Prognostic Role of p53 Immunohistochemical Status in Invasive Breast Cancer. A Retrospective Review of 1387 Cases With Luminal-Like/Her2 Negative Breast Tumors

Abstract Purpose The aim of this study was to evaluate the prognostic role of p53 immunohistochemical (IHC) expression in a large cohort of patients with hormone receptors (HR)-positive/Her2-negative primary invasive breast cancer. Methods Retrospective review of consecutive cases treated at our Breast Unit between 2003 and 2013. Patients were divided into 3 subgroups based on p53 IHC expression: null (0%), low (0.1%-49%), and high (≥50%) p53 expression. Results A total of 1387 patients were included in the study with a median follow-up of 86 months. After adjusting for age, size, node status, lymphovascular invasion, progesterone, and Ki67 expression, only null p53 immunophenotype was associated with worse disease-free survival (DFS) (OR 1.74, 95% IC, 1.11-2.71, P = .015) and distant recurrence-free survival (DRFS) (OR 1.73, 95% IC, 1.04-2.90, P = .036). Null p53 impacted significantly DFS and DRFS also in patients with early breast cancer. Conclusions p53 IHC expression affects survival and, thus can be a valuable tool in the management of patients with HR-positive/Her2-negative breast cancer


Introduction
Hormone receptors (HR)-positive/Her2-negative tumors are the most frequent subtype of breast cancer.Despite a widely accepted more favorable outcome, they also include a subgroup of high-risk tumors for which systemic therapy might be beneficial in addition to the endocrine therapy alone.][3][4] The p53 protein is a transcriptional factor codified by the TP53 gene and plays a key role in multiple cellular pathways, either with its nuclear transcriptional activity or through non-transcriptional cytoplasmatic processes.][7][8][9] TP53 mutations are the most frequent genetic alteration in human cancer and occur in 20%-30% of all primary invasive breast cancers.TP53 mutations are observed in 10%-20% of luminal-like tumors, more frequently among luminal B-like tumors. 10,117][18] Consequently, different cutoffs have been proposed to distinguish abnormal p53 expression.10% and 50% are the most frequently reported cutoffs but neither has yet been validated.][21][22][23][24][25][26][27] The aim of the study was to explore the impact of p53 IHC expression on long-term outcome in a large cohort of patients treated for HR-positive/Her2-negative breast.

Materials and Methods
The present study is a retrospective review of 1387 cases with HR-positive/Her2-negative primary breast cancer treated at Breast Unit, Policlinico di Sant'Orsola, Bologna, Italy, between January 2003 and December 2013.was considered positive (LN+) if at least one macrometastasis was observed.The new St.Gallen criteria were used to classify luminal-like tumors.Luminal B-like tumors included neoplasms with either PR expression lower than 20% or Ki67 higher than 25%. 28The expression of p53 was immunohistochemically measured using mouse monoclonal antibody to p53 (Clone DO7; Ventana) and based on the percentage of stained cells all cases were divided into 3 subgroups: null (0%), low (0.1%-49%) and high (50%-100%).Early breast cancer was defined as a tumor of 2 cm or less with negative node status and no distant metastases (T1N0M0).Follow-up was defined as time between surgery and last available follow-up and expressed as the median value.Overall recurrence rate was defined as the portion of patients developing either local or distant recurrences.The primary endpoint was to investigate the prognostic impact of p53 expression on DFS and DRFS.The secondary endpoint was to evaluate such association in patients with early breast cancer.Events for the disease-free survival analysis were defined as the first disease recurrence, either local or distant, for the primary and-point and distant recurrences for the secondary endpoint.Statistical analysis was carried out using the SPSS software (version 20.0, IBM).For comparison among groups, chisquare and Mann-Whitney tests were used as appropriate.Kaplan-Meier survival analyses were conducted to compare survival among groups.The final multivariate Cox regression model was built from the set of variables that reached P < .10 at the univariate analysis by removing predictors based on the P-value in a stepwise manner.The threshold for statistical significance of 2-tailed tests was set at P < .05.

Results
A total of 1387 cases were included in this study, of which 786 (57%) were luminal A-like and 601 (43%) were luminal B-like tumors.Invasive ductal carcinoma was the most common histological type (1144 patients, 82.4%).Luminal B-like tumors were associated with larger size, higher histologic grade and more frequent axillary lymph node involvement, presence of LVI, null p53, or high p53 expression.Null p53 and high p53 expression were observed in 59 (4.3%) and 93 (6.7%) of all cases, respectively.Postsurgical hormonal and chemotherapy (CHT) were prescribed in 92% and 40% of the cases, respectively.CHT was more likely to be administered in patients with luminal B-like tumors.Clinicopathological data are presented in Table 1.
Median follow-up was 86 months.A total of 270 patients developed either local or distant recurrences with an overall recurrence rate of 19.5%.The most frequent sites of distant metastases were the skeletal system (7%), liver (6%), lung (4%), distant lymph node stations (2%), and the central nervous system (1%).Median interval from the time of surgery to the recurrence was 44 months (56 and 35 months for luminal A-and B-like, respectively (P < .001).
Kaplan-Meier curves for DFS and DRFS are shown in Fig. 1.After adjusting for age, size, node status, histologic grade, LVI, PR and Ki67 expression, null p53 resulted an independent and significant factor associated with worse DFS and DRFS (OR 1.74, 95% IC, 1.11-2.71,P = .015and OR 1.73, 95% IC, 1.04-2.90,P = .036,respectively).Univariate and multivariate Cox regression analyses are shown in Table 2. Subsequently, we restricted our analysis to the group of patients with early breast cancer.We identified 732 cases with early breast cancer of which 489 (67%) were luminal A-like and 243 (33%) were B-like tumors.Clinicopathological data for patients with early breast cancer are shown in Table 3. Null p53 was significantly associated to a worse DFS and DRFS (OR 5.46, 95% IC, 2.52-13.24,P < .001and OR 8.48, 95% IC, 2.87-25.01,P < .001respectively).Univariate and multivariate regression analysis are shown in Table 4.
In both the analyses, all cases or early breast cancer alone, high 53 expression did not result an independent prognostic factor in the multivariate analysis.

Discussion
TP53 mutations represent the most frequent genetic abnormality in human cancer.They are observed in 20%-30% of all primary invasive breast cancer and missense mutations account for up to 90% of the cases.Missense mutations generally lead to a dysfunctional mutant p53 protein with an increased stability that tends to accumulate in the nucleus.Therefore, IHC determination of p53 expression has been proposed and used as a surrogate for TP53 status.In the series presented by Schmitt et al and Alsner et al, there was a strong relation between p53 accumulation and TP53 mutations, while Norberg et al reported a sensibility and specificity of 72.2% and 92%, respectively for IHC compared to DNA sequencing.Various authors have provided different cutoffs to define p53 overexpression, but to date no suitable value has been established.The most frequently reported cutoff values are set at either 10% (Millar et al) or 50% (Kikuchi et al).In our cohort, 50% was deemed a reasonable cutoff, as it correlated more strongly to outcome.Null mutations (nonsense and frameshift) are less common and lead to a truncated nonfunctional mutant p53 protein.They are associated with a total absence of immunostaining for p53. 12,29The biological differences and prognostic impact between missense and null mutations are still not clear.1][32][33] In the present study, we consider the surrogates of missense and null mutations separately and propose a new classification based on p53 IHC expression: null (0%), low (0.1%-49%) and high (≥50%).Such classification has not been previously reported in literature to our best knowledge.The Oncologist, 2024, Vol. 29, No. 5 The prognostic impact of p53 status in breast cancer is still not fully clarified.Wild-type TP53 has been correlated to worse outcome in patients receiving chemotherapy, while mutant-p53 has been linked to a negative prognostic impact in patients treated with endocrine therapy alone. 14The role of p53 expression determined by IHC is still controversial.Several authors have reported an association between p53 overexpression and worse outcome in patients with luminal-like breast cancer.Lee et al found such association only among luminal A-like tumors, while in the series presented by Reed et al no significant correlation between p53 accumulation and survival was observed.In our cohort, high p53 expression was associated with shorter DFS and DRFS only in the univariate analysis.On the contrary, null p53 resulted a significant and independent prognostic factor for both DFS and DRFS.Null p53 is more likely to be observed in case of nonsense or frameshift TP53 mutations for which a negative prognostic impact has already been reported.These findings were confirmed also in the analysis of patients with early breast cancer.Due to lack of complete data, we could not analyze the differences among groups based on the type of postsurgical therapy received.
As our findings suggest, patients with null p53 are at higher risk, thus systemic adjuvant therapy might prove beneficial in terms of disease-free survival in these cases, particularly among patients with early breast cancer.As a monocentric study, the strongest point of our work is the homogeneity of the sample, loco-regional treatment, and pathological evaluation, particularly in an era with fewer therapeutic options and when tumor stage played a primary role in treatment decisions rather than the molecular profile.On the other hand, it has the limits of a retrospective study and a limited sample of cases and data on postoperative therapy.Further studies are required to investigate the role of p53 status on survival distinguishing on the type of postsurgical treatment received.

Conclusion
Integrating p53 status determined by IHC with the traditional clinicopathological features in patients with HR-positive/ Her2-negative breast tumors can be a valuable tool in the hands of the clinician in the decision of the most appropriate therapy, particularly in patients with early breast cancer and null p53 expression.

Table 1 .
The study was approved by the local ethics committee (21/2015/O/OssN).Clinicopathological features in all patients.

Table 2 .
Cox proportional hazards models of DFS and DRFS in all patients.

Table 3 .
Clinicopathological features of patients with early breast cancer.
*Data not available for all patients.