is the first meta-analysis to assess the usefulness of TP53 status for predicting the response of breast cancer patients to neoadjuvant chemotherapy. Our data support TP53 status as a useful predictive factor for assessing treatment response to neoadjuvant chemotherapy in breast cancer patients. However, future prospective studies with large sample sizes and better study designs are required to confirm our findings. Moreover, the interactions of this marker with other molecular markers such as HER-2 [34] or estrogen receptor [35] remain unknown, and should be topics for further investigation.