- Email: [email protected]
Survivin As a Predictive Biomarker of Complete Pathologic Response to Neoadjuvant Chemotherapy in Patients With Stage II and Stage III Breast Cancer
Original Study
Survivin As a Predictive Biomarker of Complete Pathologic Response to Neoadjuvant Chemotherapy in Patients With Stage II and Stage III Breast Cancer Cristiane Rios Petrarca,1 Andre T. Brunetto,1 Vinicius Duval,1 Adriana Brondani,2 Gisele Pereira Carvalho,1 and Bernardo Garicochea1 Abstract Introduction: Predictive biomarkers of response to neoadjuvant chemotherapy in patients with breast cancer are needed to better characterize tumors and enable more tailored therapies. Methods: The expression levels of survivin, BCL-2, cyclin D1, ETS1, and PDEF in tumor samples obtained in the diagnostic biopsies of patients undergoing neoadjuvant chemotherapy for stage II and stage III disease were evaluated by immunohistochemistry (IHC). The mean expression score (range, 0-15) obtained by 3 different pathologists was used for analysis and correlated with complete pathologic response (pCR) and survival by standard univariate and multivariate methods. Results: Fortyfive female patients were included in this study and received preoperative standard anthracycline/taxane– based chemotherapy. The median age at diagnosis was 49 years (range, 25-70 years). Three patients (7.1%) achieved pCR. The mean expression score of survivin in the diagnostic biopsies was significantly higher (P ⫽ .01) in patients with pCR (9.3) than in those without (3.4). There was no significant association with pCR for the other biologic markers analyzed nor was there correlation with prognosis. Survivin levels were not associated with age, tumor grade, clinical stage, or receptor status. Conclusion: High expression levels of survivin in the primary tumor may be used as a potential predictive biomarker of pCR to neoadjuvant chemotherapy in patients with stage II and stage III breast cancer. Clinical Breast Cancer, Vol. 11, No. 2, 129-34 © 2011 Elsevier Inc. All rights reserved. Keywords: Breast cancer, Neoadjuvant chemotherapy, Predictive biomarker, Survivin, Complete pathologic response
Introduction Neoadjuvant chemotherapy is accepted as standard of care in locally advanced breast cancer (LABC) in an effort to improve the possibility of breast-conserving surgery and to render inoperable tumors resectable.1,2 It also has the advantage of early initiation of systemic therapy and in vivo assessment of response to treatment. The rate of pCR for preoperative chemotherapy has been confirmed as a strong prognostic marker that is significantly associated with longer progression-free survival (PFS) and overall survival (OS),3,4 1 Department of Oncology and Pathology, Hospital São Lucas, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre-RS, Brazil 2 Department of Pathology, Universidade Luterana do Brasil, Canoas-RS, Brazil
Submitted: Jul 19, 2010; Revised: Sep 24, 2010; Accepted: Oct 26, 2010 Address for correspondence: Bernardo Garicochea MD, PhD, Hospital São Lucas da PUCRS, Av. Ipiranga, 6690/708, 90610 – 000 Porto Alegre, RS, Brasil Fax: 55-51-33203319; e-mail contact: [email protected]
1526-8209/$ - see frontmatter © 2011 Elsevier Inc. All rights reserved. doi: 10.1016/j.clbc.2011.03.002
although some authors argue that this is not applicable to all subtypes of breast cancer.5 Neoadjuvant treatment is the ideal setting to analyze molecular markers that may predict pathologic response to therapy in tumor tissue. In addition, gene expression profiling may provide predictive and prognostic signatures that could help characterize tumors and enable more tailored therapies. New predictive biomarkers may allow different chemotherapy regimens or hormonal or targeted agents to be tested with a streamlined approach and therefore accelerate drug development. This study primarily aims to evaluate 2 groups of genes with distinct biologic activity as potential predictive biomarkers of response to neoadjuvant chemotherapy: survivin (BIRC5) and the ETS (E-twenty six) family, which have been implicated in the development of human cancer. The protein survivin, a member of the inhibitor of apoptosis (IAP) family, has been found in various neoplasms, including breast cancer, but is usually not detected in normal tissue.6 Survivin can participate in several cellular survival functions,
Clinical Breast Cancer April 2011
129
Survivin As a Predictive Biomarker of pCR to Neoadjuvant Chemotherapy including inhibition of apoptosis, cell cycle progression, and DNA repair,7 and there is evidence of an association between expression levels and survival in breast, esophageal, and rectal cancers8-10 The ETS family of proteins are transcriptional factors implicated in cell proliferation and differentiation, apoptosis, and oncogenesis.11,12 ETS1 and PDGF are members of this family and have been associated with invasive breast cancer, hormonal receptor status, and increased metastatic potential in breast carcinomas.11,13,14 There is limited published work looking at the association of these proteins and pathologic response to neoadjuvant chemotherapy; hence our efforts to evaluate these potential predictive markers. In parallel we also reviewed markers of proliferation, cell cycle progression, and apoptosis that have been classically associated with breast carcinogenesis: cyclin D1 and BCL-2.15,16
Methods Patient Inclusion Included in this analysis were consecutive female patients with breast cancer, TNM stages II and III (according to the 6th edition of the American Joint Committee on Cancer [AJCC]) and ages between 18 and 70 years, who were undergoing neoadjuvant chemotherapy with AC-T (doxorubicin [Adriamycin] 60 mg/m2 Day 1 and cyclophosphamide 600 mg/m2 Day1 for 4 cycles of 21 days, followed by docetaxel 75 mg/m2 D 1 for 4 cycles of 21 days) during the period between July 2004 and November 2009 in the Breast Oncology Department at Sao Lucas Hospital-PUC RS . Trastuzumab is not provided by the Brazilian Public Health System; therefore patients with overexpression of ErbB2 did not receive this agent. Exclusion criteria were metastatic or recurrent disease, comorbidities precluding the use of anthracyclines or taxanes, and history of other malignancies (except for basal cell skin carcinoma). Estrogen (ER) and progesterone (PR) status was assessed by standard immunohistochemistry (IHC) tests : nuclear staining in ⱖ 10% was considered positive (according to the indication that a significant difference in 5-year recurrence-free survival between ER-positive (ER⫹) and ER-negative (ER–) patients has been reported for a cutoff of 10%.17 ErbB2 status was analyzed by IHC and graded as 1⫹, 2⫹, or 3⫹ for positivity. ErbB2 was defined as positive when intense circumferential membrane staining was found in ⬎ 10% of the invasive carcinoma (ErbB2 ⫽ 3⫹). ErbB2 status classified as 2⫹ was not further assessed by fluorescence in situ hybridization analysis or polymerase chain reaction (PCR). Pathologic response was independently evaluated after surgical resection of the remaining tumor and nodes. The clinical measurement of the response to neoadjuvant therapy was defined according to the International Union Against Cancer (UICC) criteria.18 pCR was defined as the histologic absence of residual invasive disease in both the breast and the axilla. Informed consent was obtained from all patients before inclusion and data was collected prospectively.
Immunohistochemistry Tissue blocks were trimmed and sectioned with a microtome (Leica SM 2000R, Leica, Nussloch, Germany) to obtain 3-m sections, which were put on pretreated slides with HistoGrip (Invitrogen Corporation Grand Island, New York) at 60°C for 24 hours. The sections were then deparaffinized with xylene and rehydrated in ethanol. The inactivation of endogenous peroxidase occurred through
130
Clinical Breast Cancer April 2011
immersion in hydrogen peroxide and was thereafter put through the antigen retrieval process using the pressure cooker method. Incubation overnight at temperatures between 2° and 6°C was carried out and antibody diluents were then applied with background reducing components (Dako North America Inc., Carpinteria, CA) for the following antibodies and concentrations: survivin, 1:50 (Dako North America Inc.), BCL2, 1:200 (Invitrogen), cyclin D1, 1:50 (Dako North America Inc.), ETS1, 1:50 (Novocastra Laboratories Ltd., Newcastle Upon Tyne, UK), and PDEF, 1:25 (Santa Cruz Biotechnology, Santa Cruz, CA). Each marker was tested for external controls. The picture max system (HRP Polymer Conjugate Broad Spectrum, Invitrogen,) was used to amplify the antibody-antigen reaction, after which slides were incubated in a solution of diaminobenzidine and stained in hematoxylin. Finally slides were dehydrated in absolute ethyl alcohol and subsequently mounted with the synthetic medium Entellan (Merck, KGaA, Darmstadt, Germany). Images were then captured with a digital camera (Sony Corp., Tokyo, Japan) (magnification, 200⫻) from a Leica CME microscope (Leica Microsystems GmbH, Wetzlar, Germany).
Immunohistochemistry Evaluation Expression scores for survivin, BCL-2, cyclin D1, ETS1, and PDEF in the original diagnostic tumor biopsy samples were analyzed by 3 independent pathologists blinded to outcomes using a score described by Schmidt and colleagues.19 This score is based on 2 distinct characteristics: the percentage of positivity and the intensity grade of each marker in the slide. Positivity was measured by the following scores for percentage of positive tumor cells per slide: 0%, score ⫽ 0; ⬍ 5%, score ⫽ 1; 5% to 20%, score ⫽ 2; 21% to 50%, score ⫽ 3; 51% to 75%, score ⫽ 4, ⬎ 75%, score ⫽ 5. The intensity grade of the marker was scored as follows: absence, score ⫽ 0; weak, score ⫽ 1; moderate, score ⫽ 2; and strong, score ⫽ 3. Both scores were then multiplied, giving an intermediate score ranging from 0 to 15. The final score considered for analysis was the mean score obtained by the 3 pathologists. See details for survivin scores and IHC analysis in Figure 1.
Statistical Analysis The OS duration was the time between the date of initial diagnosis and the date of the last status report, whether the patient was alive or dead and regardless of the cause of death. The Kaplan-Meier method was applied to estimate PFS and OS. The Cox regression model was applied for the estimation of the hazard ratio (HR) and for the multivariate analysis using a forward selection. The Cox-Mantel test was used to compare survival distributions. Because many cutoff values have been assessed in the literature for biologic parameters using IHC and because various results have been achieved, we chose to give 3 different cutoffs levels for the mean score obtained: low (0-5), medium (6-10), and high (11-15). Comparison was made between low expression levels and medium/high levels for each marker by Fisher’s exact test. The Mann-Whitney test was used to compare the mean overall immunostaining score with pCR. Patients with any missing biomarker values (scores) were excluded from the biomarker analysis. The data were processed in Stata 10.0 software. Significance tests were 2-tailed and 5% significance level was used throughout. This work was approved by the local ethics committee.
Cristiane Rios Petrarca et al Figure 1 Immunohistochemistry Score: A Typical Example of Tissue Sections with Low (mean score of 0) and High (mean score of 8) Expression Levels of Survivin are Shown in A and B, Respectively (200ⴛ)
A
Table 1 Baseline Characteristics Baseline Characteristics Median age
Total of 45 Patients 49 years (range, 25-70 years)
Median Follow-up Time
3.6 years (range, 0.8-5.7 years)
Median Overall Survival
4.4 years (95% CI, 3,9-4.9 years)
Median Progression-free Survival
3.4 years (95% CI, 3.1-4.3 years)
Median Duration of Chemotherapy
4.8 months (range, 2.7-6.9 months)
Receptor Status Estrogen receptor positive
29 (64.4%)
Progesterone receptor positive
24 (53.3%)
⫹
ErbB2 (3⫹ on IHC)*
7 (15.5%)
Clinical Stage AJCC (TNM)
B
IIA
13 (29%)
IIB
10 (22%)
IIIA
13 (29%)
IIIB
4 (9%)
IIIC
4 (9%)
Response to Chemotherapy Complete pathologic response
7% (95% CI, 0%-13.9%)
Partial pathologic response
80% (95% CI, 68%-91%)
Pathologic Characteristics
This score is based on 2 distinct characteristics: the percentage of positivity and the intensity grade of each marker in the slide. Both scores were then multiplied giving an intermediate score ranging from 0 to 15. There was no major variability between pathologists when analyzing the mean expression levels for survivin (mean SD, 0.86). The survivin antibody used was the 12cY clone, which is immunogenic to full-length recombinant protein expressed in Escherichia coli. It is specific for an epitope present in the first 34 N-terminal amino acids of the survivin protein. This clone encompasses alternative splice variants of the survivin gene: the protein survivin-2B and survivin-deltaEx3.
Results Forty-five patients with stage II and stage III breast cancer were included in the analysis. The median follow-up time was 3.6 years (range, 0.8-5.7 years) and the median age at study entry was 49 years (range, 25-70 years). The median PFS and OS were 3.4 years (95% confidence interval [CI], 3.1-4.3 years) and 4.4 years (95% CI, 3.94.9 years), respectively. Fifteen patients (33%) had died and disease in 22 (49%) had progressed at the time of survival analysis. The median duration of chemotherapy was 4.8 months (range: 2.7-6.9 months). The median time from diagnostic biopsy to start of neoadjuvant chemotherapy was 3.4 weeks (range, 0.3-22.5 weeks), and the median time from last cycle of chemotherapy to definitive surgery was 4.7 weeks (range, 2.1 weeks-19.0 weeks). Baseline characteristics are shown in Table 1. Three patients (6%) were excluded from the final biomarker analysis because they had incomplete biomarker expression scores. Forty-three patients (96%) included in the study underwent surgery: 19 patients (42.2%) had modified radical mastectomy and 24 patients (53.3%) had lumpectomy/setorectomy with axillary clear-
Invasive ductal carcinoma
33 (73.33%)
Invasive lobular carcinoma
3 (6.6%)
Others/Unknown
9 (20%)
Abbreviation: AJCC ⫽ American Joint Committee on Cancer. * Human epidermal receptor protein-2 (ErbB2) status was analyzed by immunohistochemistry (ICH). The Brazilian National Health System does not fund trastuzumab.
ance or sentinel node biopsy. Two patients (4.4%) had unresectable disease. Seven percent of patients (95% CI, 0%-13.9%) had a pCR to neoadjuvant chemotherapy; 80% (95% CI, 68%-91%) had a partial response; and 13% (95% CI: 3.4%-23.2%) presented with stable or nonevaluable disease. The median expression level of survivin was 3.3 (range, 0-11). The median expression levels of survivin stratified by age were 3.6 (25-39 years), 2.6 (40-49 years), 4.3 (50-59 years) and 6,6 (⬎ 60 years). The median expression levels of the remaining markers were 7.6 (range, 0-15) for ETS1, 8.7 (range, 2.3-15) for BCL-2, 2.6 (range, 0-15) for PDEF, and 2.3 (range, 0-9) for cyclin D1. The mean overall survivin score was significantly higher for patients with pCR (9.3) compared with those without pCR (3.4; P ⫽ .01) (Table 2). In addition, one can observe in Table 3 that survivin was the only marker significantly associated with pCR when stratified by expression levels (low versus median/high levels). There was no statistically significant correlation of different expression levels of survivin for age, clinical stage, tumor grade, or ER, PR, and ErbB2 status. None of the analyzed markers was significantly associated with adverse PFS or OS distributions. Higher clinical stage and tumor grade, PR– status, and lack of response to che-
Clinical Breast Cancer April 2011
131
Survivin As a Predictive Biomarker of pCR to Neoadjuvant Chemotherapy Table 2 Median and Mean Biomarker Scores According to Pathologic Response Mean Expression Levels and SD According to pCR
Variable
Median Expression Levels
Mean Expression Levels
No
Yes
Survivin
3.3 (range, 0-11)
3.9
3.42 (0.47)
9.33 (0.88)
.01
Cyclin D1
2.3 (range, 0-9)
2.3
2.26 (0.30)
2.78 (1.16)
.48
PDEF
1.6 (range, 0-15)
2.2
2.16 (0.44)
3.66 (1.20)
.14
BCL2
8.7 (range, 2.3-15)
8.7
8.56 (0.55)
10.67 (0.33)
.24
ETS1
7.6 (range, 0-15)
7.6
7.55 (0.69)
7.89 (1.06)
.82
P Value*
Abbreviation: pCR ⫽ complete pathologic response. * Mann-Whitney test.
Table 3 Expression Levels (Low vs. Median/High) According to Percentage of Patients With Complete Pathologic Response Number of Patients
% pCR
P Value†
0-5
30
0.0
.02
6-15
12
27.3
0-5
38
8.3
6-15
3
0.0
0-5
38
5.6
6-15
3
33.3
0-5
7
0.0
6-15
32
9.7
0-5
15
0.0
6-15
27
12.0
Marker Level * Survivin
Cyclin D1 1.00
PEDF .22
BCL2 1.00
ETS1 .28
Abbreviation: pCR ⫽ complete pathologic response. * Low IHC score (0-5) and median/high IHC score (6-15); † Fisher exact test.
motherapy were significantly associated with shorter PFS in the Cox regression analysis (Table 4).
Discussion pCR to neoadjuvant chemotherapy in LABC ranges from 6% to 19%3 and is significantly associated with longer PFS and OS.4 In our study 7% of patients had a pCR to neoadjuvant anthracycline/taxane– based chemotherapy and 53% were able to undergo breastconserving surgery. The high prevalence of patients with positive ER⫹ status could have contributed to lower rates of complete pCR in our study.3 More important, high expression levels of survivin were significantly associated with pCR. This was demonstrated in a small cohort of breast cancer patients with low rates of pCR, reinforcing the strong association of survivin as a predictive biomarker of response to treatment in this unselected population.
132
Clinical Breast Cancer April 2011
The survivin gene (BIRC5) is a member of the IAP family and has several cellular implications, mainly inhibition of apoptosis but also dysregulation of mitosis, cell cycle progression, carcinogenesis, and DNA repair.7 Dysregulation of the survivin networks, uncoupled from tumor suppressors, may be required early to initiate the process of malignant transformation and later to maintain the malignant phenotype of established tumors6. Four alternative splice variants (survivin-deltaEx3, survivin-3B, survivin-2B and survivin-2B alpha) have been described, some with distinct functions.20,21 High levels of survivin have been identified in a wide range of malignancies, including breast, lung, prostate, pancreatic, and colorectal cancers, but interestingly it is not encountered in normal adult tissue.6 Studies have revealed a significant relationship with high expression levels of survivin and prognosis in breast cancer, particularly as an independent predictor of shorter duration of survival in patients with poor prognostic features.9,22,23 Boidot and associates24 analyzed, by means of real-time quantitative PCR, 60 breast carcinoma patients treated with FEC (5-fluorouracil ⫹ epirubicin ⫹ cyclophosphamide) or with docetaxel ⫹ epirubicin chemotherapy. The increased expression and ratio of survivin-3B (an alternative splice variant) after 1 course of docetaxel ⫹ epirubicin was associated with reduced disease-free survival (DFS) and OS and was significantly associated with resistance in the FEC treatment group. Interestingly, before treatment neither survivin transcript expression nor ratio was correlated with DFS or OS in any group. Span and colleagues25 evaluated patients treated with endocrine therapy; those with high survivin levels, quantified by enzyme-linked immunosorbent assay (ELISA), exhibited a significantly shorter PFS than those who had low levels. In contrast, for patients treated with chemotherapy, those with high survivin levels had a significantly longer PFS than those with low levels. In addition, survivin protein levels were associated with clinicopathologic characteristics of patients with a poor prognosis (eg, higher in high-grade tumors and in tumors from younger patients). As patients with high-grade tumors have a poorer prognosis irrespective of therapy, these data highlight that tumors with high survivin levels may respond better to chemotherapy, and this could be explained by the fact that highly proliferating cells are frequently more sensitive to chemotherapy.26 Moreover survivin showed a statistically significant negative correlation with PR status in a recent study,27 but its expression was independent of ER status. Similar observations had previously been
Cristiane Rios Petrarca et al Table 4 Cox Regression Analysis for Progression-free Survival Factor
Hazard Ratio
95% CI
P Value (Cox Regression)
TNM Clinical Stage (III vs. II)
4.05
1.17–14.0
.027
Progesterone Receptor (negative vs. positive)
3.17
1.16–8.6
.024
Response to Chemotherapy (lack of response versus pCR ⴙ pPR)
5.39
1.24–23.4
.024
Histologic Tumor Grade (grade III vs. grade I/II)
3.39
1.23–9.3
.018
Table shows that stage III disease, PR– status, grade III tumors, and lack of response to chemotherapy are associated with shorter PFS. Abbreviations: CI ⫽ confidence interval; pCR ⫽ pathologic complete response, pPR ⫽ pathologic partial response.
obtained by other investigators.28,29 Our numbers reveal no significant association of receptor status and survivin levels, although PR– status is associated with shorter PFS. PR– status has been shown to be a poor prognosticator in breast cancer and is associated with a higher response to chemotherapy.30,31 Not surprisingly, higher clinical stage, high tumor grade, and lack of response to chemotherapy were associated with shorter PFS rates in our cohort of patients. Survivin, cyclin D1, PDEF, ETS1, and BCL-2 showed no association with adverse outcome; this may be a consequence of the relatively small number of patients included, short follow-up time, and low number of events. There are many known limitations when comparing different biomarkers, including the variety of methods used (eg, IHC, Western blot, ELISA, PCR), the capacity to reproduce the correct technique, and heterogeneity when analyzing the materials and results of each biomarker in response to treatment.32 The proposed IHC method using a median score based on the positivity and intensity of staining19 is within current practice and gives a greater level of reproducibility for the results obtained, although it is unable to distinguish all splice variants that may represent diverse cellular functions. It would be interesting to explore in future studies whether different subtypes of breast cancer or chemotherapy regimens, including the use of trastuzumab in ErbB2⫹ disease, would yield similar results. Elevated expression of ErbB3 has been shown to confer paclitaxel resistance in ErbB2-overexpressing breast cancer cells through upregulation of survivin,33 and inhibition of ErbB2 by trastuzumab can reduce survivin expression in breast cancer cells.34 Several splice variants of survivin are present in breast cancer tissues22,35 and may have different subcellular localizations. Different cellular functions20,21 may possibly interfere with the interpretation of the results obtained in clinical studies when evaluating response to therapeutic agents and prognosis.
Conclusion We think that future studies in breast cancer evaluating response to treatment should incorporate survivin as a predictive biomarker in the neoadjuvant setting and that survivin may be a surrogate marker of high proliferation status. We were unable to draw any further conclusions regarding the other biomarkers analyzed.
Acknowledgments This work was supported by grants from Sanofi Aventis and Hospital Sao Lucas da PUC-RS. We acknowledge Dr. Samuel Dumith for technical assistance and statistical support.
Disclosures The authors have no other 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. No writing assistance was received in the production of this article.
References 1. Fisher B, Bryant J, Wolmark N, et al. Effect of preoperative chemotherapy on the outcome of women with operable breast cancer. J Clin Oncol 1998; 16:2672-85. 2. Buzdar AU, Singletary SE,Theriault RL, et al. Prospective evaluation of paclitaxel versus combination chemotherapy with fluorouracil, doxorubicin, and cyclophosphamide as neoadjuvant therapy in patients with operable breast cancer. J Clin Oncol 1999; 17:3412-7. 3. Goldhirsch A. Ingle JN, Gelber RD, et al. Thresholds for therapies: highlights of the St Gallen International Expert Consensus on the primary therapy of early breast cancer 2009. Ann Oncol 2009; 20:1319-29. 4. Bonadonna G, Valagussa P, Brambilla C, et al. Primary chemotherapy in operable breast cancer: Eight-year experience at the Milan Cancer Institute. J Clin Oncol 1998; 16:93-100. 5. Smith IE. Neoadjuvant/presurgical treatments. Breast Cancer Res 2008; 10(suppl 4):24. 6. Altieri DC. Survivin, cancer networks and pathway-directed drug discovery. Nat Rev Cancer 2008; 8:61-70. 7. Blanc-Brude OP, Mesri M, Wall NR, et al. Therapeutic targeting of the survivin pathway in cancer: initiation of mitochondrial apoptosis and suppression of tumorassociated angiogenesis. Clin Cancer Res 2003; 9:2683-92. 8. Hoffmann AC, Vallbohmer D, Grimminger P, et al. Preoperative survivin mRNA detection in peripheral blood is an independent predictor of outcome in esophageal carcinoma. Pharmacogenomics 2010; 11:341-7. 9. Son BH, Choi JS,Lee JH. Prognostic values of KAI1 and survivin expression in an infiltrating ductal carcinoma of the breast. Pathology 2005; 37:131-6. 10. Knutsen A, Adell G. Sun XF. Survivin expression is an independent prognostic factor in rectal cancer patients with and without preoperative radiotherapy. Int J Radiat Oncol Biol Phys 2004; 60:149-55. 11. Feldman RJ, Sementchenko VI, Gayed M, et al. Pdef expression in human breast cancer is correlated with invasive potential and altered gene expression. Cancer Res 2003; 63:4626-31. 12. Oikawa T, Yamada T. Molecular biology of the Ets family of transcription factors. Gene 2003; 303:11-34. 13. Turcotte S, Forget MA, Beauseigle D, et al. Prostate-derived Ets transcription factor overexpression is associated with nodal metastasis and hormone receptor positivity in invasive breast cancer. Neoplasia 2007; 9:788-96. 14. Katayama S, Nakayama T, Ito M, et al. Expression of the ets-1 proto-oncogene in human breast carcinoma: differential expression with histological grading and growth pattern. Histol Histopathol 2005; 20:119-26. 15. Rudas M, Lehnert M, Huynh A, et al. Cyclin D1 expression in breast cancer patients receiving adjuvant tamoxifen-based therapy. Clin Cancer Res 2008; 14:1767-74. 16. Pusztai L, Krishnamurti S, Perez Cardona J, et al. Expression of bAG-1 and BcL-2 proteins before and after neoadjuvant chemotherapy of locally advanced breast cancer. Cancer Invest 2004; 22:248-56. 17. Horii R, Akiyama F, Ito Y, Iwase T. Assessment of hormone receptor status in breast cancer. Pathol Int 2007; 57:784-90. 18. Hayward JL, Carbone PP, Rubens RD. Assessment of response to therapy in advanced breast cancer. Br J Cancer 1977; 35:292-8. 19. Schmidt M, Bachhuber A, Victor A, et al. P53 expression and resistance against paclitaxel in patients with metastatic breast cancer. J Cancer Res Clin Oncol 2003; 129:295-302. 20. Li F. Role of survivin and its splice variants in tumorigenesis. Br J Cancer 2005; 92:212-6.
Clinical Breast Cancer April 2011
133
Survivin As a Predictive Biomarker of pCR to Neoadjuvant Chemotherapy 21. Mahotka C, Liebmann J, Wenzel M, et al. Differential subcellular localization of functionally divergent survivin splice variants. Cell Death Differ 2002; 9:1334-42. 22. Ryan BM, Konecny GE, Kahlert S, et al. Survivin expression in breast cancer predicts clinical outcome and is associated with HER2, VEGF, urokinase plasminogen activator and PAI-1. Ann Oncol 2006; 17:597-604. 23. Hinnis AR, Luckett JC, Walker RA. Survivin is an independent predictor of short-term survival in poor prognostic breast cancer patients. Br J Cancer 2007; 96:639-45. 24. Boidot R, Vegran F, Lizard-Nacol S. Predictive value of survivin alternative transcript expression in locally advanced breast cancer patients treated with neoadjuvant chemotherapy. Int J Mol Med 2009; 23:285-91. 25. Span PN, Tjan-Heijnen VC, Manders P, et al. High survivin predicts a poor response to endocrine therapy, but a good response to chemotherapy in advanced breast cancer. Breast Cancer Res Treat 2006; 98:223-30. 26. Urruticoechea A, Smith Ie, Dowsett M. Proliferation marker Ki-67 in early breast cancer. J Clin Oncol 2005; 23:7212-20. 27. Radnade KJ, Nerurkar AV, Phulpagar MD, et al.. Expression of survivin and p53 proteins and their correlation with hormone receptor status in indian breast cancer patients. Indian J Med Sci 2009; 63:481-90.
134
Clinical Breast Cancer April 2011
28. Chew JS, Shew JY,,Huang CS. Immunohistochemical analysis of survivin expression in primary breast cancers. J Formos Med Assoc 2004; 103:925-31. 29. Span PN, Sweep FC, Wiegerinck ET, et al. Survivin is an independent prognostic marker for risk stratification of breast cancer patients. Clin Chem 2004; 50:1986-93. 30. Caudle AS, Gonzalez-Angulo AM, Hunt KK, et al. Predictors of tumor progression during neoadjuvant chemotherapy in breast cancer. J Clin Oncol 2010; 28:1821-8. 31. Onitilo AA,Engel JM Greenlee RT, et al. Breast cancer subtypes based on Er/Pr and Her2 expression: comparison of clinicopathologic features and survival. Clin Med Res 2009; 7:4-13. 32. Owzar K. Alternate statistical tools and limitations in genetic marker association studies in single-arm drug cancer trials. J Clin Oncol 2008; 26:1400-1. 33. Wang S, Huang X. Lee Ck, Liu B: Elevated expression of erbB3 confers paclitaxel resistance in erbB2-overexpressing breast cancer cells via upregulation of survivin. Oncogene 29:4225-36. Epub 2010 May 24. 34. Zhu H, Zhang G, Wang Y, et al. Inhibition of ErbB2 by Herceptin reduces survivin expression via the ErbB2-beta-catenin/TCF4-survivin pathway in ErbB2-overexpressed breast cancer cells. Cancer Sci 2010; 101:1156-62. 35. Ryan B, O’Donovan N, Browne B, et al. Expression of survivin and its splice variants survivin-2B and survivin-DeltaEx3 in breast cancer. Br J Cancer 2005; 92:120-4.
Survivin As a Predictive Biomarker of Complete Pathologic Response to Neoadjuvant Chemotherapy in Patients With Stage II and Stage III Breast Cancer Cristiane Rios Petrarca,1 Andre T. Brunetto,1 Vinicius Duval,1 Adriana Brondani,2 Gisele Pereira Carvalho,1 and Bernardo Garicochea1 Abstract Introduction: Predictive biomarkers of response to neoadjuvant chemotherapy in patients with breast cancer are needed to better characterize tumors and enable more tailored therapies. Methods: The expression levels of survivin, BCL-2, cyclin D1, ETS1, and PDEF in tumor samples obtained in the diagnostic biopsies of patients undergoing neoadjuvant chemotherapy for stage II and stage III disease were evaluated by immunohistochemistry (IHC). The mean expression score (range, 0-15) obtained by 3 different pathologists was used for analysis and correlated with complete pathologic response (pCR) and survival by standard univariate and multivariate methods. Results: Fortyfive female patients were included in this study and received preoperative standard anthracycline/taxane– based chemotherapy. The median age at diagnosis was 49 years (range, 25-70 years). Three patients (7.1%) achieved pCR. The mean expression score of survivin in the diagnostic biopsies was significantly higher (P ⫽ .01) in patients with pCR (9.3) than in those without (3.4). There was no significant association with pCR for the other biologic markers analyzed nor was there correlation with prognosis. Survivin levels were not associated with age, tumor grade, clinical stage, or receptor status. Conclusion: High expression levels of survivin in the primary tumor may be used as a potential predictive biomarker of pCR to neoadjuvant chemotherapy in patients with stage II and stage III breast cancer. Clinical Breast Cancer, Vol. 11, No. 2, 129-34 © 2011 Elsevier Inc. All rights reserved. Keywords: Breast cancer, Neoadjuvant chemotherapy, Predictive biomarker, Survivin, Complete pathologic response
Introduction Neoadjuvant chemotherapy is accepted as standard of care in locally advanced breast cancer (LABC) in an effort to improve the possibility of breast-conserving surgery and to render inoperable tumors resectable.1,2 It also has the advantage of early initiation of systemic therapy and in vivo assessment of response to treatment. The rate of pCR for preoperative chemotherapy has been confirmed as a strong prognostic marker that is significantly associated with longer progression-free survival (PFS) and overall survival (OS),3,4 1 Department of Oncology and Pathology, Hospital São Lucas, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre-RS, Brazil 2 Department of Pathology, Universidade Luterana do Brasil, Canoas-RS, Brazil
Submitted: Jul 19, 2010; Revised: Sep 24, 2010; Accepted: Oct 26, 2010 Address for correspondence: Bernardo Garicochea MD, PhD, Hospital São Lucas da PUCRS, Av. Ipiranga, 6690/708, 90610 – 000 Porto Alegre, RS, Brasil Fax: 55-51-33203319; e-mail contact: [email protected]
1526-8209/$ - see frontmatter © 2011 Elsevier Inc. All rights reserved. doi: 10.1016/j.clbc.2011.03.002
although some authors argue that this is not applicable to all subtypes of breast cancer.5 Neoadjuvant treatment is the ideal setting to analyze molecular markers that may predict pathologic response to therapy in tumor tissue. In addition, gene expression profiling may provide predictive and prognostic signatures that could help characterize tumors and enable more tailored therapies. New predictive biomarkers may allow different chemotherapy regimens or hormonal or targeted agents to be tested with a streamlined approach and therefore accelerate drug development. This study primarily aims to evaluate 2 groups of genes with distinct biologic activity as potential predictive biomarkers of response to neoadjuvant chemotherapy: survivin (BIRC5) and the ETS (E-twenty six) family, which have been implicated in the development of human cancer. The protein survivin, a member of the inhibitor of apoptosis (IAP) family, has been found in various neoplasms, including breast cancer, but is usually not detected in normal tissue.6 Survivin can participate in several cellular survival functions,
Clinical Breast Cancer April 2011
129
Survivin As a Predictive Biomarker of pCR to Neoadjuvant Chemotherapy including inhibition of apoptosis, cell cycle progression, and DNA repair,7 and there is evidence of an association between expression levels and survival in breast, esophageal, and rectal cancers8-10 The ETS family of proteins are transcriptional factors implicated in cell proliferation and differentiation, apoptosis, and oncogenesis.11,12 ETS1 and PDGF are members of this family and have been associated with invasive breast cancer, hormonal receptor status, and increased metastatic potential in breast carcinomas.11,13,14 There is limited published work looking at the association of these proteins and pathologic response to neoadjuvant chemotherapy; hence our efforts to evaluate these potential predictive markers. In parallel we also reviewed markers of proliferation, cell cycle progression, and apoptosis that have been classically associated with breast carcinogenesis: cyclin D1 and BCL-2.15,16
Methods Patient Inclusion Included in this analysis were consecutive female patients with breast cancer, TNM stages II and III (according to the 6th edition of the American Joint Committee on Cancer [AJCC]) and ages between 18 and 70 years, who were undergoing neoadjuvant chemotherapy with AC-T (doxorubicin [Adriamycin] 60 mg/m2 Day 1 and cyclophosphamide 600 mg/m2 Day1 for 4 cycles of 21 days, followed by docetaxel 75 mg/m2 D 1 for 4 cycles of 21 days) during the period between July 2004 and November 2009 in the Breast Oncology Department at Sao Lucas Hospital-PUC RS . Trastuzumab is not provided by the Brazilian Public Health System; therefore patients with overexpression of ErbB2 did not receive this agent. Exclusion criteria were metastatic or recurrent disease, comorbidities precluding the use of anthracyclines or taxanes, and history of other malignancies (except for basal cell skin carcinoma). Estrogen (ER) and progesterone (PR) status was assessed by standard immunohistochemistry (IHC) tests : nuclear staining in ⱖ 10% was considered positive (according to the indication that a significant difference in 5-year recurrence-free survival between ER-positive (ER⫹) and ER-negative (ER–) patients has been reported for a cutoff of 10%.17 ErbB2 status was analyzed by IHC and graded as 1⫹, 2⫹, or 3⫹ for positivity. ErbB2 was defined as positive when intense circumferential membrane staining was found in ⬎ 10% of the invasive carcinoma (ErbB2 ⫽ 3⫹). ErbB2 status classified as 2⫹ was not further assessed by fluorescence in situ hybridization analysis or polymerase chain reaction (PCR). Pathologic response was independently evaluated after surgical resection of the remaining tumor and nodes. The clinical measurement of the response to neoadjuvant therapy was defined according to the International Union Against Cancer (UICC) criteria.18 pCR was defined as the histologic absence of residual invasive disease in both the breast and the axilla. Informed consent was obtained from all patients before inclusion and data was collected prospectively.
Immunohistochemistry Tissue blocks were trimmed and sectioned with a microtome (Leica SM 2000R, Leica, Nussloch, Germany) to obtain 3-m sections, which were put on pretreated slides with HistoGrip (Invitrogen Corporation Grand Island, New York) at 60°C for 24 hours. The sections were then deparaffinized with xylene and rehydrated in ethanol. The inactivation of endogenous peroxidase occurred through
130
Clinical Breast Cancer April 2011
immersion in hydrogen peroxide and was thereafter put through the antigen retrieval process using the pressure cooker method. Incubation overnight at temperatures between 2° and 6°C was carried out and antibody diluents were then applied with background reducing components (Dako North America Inc., Carpinteria, CA) for the following antibodies and concentrations: survivin, 1:50 (Dako North America Inc.), BCL2, 1:200 (Invitrogen), cyclin D1, 1:50 (Dako North America Inc.), ETS1, 1:50 (Novocastra Laboratories Ltd., Newcastle Upon Tyne, UK), and PDEF, 1:25 (Santa Cruz Biotechnology, Santa Cruz, CA). Each marker was tested for external controls. The picture max system (HRP Polymer Conjugate Broad Spectrum, Invitrogen,) was used to amplify the antibody-antigen reaction, after which slides were incubated in a solution of diaminobenzidine and stained in hematoxylin. Finally slides were dehydrated in absolute ethyl alcohol and subsequently mounted with the synthetic medium Entellan (Merck, KGaA, Darmstadt, Germany). Images were then captured with a digital camera (Sony Corp., Tokyo, Japan) (magnification, 200⫻) from a Leica CME microscope (Leica Microsystems GmbH, Wetzlar, Germany).
Immunohistochemistry Evaluation Expression scores for survivin, BCL-2, cyclin D1, ETS1, and PDEF in the original diagnostic tumor biopsy samples were analyzed by 3 independent pathologists blinded to outcomes using a score described by Schmidt and colleagues.19 This score is based on 2 distinct characteristics: the percentage of positivity and the intensity grade of each marker in the slide. Positivity was measured by the following scores for percentage of positive tumor cells per slide: 0%, score ⫽ 0; ⬍ 5%, score ⫽ 1; 5% to 20%, score ⫽ 2; 21% to 50%, score ⫽ 3; 51% to 75%, score ⫽ 4, ⬎ 75%, score ⫽ 5. The intensity grade of the marker was scored as follows: absence, score ⫽ 0; weak, score ⫽ 1; moderate, score ⫽ 2; and strong, score ⫽ 3. Both scores were then multiplied, giving an intermediate score ranging from 0 to 15. The final score considered for analysis was the mean score obtained by the 3 pathologists. See details for survivin scores and IHC analysis in Figure 1.
Statistical Analysis The OS duration was the time between the date of initial diagnosis and the date of the last status report, whether the patient was alive or dead and regardless of the cause of death. The Kaplan-Meier method was applied to estimate PFS and OS. The Cox regression model was applied for the estimation of the hazard ratio (HR) and for the multivariate analysis using a forward selection. The Cox-Mantel test was used to compare survival distributions. Because many cutoff values have been assessed in the literature for biologic parameters using IHC and because various results have been achieved, we chose to give 3 different cutoffs levels for the mean score obtained: low (0-5), medium (6-10), and high (11-15). Comparison was made between low expression levels and medium/high levels for each marker by Fisher’s exact test. The Mann-Whitney test was used to compare the mean overall immunostaining score with pCR. Patients with any missing biomarker values (scores) were excluded from the biomarker analysis. The data were processed in Stata 10.0 software. Significance tests were 2-tailed and 5% significance level was used throughout. This work was approved by the local ethics committee.
Cristiane Rios Petrarca et al Figure 1 Immunohistochemistry Score: A Typical Example of Tissue Sections with Low (mean score of 0) and High (mean score of 8) Expression Levels of Survivin are Shown in A and B, Respectively (200ⴛ)
A
Table 1 Baseline Characteristics Baseline Characteristics Median age
Total of 45 Patients 49 years (range, 25-70 years)
Median Follow-up Time
3.6 years (range, 0.8-5.7 years)
Median Overall Survival
4.4 years (95% CI, 3,9-4.9 years)
Median Progression-free Survival
3.4 years (95% CI, 3.1-4.3 years)
Median Duration of Chemotherapy
4.8 months (range, 2.7-6.9 months)
Receptor Status Estrogen receptor positive
29 (64.4%)
Progesterone receptor positive
24 (53.3%)
⫹
ErbB2 (3⫹ on IHC)*
7 (15.5%)
Clinical Stage AJCC (TNM)
B
IIA
13 (29%)
IIB
10 (22%)
IIIA
13 (29%)
IIIB
4 (9%)
IIIC
4 (9%)
Response to Chemotherapy Complete pathologic response
7% (95% CI, 0%-13.9%)
Partial pathologic response
80% (95% CI, 68%-91%)
Pathologic Characteristics
This score is based on 2 distinct characteristics: the percentage of positivity and the intensity grade of each marker in the slide. Both scores were then multiplied giving an intermediate score ranging from 0 to 15. There was no major variability between pathologists when analyzing the mean expression levels for survivin (mean SD, 0.86). The survivin antibody used was the 12cY clone, which is immunogenic to full-length recombinant protein expressed in Escherichia coli. It is specific for an epitope present in the first 34 N-terminal amino acids of the survivin protein. This clone encompasses alternative splice variants of the survivin gene: the protein survivin-2B and survivin-deltaEx3.
Results Forty-five patients with stage II and stage III breast cancer were included in the analysis. The median follow-up time was 3.6 years (range, 0.8-5.7 years) and the median age at study entry was 49 years (range, 25-70 years). The median PFS and OS were 3.4 years (95% confidence interval [CI], 3.1-4.3 years) and 4.4 years (95% CI, 3.94.9 years), respectively. Fifteen patients (33%) had died and disease in 22 (49%) had progressed at the time of survival analysis. The median duration of chemotherapy was 4.8 months (range: 2.7-6.9 months). The median time from diagnostic biopsy to start of neoadjuvant chemotherapy was 3.4 weeks (range, 0.3-22.5 weeks), and the median time from last cycle of chemotherapy to definitive surgery was 4.7 weeks (range, 2.1 weeks-19.0 weeks). Baseline characteristics are shown in Table 1. Three patients (6%) were excluded from the final biomarker analysis because they had incomplete biomarker expression scores. Forty-three patients (96%) included in the study underwent surgery: 19 patients (42.2%) had modified radical mastectomy and 24 patients (53.3%) had lumpectomy/setorectomy with axillary clear-
Invasive ductal carcinoma
33 (73.33%)
Invasive lobular carcinoma
3 (6.6%)
Others/Unknown
9 (20%)
Abbreviation: AJCC ⫽ American Joint Committee on Cancer. * Human epidermal receptor protein-2 (ErbB2) status was analyzed by immunohistochemistry (ICH). The Brazilian National Health System does not fund trastuzumab.
ance or sentinel node biopsy. Two patients (4.4%) had unresectable disease. Seven percent of patients (95% CI, 0%-13.9%) had a pCR to neoadjuvant chemotherapy; 80% (95% CI, 68%-91%) had a partial response; and 13% (95% CI: 3.4%-23.2%) presented with stable or nonevaluable disease. The median expression level of survivin was 3.3 (range, 0-11). The median expression levels of survivin stratified by age were 3.6 (25-39 years), 2.6 (40-49 years), 4.3 (50-59 years) and 6,6 (⬎ 60 years). The median expression levels of the remaining markers were 7.6 (range, 0-15) for ETS1, 8.7 (range, 2.3-15) for BCL-2, 2.6 (range, 0-15) for PDEF, and 2.3 (range, 0-9) for cyclin D1. The mean overall survivin score was significantly higher for patients with pCR (9.3) compared with those without pCR (3.4; P ⫽ .01) (Table 2). In addition, one can observe in Table 3 that survivin was the only marker significantly associated with pCR when stratified by expression levels (low versus median/high levels). There was no statistically significant correlation of different expression levels of survivin for age, clinical stage, tumor grade, or ER, PR, and ErbB2 status. None of the analyzed markers was significantly associated with adverse PFS or OS distributions. Higher clinical stage and tumor grade, PR– status, and lack of response to che-
Clinical Breast Cancer April 2011
131
Survivin As a Predictive Biomarker of pCR to Neoadjuvant Chemotherapy Table 2 Median and Mean Biomarker Scores According to Pathologic Response Mean Expression Levels and SD According to pCR
Variable
Median Expression Levels
Mean Expression Levels
No
Yes
Survivin
3.3 (range, 0-11)
3.9
3.42 (0.47)
9.33 (0.88)
.01
Cyclin D1
2.3 (range, 0-9)
2.3
2.26 (0.30)
2.78 (1.16)
.48
PDEF
1.6 (range, 0-15)
2.2
2.16 (0.44)
3.66 (1.20)
.14
BCL2
8.7 (range, 2.3-15)
8.7
8.56 (0.55)
10.67 (0.33)
.24
ETS1
7.6 (range, 0-15)
7.6
7.55 (0.69)
7.89 (1.06)
.82
P Value*
Abbreviation: pCR ⫽ complete pathologic response. * Mann-Whitney test.
Table 3 Expression Levels (Low vs. Median/High) According to Percentage of Patients With Complete Pathologic Response Number of Patients
% pCR
P Value†
0-5
30
0.0
.02
6-15
12
27.3
0-5
38
8.3
6-15
3
0.0
0-5
38
5.6
6-15
3
33.3
0-5
7
0.0
6-15
32
9.7
0-5
15
0.0
6-15
27
12.0
Marker Level * Survivin
Cyclin D1 1.00
PEDF .22
BCL2 1.00
ETS1 .28
Abbreviation: pCR ⫽ complete pathologic response. * Low IHC score (0-5) and median/high IHC score (6-15); † Fisher exact test.
motherapy were significantly associated with shorter PFS in the Cox regression analysis (Table 4).
Discussion pCR to neoadjuvant chemotherapy in LABC ranges from 6% to 19%3 and is significantly associated with longer PFS and OS.4 In our study 7% of patients had a pCR to neoadjuvant anthracycline/taxane– based chemotherapy and 53% were able to undergo breastconserving surgery. The high prevalence of patients with positive ER⫹ status could have contributed to lower rates of complete pCR in our study.3 More important, high expression levels of survivin were significantly associated with pCR. This was demonstrated in a small cohort of breast cancer patients with low rates of pCR, reinforcing the strong association of survivin as a predictive biomarker of response to treatment in this unselected population.
132
Clinical Breast Cancer April 2011
The survivin gene (BIRC5) is a member of the IAP family and has several cellular implications, mainly inhibition of apoptosis but also dysregulation of mitosis, cell cycle progression, carcinogenesis, and DNA repair.7 Dysregulation of the survivin networks, uncoupled from tumor suppressors, may be required early to initiate the process of malignant transformation and later to maintain the malignant phenotype of established tumors6. Four alternative splice variants (survivin-deltaEx3, survivin-3B, survivin-2B and survivin-2B alpha) have been described, some with distinct functions.20,21 High levels of survivin have been identified in a wide range of malignancies, including breast, lung, prostate, pancreatic, and colorectal cancers, but interestingly it is not encountered in normal adult tissue.6 Studies have revealed a significant relationship with high expression levels of survivin and prognosis in breast cancer, particularly as an independent predictor of shorter duration of survival in patients with poor prognostic features.9,22,23 Boidot and associates24 analyzed, by means of real-time quantitative PCR, 60 breast carcinoma patients treated with FEC (5-fluorouracil ⫹ epirubicin ⫹ cyclophosphamide) or with docetaxel ⫹ epirubicin chemotherapy. The increased expression and ratio of survivin-3B (an alternative splice variant) after 1 course of docetaxel ⫹ epirubicin was associated with reduced disease-free survival (DFS) and OS and was significantly associated with resistance in the FEC treatment group. Interestingly, before treatment neither survivin transcript expression nor ratio was correlated with DFS or OS in any group. Span and colleagues25 evaluated patients treated with endocrine therapy; those with high survivin levels, quantified by enzyme-linked immunosorbent assay (ELISA), exhibited a significantly shorter PFS than those who had low levels. In contrast, for patients treated with chemotherapy, those with high survivin levels had a significantly longer PFS than those with low levels. In addition, survivin protein levels were associated with clinicopathologic characteristics of patients with a poor prognosis (eg, higher in high-grade tumors and in tumors from younger patients). As patients with high-grade tumors have a poorer prognosis irrespective of therapy, these data highlight that tumors with high survivin levels may respond better to chemotherapy, and this could be explained by the fact that highly proliferating cells are frequently more sensitive to chemotherapy.26 Moreover survivin showed a statistically significant negative correlation with PR status in a recent study,27 but its expression was independent of ER status. Similar observations had previously been
Cristiane Rios Petrarca et al Table 4 Cox Regression Analysis for Progression-free Survival Factor
Hazard Ratio
95% CI
P Value (Cox Regression)
TNM Clinical Stage (III vs. II)
4.05
1.17–14.0
.027
Progesterone Receptor (negative vs. positive)
3.17
1.16–8.6
.024
Response to Chemotherapy (lack of response versus pCR ⴙ pPR)
5.39
1.24–23.4
.024
Histologic Tumor Grade (grade III vs. grade I/II)
3.39
1.23–9.3
.018
Table shows that stage III disease, PR– status, grade III tumors, and lack of response to chemotherapy are associated with shorter PFS. Abbreviations: CI ⫽ confidence interval; pCR ⫽ pathologic complete response, pPR ⫽ pathologic partial response.
obtained by other investigators.28,29 Our numbers reveal no significant association of receptor status and survivin levels, although PR– status is associated with shorter PFS. PR– status has been shown to be a poor prognosticator in breast cancer and is associated with a higher response to chemotherapy.30,31 Not surprisingly, higher clinical stage, high tumor grade, and lack of response to chemotherapy were associated with shorter PFS rates in our cohort of patients. Survivin, cyclin D1, PDEF, ETS1, and BCL-2 showed no association with adverse outcome; this may be a consequence of the relatively small number of patients included, short follow-up time, and low number of events. There are many known limitations when comparing different biomarkers, including the variety of methods used (eg, IHC, Western blot, ELISA, PCR), the capacity to reproduce the correct technique, and heterogeneity when analyzing the materials and results of each biomarker in response to treatment.32 The proposed IHC method using a median score based on the positivity and intensity of staining19 is within current practice and gives a greater level of reproducibility for the results obtained, although it is unable to distinguish all splice variants that may represent diverse cellular functions. It would be interesting to explore in future studies whether different subtypes of breast cancer or chemotherapy regimens, including the use of trastuzumab in ErbB2⫹ disease, would yield similar results. Elevated expression of ErbB3 has been shown to confer paclitaxel resistance in ErbB2-overexpressing breast cancer cells through upregulation of survivin,33 and inhibition of ErbB2 by trastuzumab can reduce survivin expression in breast cancer cells.34 Several splice variants of survivin are present in breast cancer tissues22,35 and may have different subcellular localizations. Different cellular functions20,21 may possibly interfere with the interpretation of the results obtained in clinical studies when evaluating response to therapeutic agents and prognosis.
Conclusion We think that future studies in breast cancer evaluating response to treatment should incorporate survivin as a predictive biomarker in the neoadjuvant setting and that survivin may be a surrogate marker of high proliferation status. We were unable to draw any further conclusions regarding the other biomarkers analyzed.
Acknowledgments This work was supported by grants from Sanofi Aventis and Hospital Sao Lucas da PUC-RS. We acknowledge Dr. Samuel Dumith for technical assistance and statistical support.
Disclosures The authors have no other 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. No writing assistance was received in the production of this article.
References 1. Fisher B, Bryant J, Wolmark N, et al. Effect of preoperative chemotherapy on the outcome of women with operable breast cancer. J Clin Oncol 1998; 16:2672-85. 2. Buzdar AU, Singletary SE,Theriault RL, et al. Prospective evaluation of paclitaxel versus combination chemotherapy with fluorouracil, doxorubicin, and cyclophosphamide as neoadjuvant therapy in patients with operable breast cancer. J Clin Oncol 1999; 17:3412-7. 3. Goldhirsch A. Ingle JN, Gelber RD, et al. Thresholds for therapies: highlights of the St Gallen International Expert Consensus on the primary therapy of early breast cancer 2009. Ann Oncol 2009; 20:1319-29. 4. Bonadonna G, Valagussa P, Brambilla C, et al. Primary chemotherapy in operable breast cancer: Eight-year experience at the Milan Cancer Institute. J Clin Oncol 1998; 16:93-100. 5. Smith IE. Neoadjuvant/presurgical treatments. Breast Cancer Res 2008; 10(suppl 4):24. 6. Altieri DC. Survivin, cancer networks and pathway-directed drug discovery. Nat Rev Cancer 2008; 8:61-70. 7. Blanc-Brude OP, Mesri M, Wall NR, et al. Therapeutic targeting of the survivin pathway in cancer: initiation of mitochondrial apoptosis and suppression of tumorassociated angiogenesis. Clin Cancer Res 2003; 9:2683-92. 8. Hoffmann AC, Vallbohmer D, Grimminger P, et al. Preoperative survivin mRNA detection in peripheral blood is an independent predictor of outcome in esophageal carcinoma. Pharmacogenomics 2010; 11:341-7. 9. Son BH, Choi JS,Lee JH. Prognostic values of KAI1 and survivin expression in an infiltrating ductal carcinoma of the breast. Pathology 2005; 37:131-6. 10. Knutsen A, Adell G. Sun XF. Survivin expression is an independent prognostic factor in rectal cancer patients with and without preoperative radiotherapy. Int J Radiat Oncol Biol Phys 2004; 60:149-55. 11. Feldman RJ, Sementchenko VI, Gayed M, et al. Pdef expression in human breast cancer is correlated with invasive potential and altered gene expression. Cancer Res 2003; 63:4626-31. 12. Oikawa T, Yamada T. Molecular biology of the Ets family of transcription factors. Gene 2003; 303:11-34. 13. Turcotte S, Forget MA, Beauseigle D, et al. Prostate-derived Ets transcription factor overexpression is associated with nodal metastasis and hormone receptor positivity in invasive breast cancer. Neoplasia 2007; 9:788-96. 14. Katayama S, Nakayama T, Ito M, et al. Expression of the ets-1 proto-oncogene in human breast carcinoma: differential expression with histological grading and growth pattern. Histol Histopathol 2005; 20:119-26. 15. Rudas M, Lehnert M, Huynh A, et al. Cyclin D1 expression in breast cancer patients receiving adjuvant tamoxifen-based therapy. Clin Cancer Res 2008; 14:1767-74. 16. Pusztai L, Krishnamurti S, Perez Cardona J, et al. Expression of bAG-1 and BcL-2 proteins before and after neoadjuvant chemotherapy of locally advanced breast cancer. Cancer Invest 2004; 22:248-56. 17. Horii R, Akiyama F, Ito Y, Iwase T. Assessment of hormone receptor status in breast cancer. Pathol Int 2007; 57:784-90. 18. Hayward JL, Carbone PP, Rubens RD. Assessment of response to therapy in advanced breast cancer. Br J Cancer 1977; 35:292-8. 19. Schmidt M, Bachhuber A, Victor A, et al. P53 expression and resistance against paclitaxel in patients with metastatic breast cancer. J Cancer Res Clin Oncol 2003; 129:295-302. 20. Li F. Role of survivin and its splice variants in tumorigenesis. Br J Cancer 2005; 92:212-6.
Clinical Breast Cancer April 2011
133
Survivin As a Predictive Biomarker of pCR to Neoadjuvant Chemotherapy 21. Mahotka C, Liebmann J, Wenzel M, et al. Differential subcellular localization of functionally divergent survivin splice variants. Cell Death Differ 2002; 9:1334-42. 22. Ryan BM, Konecny GE, Kahlert S, et al. Survivin expression in breast cancer predicts clinical outcome and is associated with HER2, VEGF, urokinase plasminogen activator and PAI-1. Ann Oncol 2006; 17:597-604. 23. Hinnis AR, Luckett JC, Walker RA. Survivin is an independent predictor of short-term survival in poor prognostic breast cancer patients. Br J Cancer 2007; 96:639-45. 24. Boidot R, Vegran F, Lizard-Nacol S. Predictive value of survivin alternative transcript expression in locally advanced breast cancer patients treated with neoadjuvant chemotherapy. Int J Mol Med 2009; 23:285-91. 25. Span PN, Tjan-Heijnen VC, Manders P, et al. High survivin predicts a poor response to endocrine therapy, but a good response to chemotherapy in advanced breast cancer. Breast Cancer Res Treat 2006; 98:223-30. 26. Urruticoechea A, Smith Ie, Dowsett M. Proliferation marker Ki-67 in early breast cancer. J Clin Oncol 2005; 23:7212-20. 27. Radnade KJ, Nerurkar AV, Phulpagar MD, et al.. Expression of survivin and p53 proteins and their correlation with hormone receptor status in indian breast cancer patients. Indian J Med Sci 2009; 63:481-90.
134
Clinical Breast Cancer April 2011
28. Chew JS, Shew JY,,Huang CS. Immunohistochemical analysis of survivin expression in primary breast cancers. J Formos Med Assoc 2004; 103:925-31. 29. Span PN, Sweep FC, Wiegerinck ET, et al. Survivin is an independent prognostic marker for risk stratification of breast cancer patients. Clin Chem 2004; 50:1986-93. 30. Caudle AS, Gonzalez-Angulo AM, Hunt KK, et al. Predictors of tumor progression during neoadjuvant chemotherapy in breast cancer. J Clin Oncol 2010; 28:1821-8. 31. Onitilo AA,Engel JM Greenlee RT, et al. Breast cancer subtypes based on Er/Pr and Her2 expression: comparison of clinicopathologic features and survival. Clin Med Res 2009; 7:4-13. 32. Owzar K. Alternate statistical tools and limitations in genetic marker association studies in single-arm drug cancer trials. J Clin Oncol 2008; 26:1400-1. 33. Wang S, Huang X. Lee Ck, Liu B: Elevated expression of erbB3 confers paclitaxel resistance in erbB2-overexpressing breast cancer cells via upregulation of survivin. Oncogene 29:4225-36. Epub 2010 May 24. 34. Zhu H, Zhang G, Wang Y, et al. Inhibition of ErbB2 by Herceptin reduces survivin expression via the ErbB2-beta-catenin/TCF4-survivin pathway in ErbB2-overexpressed breast cancer cells. Cancer Sci 2010; 101:1156-62. 35. Ryan B, O’Donovan N, Browne B, et al. Expression of survivin and its splice variants survivin-2B and survivin-DeltaEx3 in breast cancer. Br J Cancer 2005; 92:120-4.