Annals of Oncology Advance Access published online on March 5, 2008
Annals of Oncology, doi:10.1093/annonc/mdn039
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
p53 status and efficacy of primary anthracyclines/alkylating agent-based regimen according to breast cancer molecular classes
,*
1 Department of Pathology, Institut Gustave Roussy, Villejuif
2 Department of Medical Oncology
3 Department of Pathology, Centre Jean Perrin, Clermont-Ferrand
4 Department of Medical Oncology and Translational Research Unit, UPRES03535, Université Paris Sud, Institut Gustave Roussy, Villejuif, France
* Correspondence to: Dr F. Andre, Department of Medical Oncology, Institut Gustave Roussy, 94805 Villejuif, France. Tel: +33142-114-371; Fax: +33142115274; E-mail: fandre{at}igr.fr
 |
Abstract |
|---|
 Top Abstract backgroundpatients and methodsresultsdiscussionfundingAcknowledgementsReferences |
|---|
Background: We hypothesized that, among molecular subclasses of breast cancer, p53 status may have a differential predictive value for the efficacy of anthracyclines/alkylating agents-based regimen. We analysed the efficacy of a preoperative combination between 5-fluorouracil, anthracyclines and cyclophosphamide according to both p53 status and molecular classification.
Patients and methods: Oestrogen receptor (ER), progesterone receptor (PgR), human epidermal growth factor receptor 2 (HER2) expression and p53 status were determined by immunohistochemistry in 293 samples from two different centres. A logistic regression model was used for multivariate analysis of predictors for pathological complete response (pCR).
Results: p53 immunostaining (54%) was associated with high grade (P = 0.002) and ER negativity (P = 0.04). p53 was detected in 59% of triple-negative tumours (ER–/PgR–/HER2–, n = 120 patients). In the overall population, pCR (9.6%) was independently predicted by high tumour grade (P = 0.002) and ER/PgR/HER2 triple negativity (P = 0.0004), but not by p53 status (P = 0.12). p53 immunostaining was associated with a trend for a higher rate of pCR in triple-negative tumours [relative risk (RR) = 2.5, 95% confidence interval (CI) = 0.8–7.5, P = 0.09], but not in non-triple-negative tumours (RR = 0.73, 95% CI = 0.16–3.3, P = 0.69).
Conclusion: p53 status may have a different predictive value for efficacy of anthracycline/alkylating agents-based regimen in each molecular subclass, a result which may explain the different results reported in literature.
anthracycline, p53, primary chemotherapy, triple-negative breast cancer
|
background |
|---|
|
TopAbstractbackgroundpatients and methodsresultsdiscussionfundingAcknowledgementsReferences |
|---|
Combination regimens which include anthracyclines (epirubicin, doxorubicin) and alkylating agents (cyclophosphamide) administered in an adjuvant setting have been shown to improve overall survival in patients with early breast cancer [1]. While effective, this treatment presents some long-term haematological and cardiac side-effects [1]. Predictive factors of response would help to assess the expected individual benefit of this treatment. p53 has been one of the mostly investigated predictive biomarker for the efficacy of anthracyclines-containing regimen in the last two decades [2–5]. Nevertheless, studies did not provide consistent results to conclude about the predictive value of p53 for the efficacy of chemotherapy in breast cancer. There is no unique explanation to date to account for such high inconsistencies across studies.
p53, the product of the TP53 tumour suppressor gene, is a nuclear protein and, when activated, is responsible for the cellular response to DNA damages: cell cycle arrest, DNA repair and/or apoptosis triggering [6]. DNA-damaging drugs function in a p53-dependent manner and p53 disruption makes numerous human cancer cells more sensitive to apoptosis induced by adriamycin in vitro [7, 8]. Inactivation of p53 function is more likely to occur in tumours with BRCA1 mutations that exhibit a basal-like phenotype [9–11]. Interestingly, anthracycline-induced apoptosis has been reported to be p53 mediated in luminal, but not in basal-like tumours [12]. When associated with BRCA1 mutations, p53 mutations were more likely to predict high sensitivity to anthracyclines in vitro [13]. In addition, it must be emphasized that BRCA1-mutated tumours have been reported to be highly sensitive to alkylating agents [14]. Altogether, these data indicate that the predictive value of p53 for the efficacy of anthracycline/cyclophosphamide regimen in breast cancer might differ according to the presence of BRCA1 mutations.
Breast cancers have been recently classified in genetically homogeneous subgroups that are thought to derive from different progenitors [15]. Particularly, basal-like breast cancers refer to a molecular subgroup that does not express oestrogen receptor (ER), progesterone receptor (PgR) and human epidermal growth factor receptor 2 (HER2), and does express cytokeratin 5/6 or epidermal growth factor receptor [16]. This subgroup roughly corresponds to clinically described triple-negative tumours that exhibit a negative immunostaining for ER, PgR and HER2 and a higher chemosensitivity [17]. Interestingly, this latter molecular group is more likely to carry BRCA1 mutations, together with TP53 gene mutations.
We previously hypothesized that the predictive value of a single biomarker could rely on the genetic background on the tumour and that different breast cancers subgroups may have different predictive markers of response to chemotherapy [12, 18]. In the present study, we have evaluated the predictive value of p53 expression for the efficacy of a preoperative 5-fuorouracil, epirubicin and cyclophosphamide (FEC) or 5-fuorouracil, doxorubicin and cyclophosphamide (FAC) regimen according to the molecular classes defined using ER, PgR and HER2 expressions.
|
patients and methods |
|---|
|
TopAbstractbackgroundpatients and methodsresultsdiscussionfundingAcknowledgementsReferences |
|---|
patients and treatment
Patients were retrospectively selected to have presented a breast adenocarcinoma treated with a preoperative chemotherapy that combined FEC or FAC regimen, intravenously every 3 weeks for 4–6 cycles in two French Cancer Centres (Centre Jean Perrin, Clermont Ferrand, and Institut Gustave Roussy, Villejuif). In addition, immunohistochemical determination of p53, ER, PgR and HER2 status of the primary tumour were required to include the patients in the present analysis.
histopathological analyses
Immunostaining has been carried out with automated immunostainers (ventana Benchmark). Tumour grade was defined on tumour biopsy (core needle biopsy) before chemotherapy. ER, PgR and HER2 expression were routinely determined by immunohistochemistry according to local guidelines or, when unavailable, on residual cancer tissue after mastectomy (n = 16 patients). Tumours were classified as ER or PgR positive if they exhibited at least 10% of stained cancer cells. The HercepTest scoring method was used for the determination of c-erbB-2 status (0, 1+: negative; 2+, 3+: positive [19]). Evaluation of TP53 accumulation was done with the DO7 clone (Novocastra, UK; dilution 1 : 250 at the Institut Gustave Roussy [20], and 1 : 200 at the Centre Jean Perrin). This antibody has been previously reported to detect most of the p53 point mutations [21–23]. Tumours were classified as p53 positive if they exhibited at least 1% of stained cells. Tumour response to chemotherapy was classified by histopathologic analysis according to Chevallier's classification. In our study, pathological complete responses (pCRs) were defined as Chevallier level 1 (complete disappearance of any tumour cell in the breast or axilla) or level 2 (persistence of isolated in situ carcinoma) [24].
statistical analysis
For statistical analysis, differences in proportions were evaluated by the chi-square test or Fisher's exact test. We used a logistic regression model to approximate the risk ratio of achieving a pCR first in univariate analyses and then in multiple regression analysis according to baseline factors (clinical tumour size before chemotherapy (cT), tumour grade, ER, PgR, HER2 and p53 expression).
|
results |
|---|
|
TopAbstractbackgroundpatients and methodsresultsdiscussionfundingAcknowledgementsReferences |
|---|
patients and tumours characteristics
Overall, 293 breast cancer patients (median age 51 years) met the inclusion criteria and have been analysed. Primary chemotherapy consisted in 4–6 cycles of FEC100 regimen (5-fuorouracil 500 mg/m2, epirubicin 100 mg/m2, cyclophosphamide 500 mg/m2 on day 1, every 21 days) or FAC60 (5-fuorouracil 500 mg/m2, doxorubicin 60 mg/m2, cyclophosphamide 500 mg/m2) for 273 patients. Reduced doses of epirubicin, 50 or 75 mg/m2, were administered to 14 and 6 patients, respectively. After chemotherapy, all patients underwent breast surgery. Patient characteristics are reported in Table 1. Briefly, 184 patients (63%) had a clinical T2 tumour and 100 patients (34%) had a grade III tumour. A triple-negative phenotype was found in 120 tumours (41%). It must be pointed out that this rate is higher than expected due to the fact that (i) patients were retrospectively selected to have received a preoperative chemotherapy and (ii) the p53 staining was carried out in only 173 non-triple-negative cases since the goal was to compare predictive value of p53 staining according to molecular classes. A p53-positive immunohistostaining was detected in 158 tumours (54%) and was associated with high tumour grade (P = 0.002, Table 1) and ER negativity (P = 0.04). As p53 staining was highly heterogeneous within tumoural samples, no other cut-off has been studied.
|
predictive parameters for pCR
From 1987 to 1995, 293 patients had their needle biopsy studied. pCR occurred in 28 of the 293 cases (9.6%). The pCR rates according to clinicopathological characteristics are reported in Table 2. High grade [relative risk (RR) = 3.8, 95% confidence interval (CI) = 1.6–8.9, P = 0.002], ER negativity (RR = 3.4, 95% CI = 1.4–8.2, P = 0.007), PgR negativity (RR = 4.0, 95% CI = 1.3–11.9, P = 0.02) and triple negativity (RR = 5.0, 95% CI = 2.0–12.2, P = 0.0004) were good predictors of pCR achievement in the univariate analysis (Table 2). p53-positive immunostaining was not associated with a significantly higher rate of pCR (RR = 1.9, 95% CI = 0.8–4.3, P = 0.12). In the multivariate analysis, high grade (RR = 2.6, 95% CI = 1.1––6.2, P = 0.04) and triple-negative phenotype (RR = 3.9, 95% CI = 1.4–10.4, P = 0.007) were statistically associated with pCR.
|
predictive value of p53 immunostaining according to molecular classes
We next evaluated the pCR rates according to p53 immunostainings and molecular classes (Table 3). Interestingly, the association between p53-positive immunostaining in triple-negative tumours and pCR was almost significant (RR = 2.5, 95% CI = 0.9–7.5, P = 0.08), where the pCR proportions were 22% and 10% in p53+ and p53– triple-negative tumours, respectively. At the opposite, pCR proportions were 3% and 4% in patients with p53+ and p53– non-triple-negative tumours, respectively (RR = 0.7, 95% CI = 0.2–3.3, P = 0.69). p53+/triple-negative tumours exhibited a 5.1-fold increase in the probability of pCR as compared with other phenotypes (95% CI = 2.2–11.3, P < 0.001). There was a trend for an interaction between p53 and triple-negative status (ratio for interaction: 3.5, P = 0.19).
|
|
discussion |
|---|
|
TopAbstractbackgroundpatients and methodsresultsdiscussionfundingAcknowledgementsReferences |
|---|
In the present study, we have reported that the predictive value of p53 immunostaining for the efficacy of primary FEC regimen may differ according to molecular subclasses. p53+/triple-negative tumours exhibited a higher rate of pCR (22%) as compared with both p53–/triple-negative (10%) and non-triple-negative tumours (4%). As previously stated, the predictive value of p53 for the efficacy of chemotherapy is a matter of controversies. One of the main explanation of the discrepancies reported in results among studies is the various methods used to assess the p53 status. Interestingly, studies which aimed at determining the predictive value of p53 were also very heterogeneous in terms of patient characteristics and drug regimen. As an illustration, in the study reported by Geisler et al. [25] most patients (72%) had low- or intermediate-grade tumours which are more likely to belong to luminal-A/BRCA1-nonmutated tumours; this study concluded that TP53 mutations were associated with primary resistance to neoadjuvant doxorubicin therapy. At the opposite, in the study reported by Bertheau et al. [5], most patients had ER-negative and high-grade tumours, which are more likely to be basal like/BRCA1 mutated. Unsurprisingly, TP53 status was reported to be a predictive factor for response to the dose-dense neoadjuvant epirubicin–cyclophosphamide regimen used in this study. Although our present study is underpowered due to the small number of patients, we would speculate that p53 mutations were associated with resistance in the first study and associated with sensitivity to anthracycline-based chemotherapy in the second one. These data together with the present analysis would indicate that the heterogeneity of breast cancer molecular subclasses among studies could account for the heterogeneity of results when the predictive value of a single biomarker is investigated.
It must be pointed out that studies which previously investigated the predictive value of p53 mutations/expressions were heterogeneous not only in terms of molecular classes but also regarding the use of alkylating agents. Interestingly, it has been indicated that the BRCA1–/p53– phenotype is associated with a high sensitivity to alkylating agents [14]. On the basis of this background, since somatic p53 mutations are more frequent in BRCA1-mutated tumours, one might expect that p53 mutations may be associated with a higher sensitivity to regimen that includes high dose of alkylating agents. As an illustration, the team that reported that p53 mutations were associated with a higher rate of pCR used high doses of cyclophosphamide in combination with anthracyclines [26]. On the opposite, studies that reported a correlation between p53 mutations and resistance to anthracyclines-based chemotherapy did not include alkylating agents in their regimen [3, 25]. In the present study, it was not possible to assess the role of alkylating agents on the p53 predictive value. Nevertheless, patients received 500 mg/m2 cyclophosphamide per cycle. Using this dosage, we found a trend for a higher rate of pCR in p53-positive tumours.
Overall, our study and previous reports indicate that the predictive role of p53 is a complex interplay between genetic background (BRCA1 mutations), molecular classification and the use of alkylating agents. This indicates that the predictive value of p53 status should be determined in more homogenous molecular classes using homogenous drug regimen. Nevertheless, the sample size from our study was not large enough to detect a possible interaction between molecular classes and p53 staining. This result may be also due to discrepancies previously described between p53 function, p53 accumulation and TP53 sequencing [8, 21–23]. Beyond p53 status, the DNA damage signalling may be also disrupted by p63, p73 or ATM (product of the ataxia-telangiectasia gene) dysfunction in the triple-negative subgroup [27, 28].
Interestingly, p53 is not the only biomarker to exhibit a class-specific predictive value for chemotherapy efficacy. TOP2A gene amplification has also been described to occur almost exclusively in HER2-overexpressing tumours and to be predictive for the efficacy of anthracyclines within this molecular class [29]. In addition, microtubule-associated protein Tau (MAP-Tau), a protein which mediates paclitaxel resistance, is low in almost all ER-negative tumours, but heterogeneous in ER-positive tumours, and may present a clinical usefulness in this latter molecular group [30]. Overall, the present study indicates that a single biomarker may exhibit a differential predictive value according to molecular classes. While on the basis of a limited number of cases, this observation, which did not reached the statistical significance threshold (P = 0.08), indicates that further investigations on predictive value of drug-specific biomarkers should be carried out in homogenous molecular classes that can be defined on the basis of immunohistochemistry [31, 32]. Finally, larger studies are therefore needed to validate our hypothesis of differential predictive value of p53 by interaction tests.
|
funding |
|---|
|
TopAbstractbackgroundpatients and methodsresultsdiscussionfundingAcknowledgementsReferences |
|---|
Centre Jean Perrin and Institut Gustave Roussy.
|
Acknowledgements |
|---|
|
TopAbstractbackgroundpatients and methodsresultsdiscussionfundingAcknowledgementsReferences |
|---|
Conflict of interest: none.
|
Footnotes |
|---|
These two authors equally contributed to this work as senior author. 
Received for publication December 4, 2007. Revision received January 22, 2008. Accepted for publication January 24, 2008.
|
References |
|---|
|
TopAbstractbackgroundpatients and methodsresultsdiscussionfundingAcknowledgementsReferences |
|---|
1. Early Breast Cancer Trialists’ Collaborative Group (EBCTCG). Effects of chemotherapy and hormonal therapy for early breast cancer on recurrence and 15-year survival: an overview of the randomised trials. Lancet (2005) 365:1687–1717.[CrossRef][Web of Science][Medline]
2. Rozan S, Vincent-Salomon A, Zafrani B, et al. No significant predictive value of c-erbB-2 or p53 expression regarding sensitivity to primary chemotherapy or radiotherapy in breast cancer. Int J Cancer (1998) 79:27–33.[CrossRef][Web of Science][Medline]
3. Bottini A, Berruti A, Bersiga A, et al. p53 but not bcl-2 immunostaining is predictive of poor clinical complete response to primary chemotherapy in breast cancer patients. Clin Cancer Res (2000) 6:2751–2758.
4. Berns EM, Foekens JA, Vossen R, et al. Complete sequencing of TP53 predicts poor response to systemic therapy of advanced breast cancer. Cancer Res (2000) 60:2155–2162.
5. Bertheau P, Turpin E, Rickman DS, et al. Exquisite sensitivity of TP53 mutant and basal breast cancers to a dose-dense epirubicin-cyclophosphamide regimen. PLoS Med (2007) 4:e90.[CrossRef][Medline]
6. Vousden KH, Lu X. Live or let die: the cell's response to p53. Nat Rev Cancer (2002) 2:594–604.[CrossRef][Web of Science][Medline]
7. Bunz F, Hwang PM, Torrance C, et al. Disruption of p53 in human cancer cells alters the responses to therapeutic agents. J Clin Invest (1999) 104:263–269.[Web of Science][Medline]
8. O'Connor PM, Jackman J, Bae I, et al. Characterization of the p53 tumor suppressor pathway in cell lines of the National Cancer Institute anticancer drug screen and correlations with the growth-inhibitory potency of 123 anticancer agents. Cancer Res (1997) 57:4285–4300.
9. Liu X, Holstege H, van der Gulden H, et al. Somatic loss of BRCA1 and p53 in mice induces mammary tumors with features of human BRCA1-mutated basal-like breast cancer. Proc Natl Acad Sci USA (2007) 104:12111–12116.
10. Foulkes WD, Brunet JS, Stefansson IM, et al. The prognostic implication of the basal-like (cyclin E high/p27 low/p53+/glomeruloid-microvascular-proliferation+) phenotype of BRCA1-related breast cancer. Cancer Res (2004) 64:830–835.
11. Troester MA, Herschkowitz JI, Oh DS, et al. Gene expression patterns associated with p53 status in breast cancer. BMC Cancer (2006) 6:276.[CrossRef][Medline]
12. Andre F, Pusztai L. Heterogeneity of breast cancer among patients and implications for patient selection for adjuvant chemotherapy. Pharm Res (2006) 23:1951–1958.[CrossRef][Web of Science][Medline]
13. Fedier A, Moawad A, Haller U, et al. p53-deficient cells display increased sensitivity to anthracyclines after loss of the catalytic subunit of the DNA-dependent protein kinase. Int J Oncol (2003) 23:1431–1437.[Web of Science][Medline]
14. Garber JE, Richardson A, Harris LN, et al. Neo-adjuvant cisplatin in triple-negative breast cancer (2006) San Antonio Breast Cancer Symposium Poster No. 3074.
15. Sorlie T, Perou CM, Tibshirani R, et al. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci USA (2001) 98:10869–10874.
16. Nielsen TO, Hsu FD, Jensen K, et al. Immunohistochemical and clinical characterization of the basal-like subtype of invasive breast carcinoma. Clin Cancer Res (2004) 10:5367–5374.
17. Rouzier R, Perou CM, Symmans WF, et al. Breast cancer molecular subtypes respond differently to preoperative chemotherapy. Clin Cancer Res (2005) 11:5678–5685.
18. Andre F, Pusztai L. Molecular classification of breast cancer: implications for selection of adjuvant chemotherapy. Nat Clin Pract Oncol (2006) 3:621–632.[CrossRef][Web of Science][Medline]
19. Jacobs TW, Gown AM, Yaziji H, et al. Specificity of HercepTest in determining HER-2/neu status of breast cancers using the United States Food and Drug Administration-approved scoring system. J Clin Oncol (1999) 17:1983–1987.
20. Mathieu MC, Rouzier R, Llombart-Cussac A, et al. The poor responsiveness of infiltrating lobular breast carcinomas to primary chemotherapy can be explained by their biological profile. Eur J Cancer (2004) 40:342–351.[CrossRef][Web of Science][Medline]
21. Bonsing BA, Corver WE, Gorsira MC, et al. Specificity of seven monoclonal antibodies against p53 evaluated with western blot, immunohistochemistry, confocal laser scanning microscopy, and flow cytometry. Cytometry (1997) 28:11–24.[CrossRef][Web of Science][Medline]
22. Sjogren S, Inganas M, Norberg T, et al. The p53 gene in breast cancer: prognostic value of complementary DNA sequencing versus immunohistochemistry. J Natl Cancer Inst (1996) 88:173–182.
23. Jacquemier J, Moles JP, Penault-Llorca F, et al. p53 immunohistochemical analysis in breast cancer with four monoclonal antibodies: comparison of staining and PCR-SSCP results. Br J Cancer (1994) 69:846–852.[Web of Science][Medline]
24. Chevallier B, Roche H, Olivier JP, et al. Inflammatory breast cancer. Pilot study of intensive induction chemotherapy (FEC-HD) results in a high histologic response rate. Am J Clin Oncol (1993) 16:223–228.[Web of Science][Medline]
25. Geisler S, Lønning PE, Aas T, et al. Influence of TP53 gene alterations and c-erbB-2 expression on the response to treatment with doxorubicin in locally advanced breast cancer. Cancer Res (2001) 61:2505–2512.
26. Bertheau P, Plassa F, Espie M, et al. Effect of mutated TP53 on response of advanced breast cancers to high-dose chemotherapy. Lancet (2002) 360:852–854.[CrossRef][Web of Science][Medline]
27. Tommiska J, Bartkova J, Heinonen M, et al. The DNA damage signalling kinase ATM is aberrantly reduced or lost in BRCA1/BRCA2-deficient and ER/PR/ERBB2-triple-negative breast cancer. Oncogene (2007) November 5; [Epub ahead of print].
28. Leong CO, Vidnovic N, DeYoung MP, et al. The p63/p73 network mediates chemosensitivity to cisplatin in a biologically defined subset of primary breast cancers. J Clin Invest (2007) 117:1370–1380.[CrossRef][Web of Science][Medline]
29. Tanner M, Isola J, et al, Scandinavian Breast Group Trial 9401. Topoisomerase IIalpha gene amplification predicts favorable treatment response to tailored and dose-escalated anthracycline-based adjuvant chemotherapy in HER-2/neu-amplified breast cancer: Scandinavian Breast Group Trial 9401. J Clin Oncol (2006) 24:2428–2436.
30. Andre F, Hatzis C, Anderson K, et al. Microtubule-associated protein-tau is a bifunctional predictor of endocrine sensitivity and chemotherapy resistance in estrogen receptor-positive breast cancer. Clin Cancer Res (2007) 13:2061–2067.
31. Conforti R, Boulet T, Tomasic G, et al. Breast cancer molecular subclassification and estrogen receptor expression to predict efficacy of adjuvant anthracyclines-based chemotherapy: a biomarker study from two randomized trials. Ann Oncol (2007) 18:1477–1483.
32. Bidard FC, Conforti R, Boulet T, et al. Does triple-negative phenotype accurately identify basal-like tumour? An immunohistochemical analysis based on 143 ‘triple-negative’ breast cancers. Ann Oncol (2007) 18:1285–1286.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  O. Gluz, C. Liedtke, N. Gottschalk, L. Pusztai, U. Nitz, and N. Harbeck Triple-negative breast cancer--current status and future directions Ann. Onc., December 1, 2009; 20(12): 1913 - 1927. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. T. Ionta, F. Atzori, M. C. Deidda, V. Pusceddu, S. Palmeri, B. Frau, M. Murgia, M. Barca, L. Minerba, and B. Massidda Long-Term Outcomes in Stage IIIB Breast Cancer Patients Who Achieved Less Than a Pathological Complete Response ( Oncologist, November 1, 2009; 14(11): 1051 - 1060. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. J. Chae, J. S. Bae, A. Lee, W. C. Park, Y. J. Seo, B. J. Song, J. S. Kim, and S. S. Jung p53 as a Specific Prognostic Factor in Triple-negative Breast Cancer Jpn. J. Clin. Oncol., April 1, 2009; 39(4): 217 - 224. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||