Exquisite sensitivity of TP53 mutant and basal breast cancers to a dose-dense epirubicin-cyclophosphamide regimen

Abstract

Background: In breast cancers, only a minority of patients fully benefit from the different chemotherapy regimens currently in use. Identification of markers that could predict the response to a particular regimen would thus be critically important for patient care. In cell lines or animal models, tumor protein p53 (TP53) plays a critical role in modulating the response to genotoxic drugs. TP53 is activated in response to DNA damage and triggers either apoptosis or cell-cycle arrest, which have opposite effects on cell fate. Yet, studies linking TP53 status and chemotherapy response have so far failed to unambiguously establish this paradigm in patients. Breast cancers with a TP53 mutation were repeatedly shown to have a poor outcome, but whether this reflects poor response to treatment or greater intrinsic aggressiveness of the tumor is unknown.

Methods and findings: In this study we analyzed 80 noninflammatory breast cancers treated by frontline (neoadjuvant) chemotherapy. Tumor diagnoses were performed on pretreatment biopsies, and the patients then received six cycles of a dose-dense regimen of 75 mg/m(2) epirubicin and 1,200 mg/m(2) cyclophosphamide, given every 14 days. After completion of chemotherapy, all patients underwent mastectomies, thus allowing for a reliable assessment of chemotherapy response. The pretreatment biopsy samples were used to determine the TP53 status through a highly efficient yeast functional assay and to perform RNA profiling. All 15 complete responses occurred among the 28 TP53-mutant tumors. Furthermore, among the TP53-mutant tumors, nine out of ten of the highly aggressive basal subtypes (defined by basal cytokeratin [KRT] immunohistochemical staining) experienced complete pathological responses, and only TP53 status and basal subtype were independent predictors of a complete response. Expression analysis identified many mutant TP53-associated genes, including CDC20, TTK, CDKN2A, and the stem cell gene PROM1, but failed to identify a transcriptional profile associated with complete responses among TP53 mutant tumors. In patients with unresponsive tumors, mutant TP53 status predicted significantly shorter overall survival. The 15 patients with responsive TP53-mutant tumors, however, had a favorable outcome, suggesting that this chemotherapy regimen can overcome the poor prognosis generally associated with mutant TP53 status.

Conclusions: This study demonstrates that, in noninflammatory breast cancers, TP53 status is a key predictive factor for response to this dose-dense epirubicin-cyclophosphamide regimen and further suggests that the basal subtype is exquisitely sensitive to this association. Given the well-established predictive value of complete responses for long-term survival and the poor prognosis of basal and TP53-mutant tumors treated with other regimens, this chemotherapy could be particularly suited for breast cancer patients with a mutant TP53, particularly those with basal features.

Figure 1. Response to Chemotherapy

(A) Top: distribution of the pathological response to chemotherapy of the 80 patients according to TP53 status as well as other clinical or biological annotations. T, T stage from the TNM classification. p-Values are below each contingency table. Bottom: results from adjusted Chi² tests. The variables tested for the prediction of complete response are shown in the columns; the stratification variable is listed in the first column. The p-values are color coded (dark yellow and bold type, p < 0.0001; light yellow, 0.0001 < p < 0.01). (B) Left graph: Kaplan-Meier analysis of the event-free survival of the patients without metastatic extension at diagnosis, all treated with the same regimen (including patient P80, see Table S1). Note that all five relapses in the TP53 mutant group occurred in the first 18 months, while in the TP53 wild-type group, relapses were still observed when survival of many patients was censored. Right graph: overall survival stratified by TP53 status and complete response.

Figure 2. Microarray Data

(A) Hierarchical clustering based on the 990 most varying genes in 37 tumors with Affymetrix-grade RNA. C1, C2, and C3 denote the three tumor clusters. Annotations: TP53 status (red, mutant; yellow, wild-type); ESR1 (immunohistochemistry) (blue, positive; green, negative); basal cytokeratins (KRT5/6 or 17, immunohistochemistry) (orange, positive; gray, negative); ERBB2 (RT-PCR) (pink, positive; gray, negative); complete pathological response to chemotherapy (blue, complete; red, incomplete); and tumor grade (green, grade 3; purple, grade 1 or 2). For chemotherapy response, patients treated with other regimens are indicated by a question mark. P1, P2, etc. refer to the patient's references in Table S1. (B) Genes linked to TP53 status (t-tests): the top and bottom genes (classified by fold changes [FC]) are shown.

Figure 3. Validation of Mutant TP53 Profile in Breast Cancers

For each of the 12 most varying genes (11 overexpressed and one underexpressed), four box plots are shown, representing the distribution of the ddCt values for the four following groups of samples: (i) TP53 mutant from the training set (empty red box), (ii) TP53 wild type from the training set (empty blue box), (iii) TP53 mutant from the validation set (filled red box), and (iv) TP53 wild type from the validation set (filled blue box). *p < 0.05; **p < 0.01, ***p < 0.001 (t-tests). Small circles indicate outliers.