- Email: [email protected]
Proliferation Determined by Ki-67 Defines Different Pathologic Response to Neoadjuvant Trastuzumab-Based Chemotherapy in HER2-Positive Breast Cancer
Original Study
Proliferation Determined by Ki-67 Defines Different Pathologic Response to Neoadjuvant Trastuzumab-Based Chemotherapy in HER2-Positive Breast Cancer Alfonso Sánchez-Muñoz,1 Victor Navarro-Perez,1 Yessica Plata-Fernández,2 Angela Santonja,1 Ignacio Moreno,1 Nuria Ribelles,1 Emilio Alba1 Abstract Pathologic complete response (pCR) to trastuzumab-based chemotherapy is a potential surrogate for survival in patients with human epidermal growth factor receptor 2 (HER2)-positive (HER2D) breast cancer (BC). A Ki-67 index ‡ 50% was an independent predictive factor for pCR to trastuzumab-based chemotherapy in patients with HER2D BC. Background: This study aimed to assess the role of proliferation measured by Ki-67 as a predictive factor for pathologic complete response (pCR) to trastuzumab-based chemotherapy in patients with human epidermal growth factor receptor 2 (HER2)-positive (HER2þ) breast cancer (BC). Methods: A total of 81 patients with HER2þ BC were treated with a sequential schedule consisting of 4 cycles of cyclophosphamide (600 mg/m2) and doxorubicin (60 mg/m2) every 3 weeks, followed by 4 cycles of weekly paclitaxel (80 mg/m2) or docetaxel (100 mg/m2) every 3 weeks combined with trastuzumab (loading dose of 8 mg/kg and then 6 mg/kg every 3 weeks) as neoadjuvant treatment. Histologic subgroups classified by hormone receptor (HR) expression and Ki-67 index were 17% HRþ/ Ki-67 50%, 41% HRþ/Ki-67 < 50%, 25% HRenegative (HR) Ki-67 50%, and 17% HR/Ki-67 < 50%. Results: pCR, defined as the absence of invasive cells in the breast and axillary lymph node, was achieved in 33 patients (41%). The median Ki-67 expression was significantly higher in tumors with pCR (53%) compared with tumors without pCR (30%) (P < .001). Receiver operating characteristic (ROC) curve methodology suggested that 50% was the optimal Ki-67 cutoff point to best identify patients who achieved a pCR. The pCR rate was significantly different between histologic subgroups: HR/Ki-67 50% (70%), HRþ/Ki-67 50% (71%), HR/Ki-67 < 50% (22%), and HRþ/ Ki-67 < 50% (18%) (P < .001). A multivariate analysis revealed that a Ki-67 marker 50% was the only independent predictive factor of pCR (P ¼ .003; odds ratio [OR], 0.133; 95% confidence interval [CI], 0.036-0.5). The median followup was 32 months (range, 14-48 months). Patients who achieved a pCR had significantly lower recurrence (P ¼ .001) and higher overall survival (OS) (P ¼ .013) compared with those who did not. There were no statistically significant differences in disease-free survival (DFS) and OS in relation to HRs, the Ki-67 marker as a continuous or categorical variable, and histologic subgroups. Conclusion: Proliferation determined by Ki-67 expression 50% was an independent predictive factor for pCR in patients with HER2þ BC treated with trastuzumab-based chemotherapy. Clinical Breast Cancer, Vol. 15, No. 5, 343-7 ª 2015 Elsevier Inc. All rights reserved. Keywords: HER2-positive breast cancer, Ki-67 marker, Neoadjuvant trastuzumab-based chemotherapy, Pathologic complete response, Predictive factor
Introduction Amplification of the human epidermal growth factor receptor 2 (HER2) gene, and corresponding HER2 receptor overexpression, 1 Medical Oncology Service, Hospital Universitario Virgen de la Victoria of Málaga, Málaga, Spain 2 Medical Oncology Service, Complejo Hospitalario de Jaén, Jaén, Spain
occurs in roughly 20% of breast cancer (BC) cases and is associated with a more aggressive clinical course and a poorer outcome.1 However, the natural history of this disease changed with the Address for correspondence: Alfonso Sánchez Muñoz, PhD, Medical Oncology Service, Hospital Universitario Virgen de la Victoria, Campus Teatinos s/n. 29010-Málaga, Spain Fax: þ34-95-227-94-07; e-mail contact: [email protected]
Submitted: Oct 28, 2014; Accepted: Jan 14, 2015; Epub: Jan 21, 2015
1526-8209/$ - see frontmatter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clbc.2015.01.005
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Ki-67 and Trastuzumab Therapy in HER+ Breast Cancer development of anti-HER2 therapy in patients with both early and metastatic HER2-positive (HER2þ) BC. Neoadjuvant chemotherapy is the standard treatment for patients with locally advanced or inoperable BC and is administered with the intention of downstaging the tumor and potentially improving the surgical options. A pathologic complete response (pCR) to neoadjuvant chemotherapy in combination with anti-HER2 therapy is a potential surrogate for survival in patients with HER2þ BC.2 Several large phase III clinical trials have reported a pCR rate of about 40% in patients with HER2þ BC.2-4 Conversely, there are still about 50% to 60% patients with HER2þ BC who cannot achieve pCR from trastuzumab-containing neoadjuvant chemotherapy. Therefore, tumor eradication is different between HER2 tumors, indicating intrinsic differences in their biological characteristics. We are uncertain whether clinical and pathologic parameters can be used to stratify HER2þ BC into subgroups with different pathologic responses. Consequently, the discovery of a pretreatment predictive factor may have an important role in patient selection and management. The Ki-67 proliferation marker as measured by immunohistochemical analysis has been studied as a prognostic and predictive factor in early BC. Tumors with a high expression of proliferation-related genes are associated with high pCR rates after neoadjuvant chemotherapy,5-8 lending value to the relationship between proliferation and chemosensitivity. Nonetheless, the value of the Ki-67 proliferation biomarker as a predictive factor in HER2þ BC is less clear and has not been adequately investigated. In this study, we retrospectively investigated whether proliferation measured by the Ki-67 index can be used for classification of HER2þ BC in 2 subtypes with different pathologic responses.
Patients and Methods Study Population We analyzed the data from a retrospective cohort of patients with newly diagnosed pathologically confirmed invasive clinical stage II or III BC, including inflammatory BC, between 2008 and 2013. Clinicopathologic characteristics, hormone receptor (HR) status, HER2 status, Ki-67 index, pathologic response, and follow-up data were collected. All patients provided written informed consent before neoadjuvant treatment began. The study protocols were approved by our institutional review board, and the studies were conducted in accordance with the principles of the Declaration of Helsinki.
Immunohistochemical Studies
344
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Biopsy using a Tru-Cut needle was performed in all eligible patients before treatment. The immunohistochemical assessment was carried out at the time of diagnosis in every tumor block. Paraffin-embedded tumor specimens were analyzed centrally by a pathologist who specializes in BC using immunohistochemical methods to measure HR status, HER2 status, and the Ki-67 index. HER2 status was determined by the HercepTest (Dako, Carpinteria, CA) as well as by a fluorescence in situ hybridization (FISH) test in biopsy specimens with a 2þ score by immunohistochemical analysis. Tumors were classified as positive with a 3þ score by immunohistochemical analysis or a 2þ score by
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immunohistochemical analysis with a positive fluorescence in situ hybridization analysis. Estrogen and progesterone receptors were measured using mononuclear antibodies (clone 6F11, prediluted and 1A6, prediluted, respectively; both from Master Diagnostica, Granada, Spain). Tumors were classified as HRþ (estrogen or progesterone, or both) if 1% of tumor cell nuclei stained positive. Ki-67 expression was evaluated with a mononuclear antibody (clone SP6, prediluted; Master Diagnostica). The semiquantitative scale imaging recommended by the American College of Pathologists was used for quantification of the Ki-67 index. The most peripheral tumor areas were selected because they constitute the line infiltration of the neoplasm. Data on HR and Ki-67 marker expression were used to classify the histologic subgroups as HRþ/Ki-67þ, HRþ/Ki-67, HR/Ki-67þ, and HR/Ki-67.
Treatment Plan and Follow-Up Patients were treated with a sequential schedule of 4 cycles of cyclophosphamide (600 mg/m2) and doxorubicin (60 mg/m2) every 3 weeks, followed by 4 cycles of weekly paclitaxel (80 mg/m2) or docetaxel (100 mg/m2) every 3 weeks combined with trastuzumab (loading dose of 8 mg/kg and then 6 mg/kg every 3 weeks) as neoadjuvant treatment. Subsequently, patients underwent surgery within 4 to 6 weeks of the last course of chemotherapy. Modified radical mastectomy or conservative surgery with axillary lymph node dissection or sentinel lymph node biopsy was performed according to the surgeons’ criteria. Patients treated with breast-conserving surgery received adjuvant radiotherapy to the whole breast after surgery. Patients who underwent mastectomy received radiation therapy to the chest wall and axillary area after surgery when they had an initial tumor 5 cm, inflammatory BC, or 4 positive nodes after neoadjuvant chemotherapy. Hormone treatment with tamoxifen or aromatase inhibitors was given in postmenopausal women with HRþ (estrogen or progesterone, or both) tumors. Premenopausal women with HR positivity were treated with tamoxifen and goserelin if they remained premenopausal after chemotherapy. No additional adjuvant chemotherapy was prescribed after surgery. Patients received adjuvant trastuzumab to complete 1 year of treatment. Once treatment was completed, patients were followed for evaluation of disease status and late-onset toxicity every 3 months during the first 2 years and every 6 months for the following 3 years or until disease progression or death.
Statistical Analysis Continuous variables were described using measures of central tendency, mean or median, measures of dispersion, standard deviation, interquartile, and range. Categorical variables were described as frequency and percentage. The comparison of proportions and significance were estimated using the c2 test. pCR was defined as the absence of invasive cells in breast and lymph nodes. For statistical analysis, the following variables were used as predictive factors of pCR: HR expression, HER2 status, Ki-67 index, age, tumor size, and histologic grade. Initially, a univariate analysis was performed for each of the variables using the log-rank test. Subsequently, a multivariate analysis was performed using the Cox proportional risk model with all the variables that had been significant in the univariate analysis.
Alfonso Sánchez-Muñoz et al The receiver operating characteristic (ROC) curve analysis was used to determine the optimal cutoff point for Ki-67 by calculating sensitivity and specificity indices corresponding to Ki-67 cutoff points selected every 10 units (range, 1-100). Cutoff points divided the study population into groups of high and low expression, which we associated with pCR achievement. We considered a cutoff point as optimal when it had the highest combined sensitivity and specificity values. Disease-free survival (DFS) was measured from the date of surgery to the documented date of progression or death from any cause or as the date of the last follow-up. Overall survival (OS) was measured from the date of surgery to the documented date of death from any cause or as the date of the last follow-up. For the calculation of DFS and OS, the Kaplan-Meier method was used, and the data were compared using the log-rank test. We studied the impact of the prognostic variables (Ki-67 index, HR expression, histologic subgroups, and pCR rate) on DFS and OS with the Cox proportional risk model. SPSS, version 14.0 (SPSS, Chicago, IL) was used for all statistical analyses.
Results Patient Characteristics
A total of 81 patients with newly diagnosed stage II/III HER2þ BC were included in the present study. Baseline clinical and pathologic characteristics are summarized in Table 1. The median age of the patients was 50 years (range, 26-73 years). Results based on ROC curves suggested that the optimal Ki-67 cutoff point that best identified patients who responded to neoadjuvant therapy was a percentage of 50% positive nuclei. Thirty-four patients (42%) had HR disease and 34 (42%) patients had a Ki-67 marker 50%. Data on HR and Ki-67 expression were used to classify the patients with HER2þ BC, resulting in 14 (17%) cases of HRþ/Ki-67 50%, 33 (41%) cases of HRþ/Ki-67 < 50%, 20 (25%) cases of HR/Ki-67 50%, and 14 (17%) cases of HR/Ki-67 < 50%.
Table 1 Baseline Clinical and Pathologic Characteristics of the Study Population (N [ 81) Characteristics Age, years, median (range)
N (%) 50 (26-73)
Histopathologic Type Ductal
73 (90)
Lobular
1 (1)
Other
7 (9)
Hormonal Status Premenopausal
55 (67)
Postmenopausal
26 (33)
Tumor Stage T1
6 (8)
T2
47 (58)
T3
14 (17)
T4
14 (17)
Hormone Receptors
N (%)
Positive
47 (58)
Negative
34 (42)
Ki-67 Index 50%
34 (42)
<50%
47 (58)
Tumor Grade I
8 (10)
II
22 (27)
III
29 (36)
NA
22 (27)
Histologic Subgroups HRþ/Ki-67 50%
14 (17)
HRþ/Ki-67 <50%
33 (41)
HR/Ki-67 50%
20 (25)
HR/Ki-67 <50%
14 (17)
Efficacy A total of 42 patients (60%) underwent conservative surgery, whereas the remaining 39 patients (40%) underwent radical surgery. pCR was achieved in 33 patients (41%). The median Ki-67 index was significantly higher in tumors with pCR (53%) compared with tumors without pCR (30%) (P < .001). The pCR rate was significantly different between the histologic subgroups of HER2þ BC, being higher in HR/Ki-67 50% (70%) and HRþ/ Ki-67 50% (71%) compared with HR/Ki-67 < 50% (22%) and HRþ/Ki-67 < 50% (18%) (P < .001). Univariate analysis showed that a Ki-67 index 50% or Ki-67 as a categorical variable, poorly differentiated tumors and histologic subgroups defined by Ki-67, and HR expression exhibited an independent and statistically significant association with pCR. HR expression was associated with a higher trend of pCR but did not reach statistical significance. A multivariate analysis based on histologic subgroups, grade, and Ki-67 expression revealed that Ki67 50% was the only independent predictive factor for pCR (P ¼ .003; odds ratio [OR], 0.133; 95% confidence interval [CI], 0.036-0.5) (Table 2). Over a median follow-up of 32 months (range, 14-48 months), 25 (31%) patients had disease relapse and 15 (18%) patients died of
tumor progression. Patients who achieved a pCR had a significantly lower recurrence rate (P ¼ .001) and higher OS (P ¼ .013) compared with those who did not. However, there were no statistically significant differences in DFS and OS in relation to HR expression, Ki-67 index as a continuous or categorical variable, and histologic subgroups of HER2þ tumors.
Discussion This study assessed the role of proliferation measured by the Ki-67 index as a predictive factor for pCR to trastuzumab-based chemotherapy in HER2þ BC. Results showed a pCR rate of
Table 2 Multivariate Analysis of Biological Markers and Pathologic Complete Response Variable Ki-67 50% versus Ki-67 <50% Histologic subgroups Grade I/II versus grade III
Odds Ratio (95% CI)
P Value
0.133 (0.036-0.5) 0.39 (0.064-2.46 0.306 (0.79-1.18)
.003 .32 .086
Abbreviation: CI ¼ confidence interval.
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40%, similar to that reported in several large phase III clinical trials.2-4 The pCR rate was higher in poorly differentiated and Ki-67 50% tumors. The HR expression was associated with a higher trend of pCR but did not reach statistical significance. The occurrence of pCR was significantly different between histologic subgroups, being higher (> 3 times) in HR/Ki-67 50% and HRþ/Ki-67 50% tumors compared with HRþ/Ki-67 < 50% and HRþ/Ki-67 < 50% tumors. These results suggest that high proliferation measured by Ki-67 50% is an independent predictive factor for pCR in patients with HER2þ BC treated with trastuzumab-based chemotherapy. Regarding HR expression in pCR prediction, interpretation should be cautious because results in this field are inconsistent. The combination of anti-HER2 therapy and neoadjuvant chemotherapy showed that HR tumors had higher pCR rates in the GeparQuattro, Neo-ALTTO (Lapatinib with Trastuzumab for HER2Positive Early Breast Cancer) and CHER-LOB (Chemotherapy, Herceptin and Lapatinib in Operable Breast Cancer) trials.4,9,10 In contrast, results from the TECHNO (Taxol Epirubicin Cyclophosphamide Herceptin Neoadjuvant) study showed that no clinical and histologic parameters, including HR status, were capable of predicting pCR.2 The NeoSphere trial reported that HR disease had higher pCR rates than did HRþ disease, but the difference was statistically significant only in the 2 dual HER2 targeting groups and not in the 2 single HER2 targeting groups.11 Those findings do not allow one to exclude a potential benefit of trastuzumab in HRþ tumors. In fact, trastuzumab-based therapy in the adjuvant setting has been shown to reduce the risk of recurrence across all subgroups, although the magnitude of benefit seemed to be lower in HRþ tumors than in HR tumors.12 Cell proliferation in cancer has not been assessed in combination with HR expression. Taking these data together, the value of HR expression in predicting pCR for HER2þ tumors needs to be ascertained by further investigations. Ki-67 is a well-established cell proliferation marker in cancer. Previous data underscore the importance of proliferation and support its incorporation into therapy decisions. First, the results of several meta-analyses suggested that a high Ki-67 index confers a higher risk of relapse and a worse survival in patients with early BC.13-15 Second, proliferation has been identified as the main component and common denominator for predicting clinical outcome in several gene signatures. In this respect, a meta-analysis of publicly available BC gene expression data revealed that > 70% of the genes associated with patient survival were correlated with the proliferation pathway, whereas 26% of the genes were related to estrogen receptor signaling and only 2% to HER2 gene amplification. The expression of genes related to proliferation was constitutively high in HR/HER2 and HER2þ tumors.16 Nonetheless, the value of the Ki-67 proliferation biomarker as a predictive factor in HER2þ BC is less clear. Gene expression analysis in HER2þ disease identifies all the main intrinsic molecular subtypes of BC (luminal A, luminal B, HER2þ, and basal-like) and are not fully concordant with pathologic markers.17,18 Among them, the Cancer Genome Atlas project reported a subset of HER2þ tumors with a prediction analysis of microarray (PAM50) HER2-enriched (HER2-E) gene expression characterized by the highest activation of the HER2-regulated genes and proliferation/ cell-cycleerelated genes, suggesting that they may benefit the most
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from anti-HER2 therapies.19 A retrospective exploratory analysis of 114 patients with HER2þ tumors enrolled in the NOAH (Neoadjuvant Herceptin) trial showed that HER2-E tumors with a high proliferation score had the highest benefit from trastuzumabbased chemotherapy.20 Patients who achieved a pCR had significantly fewer recurrences and higher OS. However, when we compared the impact of pCR on prognosis after neoadjuvant therapy in the histologic subgroups of HER2þ BC, no significant difference in DFS and OS was detected: there was no significant difference in DFS and OS between patients with Ki-67 50% and Ki-67 < 50% tumors. This may result from the small case number in subgroups and short follow-up duration that could mask the potential associations, precluding the ability to reach a definitive conclusion. Another retrospective study including 102 cases of HER2þ BC reported that HR tumors and high Ki-67 expression were predictors for pCR but not for DFS and OS, although different neoadjuvant chemotherapy regimens combined with trastuzumab were administered, and ROC curve methodology was not used to find the optimal Ki-67 cutoff point.21 Notably, Von Minckwitz et al, in a large cohort of 6377 patients,22 demonstrated that a pCR after neoadjuvant chemotherapy was associated with an improved DFS only in patients with HER2þ (nonluminal) and triple-negative tumors, but not for luminal B/HER2þ and luminal A tumors. Our study does have certain limitations. First, it was a retrospective study with a relatively small number of patients and short follow-up. Second, it may be difficult to reproduce the centralized Ki-67 expression analysis rate in daily practice. However, we believe that our data can provide useful information for clinical practice because of the homogeneity of the treatment received by our patients in the neoadjuvant setting. An ROC curve methodology was used to obtain the Ki-67 cutoff that best identified patients who responded to trastuzumab-based chemotherapy. In conclusion, HER2þ BC is a biologically heterogeneous disease, and not all patients benefit from trastuzumab-based chemotherapy to the same extent. Although next-generation tumor sequencing may identify determinants of exquisite sensitivity to anti-HER2 therapy with or without chemotherapy, the proliferation measured by the Ki-67 marker could be a useful tool to better discriminate which patients will achieve a pCR.
Clinical Practice Points It is still unknown whether clinical and pathologic parameters
can be used to stratify HER2þ BC into subgroups with a different pathologic response. If we find a pretreatment predictive factor, it would have an important role in patient selection and management. Ki-67 is a well-established cell proliferation marker. Previous data underscore the importance of proliferation and support its incorporation into therapy decisions. Several meta-analyses suggested that a high Ki-67 index confers a higher risk of relapse and worse survival in patients with early BC. Furthermore, proliferation has been identified as the main component and common denominator for predicting clinical outcome in several gene signatures. Tumors with a high expression of proliferation-related genes are associated with high pCR rates after neoadjuvant chemotherapy, lending value to the relationship between proliferation and
Alfonso Sánchez-Muñoz et al chemosensitivity. However, the value of the Ki-67 proliferation biomarker as a predictive factor in HER2þ BC is less clear and has not been adequately investigated. We analyzed if proliferation measured by the Ki-67 index can be used for classification of HER2þ BC in subtypes with different pathologic responses. This study assessed the role of proliferation measured by the Ki-67 index as a predictive factor for pCR to trastuzumab-based chemotherapy in HER2þ BC. In our study, the occurrence of pCR was significantly different between histologic subgroups, being higher in HR/Ki-67 50% and HRþ/Ki-67 50% tumors compared with HRþ/ Ki-67 < 50% and HRþ/Ki-67 < 50% tumors. These results suggest that high proliferation measured by Ki-67 50% is an independent predictive factor for pCR in patients with HER2þ BC treated with trastuzumab-based chemotherapy. The impact on clinical practice could be to use the proliferation measured by the Ki-67 marker as a useful tool to better discriminate which patients will achieve a pCR.
Disclosure The authors have stated that they have no conflicts of interest.
References 1. Slamon DJ, Clark GM, Wong SG, et al. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 1987; 235:177-82. 2. Untch M, Fasching PA, Konecny GE, et al. Pathologic complete response after neoadjuvant chemotherapy plus trastuzumab predicts favorable survival in human epidermal growth factor receptor 2-overexpressing breast cancer: results from the TECHNO trial of the AGO and GBG study groups. J Clin Oncol 2011; 29:3351-7. 3. Gianni L, Eiermann W, Semiglazov V, et al. Neoadjuvant chemotherapy with trastuzumab followed by adjuvant trastuzumab versus neoadjuvant chemotherapy alone, in patients with HER2-positive locally advanced breast cancer (the NOAH trial): a randomised controlled superiority trial with a parallel HER2-negative cohort. Lancet 2010; 375:377-84. 4. Untch M, Rezai M, Loibl S, et al. Neoadjuvant treatment with trastuzumab in HER2-positive breast cancer: results from the GeparQuattro study. J Clin Oncol 2010; 28:2024-31.
5. Guarneri V, Broglio K, Kau SW, et al. Prognostic value of pCR after primary chemotherapy in relation to hormone receptor status and other factors. J Clin Oncol 2006; 24:1037-44. 6. Sánchez-Muñoz A, García-Tapiador AM, Martínez-Ortega E, et al. Tumour molecular subtyping according to hormone receptors and HER2 status defines different pCR to neoadjuvant chemotherapy in patients with locally advanced breast cancer. Clin Transl Oncol 2008; 10:646-53. 7. Chang J, Ormerod M, Powles TJ, et al. Apoptosis and proliferation as predictors of chemotherapy response in patients with breast carcinoma. Cancer 2000; 89:2145-52. 8. Faneyte IF, Schrama JG, Peterse JL, et al. Breast cancer response to neoadjuvant chemotherapy: predictive markers and relation with outcome. Br J Cancer 2003; 88:406-12. 9. Guarneri V, Frassoldati A, Bottini A, et al. Preoperative chemotherapy plus trastuzumab, lapatinib, or both in human epidermal growth factor receptor 2-positive operable breast cancer: results of the randomized phase II CHER-LOB study. J Clin Oncol 2012; 30:1989-95. 10. Baselga J, Bradbury I, Eidtmann H, et al. Lapatinib with trastuzumab for HER2positive early breast cancer (NeoALTTO): a randomised, open-label, multicentre, phase 3 trial. Lancet 2012; 379:633-40. 11. Gianni L, Pienkowski T, Im YH, et al. Efficacy and safety of neoadjuvant pertuzumab and trastuzumab in women with locally advanced, inflammatory, or early HER2-positive breast cancer (NeoSphere): a randomised multicentre, open-label, phase 2 trial. Lancet Oncol 2012; 13:25-32. 12. Untch M, Gelber RD, Jackisch C, et al. Estimating the magnitude of trastuzumab effects within patient subgroups in the HERA trial. Ann Oncol 2008; 19:1090-6. 13. Stuart-Harris R, Caldas C, Pinder SE, Pharoah P. Proliferation markers and survival in early breast cancer: a systematic review and meta-analysis of 85 studies in 32,825 patients. Breast 2008; 17:323-34. 14. de Azambuja E, Cardoso F, de Castro G Jr, et al. Ki-67 as prognostic marker in early breast cancer: a meta-analysis of published studies involving 12,155 patients. Br J Cancer 2007; 96:1504-13. 15. Urruticoechea A, Smith IE, Dowsett M. Proliferation marker Ki-67 in early breast cancer. J Clin Oncol 2005; 23:7212-20. 16. Wirapati P, Sotiriou C, Kunkel S, et al. Meta-analysis of gene expression profiles in breast cancer: toward a unified understanding of breast cancer subtyping and prognosis signatures. Breast Cancer Res 2008; 10:R65. 17. Rivenbark AG, O’Connor SM, Coleman WB. Molecular and cellular heterogeneity in breast cancer: challenges for personalized medicine. Am J Pathol 2013; 183:1113-24. 18. Harrell JC, Prat A, Parker JS, et al. Genomic analysis identifies unique signatures predictive of brain, lung, and liver relapse. Breast Cancer Res Treat 2012; 132:523-35. 19. Cancer Genome Atlas Network. Comprehensive molecular portraits of human breast tumours. Nature 2012; 490:61-70. 20. Prat A, Bianchini G, Thomas M, et al. Research-based PAM50 subtype predictor identifies higher responses and improved survival outcomes in HER2-positive breast cancer in the NOAH study. Clin Cancer Res 2014; 20:511-21. 21. Zhang GC, Qian XK, Guo ZB, et al. Pre-treatment hormonal receptor status and Ki-67 index predict pCR to neoadjuvant trastuzumab/taxanes but not disease-free survival in HER2-positive breast cancer patients. Med Oncol 2012; 29:3222-31. 22. Von Minckwitz G, Untch M, Blohmer JU, et al. Definition and impact of pCR on prognosis after neoadjuvant chemotherapy in various intrinsic breast cancer subtypes. J Clin Oncol 2012; 30:1796-804.
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Proliferation Determined by Ki-67 Defines Different Pathologic Response to Neoadjuvant Trastuzumab-Based Chemotherapy in HER2-Positive Breast Cancer Alfonso Sánchez-Muñoz,1 Victor Navarro-Perez,1 Yessica Plata-Fernández,2 Angela Santonja,1 Ignacio Moreno,1 Nuria Ribelles,1 Emilio Alba1 Abstract Pathologic complete response (pCR) to trastuzumab-based chemotherapy is a potential surrogate for survival in patients with human epidermal growth factor receptor 2 (HER2)-positive (HER2D) breast cancer (BC). A Ki-67 index ‡ 50% was an independent predictive factor for pCR to trastuzumab-based chemotherapy in patients with HER2D BC. Background: This study aimed to assess the role of proliferation measured by Ki-67 as a predictive factor for pathologic complete response (pCR) to trastuzumab-based chemotherapy in patients with human epidermal growth factor receptor 2 (HER2)-positive (HER2þ) breast cancer (BC). Methods: A total of 81 patients with HER2þ BC were treated with a sequential schedule consisting of 4 cycles of cyclophosphamide (600 mg/m2) and doxorubicin (60 mg/m2) every 3 weeks, followed by 4 cycles of weekly paclitaxel (80 mg/m2) or docetaxel (100 mg/m2) every 3 weeks combined with trastuzumab (loading dose of 8 mg/kg and then 6 mg/kg every 3 weeks) as neoadjuvant treatment. Histologic subgroups classified by hormone receptor (HR) expression and Ki-67 index were 17% HRþ/ Ki-67 50%, 41% HRþ/Ki-67 < 50%, 25% HRenegative (HR) Ki-67 50%, and 17% HR/Ki-67 < 50%. Results: pCR, defined as the absence of invasive cells in the breast and axillary lymph node, was achieved in 33 patients (41%). The median Ki-67 expression was significantly higher in tumors with pCR (53%) compared with tumors without pCR (30%) (P < .001). Receiver operating characteristic (ROC) curve methodology suggested that 50% was the optimal Ki-67 cutoff point to best identify patients who achieved a pCR. The pCR rate was significantly different between histologic subgroups: HR/Ki-67 50% (70%), HRþ/Ki-67 50% (71%), HR/Ki-67 < 50% (22%), and HRþ/ Ki-67 < 50% (18%) (P < .001). A multivariate analysis revealed that a Ki-67 marker 50% was the only independent predictive factor of pCR (P ¼ .003; odds ratio [OR], 0.133; 95% confidence interval [CI], 0.036-0.5). The median followup was 32 months (range, 14-48 months). Patients who achieved a pCR had significantly lower recurrence (P ¼ .001) and higher overall survival (OS) (P ¼ .013) compared with those who did not. There were no statistically significant differences in disease-free survival (DFS) and OS in relation to HRs, the Ki-67 marker as a continuous or categorical variable, and histologic subgroups. Conclusion: Proliferation determined by Ki-67 expression 50% was an independent predictive factor for pCR in patients with HER2þ BC treated with trastuzumab-based chemotherapy. Clinical Breast Cancer, Vol. 15, No. 5, 343-7 ª 2015 Elsevier Inc. All rights reserved. Keywords: HER2-positive breast cancer, Ki-67 marker, Neoadjuvant trastuzumab-based chemotherapy, Pathologic complete response, Predictive factor
Introduction Amplification of the human epidermal growth factor receptor 2 (HER2) gene, and corresponding HER2 receptor overexpression, 1 Medical Oncology Service, Hospital Universitario Virgen de la Victoria of Málaga, Málaga, Spain 2 Medical Oncology Service, Complejo Hospitalario de Jaén, Jaén, Spain
occurs in roughly 20% of breast cancer (BC) cases and is associated with a more aggressive clinical course and a poorer outcome.1 However, the natural history of this disease changed with the Address for correspondence: Alfonso Sánchez Muñoz, PhD, Medical Oncology Service, Hospital Universitario Virgen de la Victoria, Campus Teatinos s/n. 29010-Málaga, Spain Fax: þ34-95-227-94-07; e-mail contact: [email protected]
Submitted: Oct 28, 2014; Accepted: Jan 14, 2015; Epub: Jan 21, 2015
1526-8209/$ - see frontmatter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clbc.2015.01.005
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Ki-67 and Trastuzumab Therapy in HER+ Breast Cancer development of anti-HER2 therapy in patients with both early and metastatic HER2-positive (HER2þ) BC. Neoadjuvant chemotherapy is the standard treatment for patients with locally advanced or inoperable BC and is administered with the intention of downstaging the tumor and potentially improving the surgical options. A pathologic complete response (pCR) to neoadjuvant chemotherapy in combination with anti-HER2 therapy is a potential surrogate for survival in patients with HER2þ BC.2 Several large phase III clinical trials have reported a pCR rate of about 40% in patients with HER2þ BC.2-4 Conversely, there are still about 50% to 60% patients with HER2þ BC who cannot achieve pCR from trastuzumab-containing neoadjuvant chemotherapy. Therefore, tumor eradication is different between HER2 tumors, indicating intrinsic differences in their biological characteristics. We are uncertain whether clinical and pathologic parameters can be used to stratify HER2þ BC into subgroups with different pathologic responses. Consequently, the discovery of a pretreatment predictive factor may have an important role in patient selection and management. The Ki-67 proliferation marker as measured by immunohistochemical analysis has been studied as a prognostic and predictive factor in early BC. Tumors with a high expression of proliferation-related genes are associated with high pCR rates after neoadjuvant chemotherapy,5-8 lending value to the relationship between proliferation and chemosensitivity. Nonetheless, the value of the Ki-67 proliferation biomarker as a predictive factor in HER2þ BC is less clear and has not been adequately investigated. In this study, we retrospectively investigated whether proliferation measured by the Ki-67 index can be used for classification of HER2þ BC in 2 subtypes with different pathologic responses.
Patients and Methods Study Population We analyzed the data from a retrospective cohort of patients with newly diagnosed pathologically confirmed invasive clinical stage II or III BC, including inflammatory BC, between 2008 and 2013. Clinicopathologic characteristics, hormone receptor (HR) status, HER2 status, Ki-67 index, pathologic response, and follow-up data were collected. All patients provided written informed consent before neoadjuvant treatment began. The study protocols were approved by our institutional review board, and the studies were conducted in accordance with the principles of the Declaration of Helsinki.
Immunohistochemical Studies
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Biopsy using a Tru-Cut needle was performed in all eligible patients before treatment. The immunohistochemical assessment was carried out at the time of diagnosis in every tumor block. Paraffin-embedded tumor specimens were analyzed centrally by a pathologist who specializes in BC using immunohistochemical methods to measure HR status, HER2 status, and the Ki-67 index. HER2 status was determined by the HercepTest (Dako, Carpinteria, CA) as well as by a fluorescence in situ hybridization (FISH) test in biopsy specimens with a 2þ score by immunohistochemical analysis. Tumors were classified as positive with a 3þ score by immunohistochemical analysis or a 2þ score by
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immunohistochemical analysis with a positive fluorescence in situ hybridization analysis. Estrogen and progesterone receptors were measured using mononuclear antibodies (clone 6F11, prediluted and 1A6, prediluted, respectively; both from Master Diagnostica, Granada, Spain). Tumors were classified as HRþ (estrogen or progesterone, or both) if 1% of tumor cell nuclei stained positive. Ki-67 expression was evaluated with a mononuclear antibody (clone SP6, prediluted; Master Diagnostica). The semiquantitative scale imaging recommended by the American College of Pathologists was used for quantification of the Ki-67 index. The most peripheral tumor areas were selected because they constitute the line infiltration of the neoplasm. Data on HR and Ki-67 marker expression were used to classify the histologic subgroups as HRþ/Ki-67þ, HRþ/Ki-67, HR/Ki-67þ, and HR/Ki-67.
Treatment Plan and Follow-Up Patients were treated with a sequential schedule of 4 cycles of cyclophosphamide (600 mg/m2) and doxorubicin (60 mg/m2) every 3 weeks, followed by 4 cycles of weekly paclitaxel (80 mg/m2) or docetaxel (100 mg/m2) every 3 weeks combined with trastuzumab (loading dose of 8 mg/kg and then 6 mg/kg every 3 weeks) as neoadjuvant treatment. Subsequently, patients underwent surgery within 4 to 6 weeks of the last course of chemotherapy. Modified radical mastectomy or conservative surgery with axillary lymph node dissection or sentinel lymph node biopsy was performed according to the surgeons’ criteria. Patients treated with breast-conserving surgery received adjuvant radiotherapy to the whole breast after surgery. Patients who underwent mastectomy received radiation therapy to the chest wall and axillary area after surgery when they had an initial tumor 5 cm, inflammatory BC, or 4 positive nodes after neoadjuvant chemotherapy. Hormone treatment with tamoxifen or aromatase inhibitors was given in postmenopausal women with HRþ (estrogen or progesterone, or both) tumors. Premenopausal women with HR positivity were treated with tamoxifen and goserelin if they remained premenopausal after chemotherapy. No additional adjuvant chemotherapy was prescribed after surgery. Patients received adjuvant trastuzumab to complete 1 year of treatment. Once treatment was completed, patients were followed for evaluation of disease status and late-onset toxicity every 3 months during the first 2 years and every 6 months for the following 3 years or until disease progression or death.
Statistical Analysis Continuous variables were described using measures of central tendency, mean or median, measures of dispersion, standard deviation, interquartile, and range. Categorical variables were described as frequency and percentage. The comparison of proportions and significance were estimated using the c2 test. pCR was defined as the absence of invasive cells in breast and lymph nodes. For statistical analysis, the following variables were used as predictive factors of pCR: HR expression, HER2 status, Ki-67 index, age, tumor size, and histologic grade. Initially, a univariate analysis was performed for each of the variables using the log-rank test. Subsequently, a multivariate analysis was performed using the Cox proportional risk model with all the variables that had been significant in the univariate analysis.
Alfonso Sánchez-Muñoz et al The receiver operating characteristic (ROC) curve analysis was used to determine the optimal cutoff point for Ki-67 by calculating sensitivity and specificity indices corresponding to Ki-67 cutoff points selected every 10 units (range, 1-100). Cutoff points divided the study population into groups of high and low expression, which we associated with pCR achievement. We considered a cutoff point as optimal when it had the highest combined sensitivity and specificity values. Disease-free survival (DFS) was measured from the date of surgery to the documented date of progression or death from any cause or as the date of the last follow-up. Overall survival (OS) was measured from the date of surgery to the documented date of death from any cause or as the date of the last follow-up. For the calculation of DFS and OS, the Kaplan-Meier method was used, and the data were compared using the log-rank test. We studied the impact of the prognostic variables (Ki-67 index, HR expression, histologic subgroups, and pCR rate) on DFS and OS with the Cox proportional risk model. SPSS, version 14.0 (SPSS, Chicago, IL) was used for all statistical analyses.
Results Patient Characteristics
A total of 81 patients with newly diagnosed stage II/III HER2þ BC were included in the present study. Baseline clinical and pathologic characteristics are summarized in Table 1. The median age of the patients was 50 years (range, 26-73 years). Results based on ROC curves suggested that the optimal Ki-67 cutoff point that best identified patients who responded to neoadjuvant therapy was a percentage of 50% positive nuclei. Thirty-four patients (42%) had HR disease and 34 (42%) patients had a Ki-67 marker 50%. Data on HR and Ki-67 expression were used to classify the patients with HER2þ BC, resulting in 14 (17%) cases of HRþ/Ki-67 50%, 33 (41%) cases of HRþ/Ki-67 < 50%, 20 (25%) cases of HR/Ki-67 50%, and 14 (17%) cases of HR/Ki-67 < 50%.
Table 1 Baseline Clinical and Pathologic Characteristics of the Study Population (N [ 81) Characteristics Age, years, median (range)
N (%) 50 (26-73)
Histopathologic Type Ductal
73 (90)
Lobular
1 (1)
Other
7 (9)
Hormonal Status Premenopausal
55 (67)
Postmenopausal
26 (33)
Tumor Stage T1
6 (8)
T2
47 (58)
T3
14 (17)
T4
14 (17)
Hormone Receptors
N (%)
Positive
47 (58)
Negative
34 (42)
Ki-67 Index 50%
34 (42)
<50%
47 (58)
Tumor Grade I
8 (10)
II
22 (27)
III
29 (36)
NA
22 (27)
Histologic Subgroups HRþ/Ki-67 50%
14 (17)
HRþ/Ki-67 <50%
33 (41)
HR/Ki-67 50%
20 (25)
HR/Ki-67 <50%
14 (17)
Efficacy A total of 42 patients (60%) underwent conservative surgery, whereas the remaining 39 patients (40%) underwent radical surgery. pCR was achieved in 33 patients (41%). The median Ki-67 index was significantly higher in tumors with pCR (53%) compared with tumors without pCR (30%) (P < .001). The pCR rate was significantly different between the histologic subgroups of HER2þ BC, being higher in HR/Ki-67 50% (70%) and HRþ/ Ki-67 50% (71%) compared with HR/Ki-67 < 50% (22%) and HRþ/Ki-67 < 50% (18%) (P < .001). Univariate analysis showed that a Ki-67 index 50% or Ki-67 as a categorical variable, poorly differentiated tumors and histologic subgroups defined by Ki-67, and HR expression exhibited an independent and statistically significant association with pCR. HR expression was associated with a higher trend of pCR but did not reach statistical significance. A multivariate analysis based on histologic subgroups, grade, and Ki-67 expression revealed that Ki67 50% was the only independent predictive factor for pCR (P ¼ .003; odds ratio [OR], 0.133; 95% confidence interval [CI], 0.036-0.5) (Table 2). Over a median follow-up of 32 months (range, 14-48 months), 25 (31%) patients had disease relapse and 15 (18%) patients died of
tumor progression. Patients who achieved a pCR had a significantly lower recurrence rate (P ¼ .001) and higher OS (P ¼ .013) compared with those who did not. However, there were no statistically significant differences in DFS and OS in relation to HR expression, Ki-67 index as a continuous or categorical variable, and histologic subgroups of HER2þ tumors.
Discussion This study assessed the role of proliferation measured by the Ki-67 index as a predictive factor for pCR to trastuzumab-based chemotherapy in HER2þ BC. Results showed a pCR rate of
Table 2 Multivariate Analysis of Biological Markers and Pathologic Complete Response Variable Ki-67 50% versus Ki-67 <50% Histologic subgroups Grade I/II versus grade III
Odds Ratio (95% CI)
P Value
0.133 (0.036-0.5) 0.39 (0.064-2.46 0.306 (0.79-1.18)
.003 .32 .086
Abbreviation: CI ¼ confidence interval.
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40%, similar to that reported in several large phase III clinical trials.2-4 The pCR rate was higher in poorly differentiated and Ki-67 50% tumors. The HR expression was associated with a higher trend of pCR but did not reach statistical significance. The occurrence of pCR was significantly different between histologic subgroups, being higher (> 3 times) in HR/Ki-67 50% and HRþ/Ki-67 50% tumors compared with HRþ/Ki-67 < 50% and HRþ/Ki-67 < 50% tumors. These results suggest that high proliferation measured by Ki-67 50% is an independent predictive factor for pCR in patients with HER2þ BC treated with trastuzumab-based chemotherapy. Regarding HR expression in pCR prediction, interpretation should be cautious because results in this field are inconsistent. The combination of anti-HER2 therapy and neoadjuvant chemotherapy showed that HR tumors had higher pCR rates in the GeparQuattro, Neo-ALTTO (Lapatinib with Trastuzumab for HER2Positive Early Breast Cancer) and CHER-LOB (Chemotherapy, Herceptin and Lapatinib in Operable Breast Cancer) trials.4,9,10 In contrast, results from the TECHNO (Taxol Epirubicin Cyclophosphamide Herceptin Neoadjuvant) study showed that no clinical and histologic parameters, including HR status, were capable of predicting pCR.2 The NeoSphere trial reported that HR disease had higher pCR rates than did HRþ disease, but the difference was statistically significant only in the 2 dual HER2 targeting groups and not in the 2 single HER2 targeting groups.11 Those findings do not allow one to exclude a potential benefit of trastuzumab in HRþ tumors. In fact, trastuzumab-based therapy in the adjuvant setting has been shown to reduce the risk of recurrence across all subgroups, although the magnitude of benefit seemed to be lower in HRþ tumors than in HR tumors.12 Cell proliferation in cancer has not been assessed in combination with HR expression. Taking these data together, the value of HR expression in predicting pCR for HER2þ tumors needs to be ascertained by further investigations. Ki-67 is a well-established cell proliferation marker in cancer. Previous data underscore the importance of proliferation and support its incorporation into therapy decisions. First, the results of several meta-analyses suggested that a high Ki-67 index confers a higher risk of relapse and a worse survival in patients with early BC.13-15 Second, proliferation has been identified as the main component and common denominator for predicting clinical outcome in several gene signatures. In this respect, a meta-analysis of publicly available BC gene expression data revealed that > 70% of the genes associated with patient survival were correlated with the proliferation pathway, whereas 26% of the genes were related to estrogen receptor signaling and only 2% to HER2 gene amplification. The expression of genes related to proliferation was constitutively high in HR/HER2 and HER2þ tumors.16 Nonetheless, the value of the Ki-67 proliferation biomarker as a predictive factor in HER2þ BC is less clear. Gene expression analysis in HER2þ disease identifies all the main intrinsic molecular subtypes of BC (luminal A, luminal B, HER2þ, and basal-like) and are not fully concordant with pathologic markers.17,18 Among them, the Cancer Genome Atlas project reported a subset of HER2þ tumors with a prediction analysis of microarray (PAM50) HER2-enriched (HER2-E) gene expression characterized by the highest activation of the HER2-regulated genes and proliferation/ cell-cycleerelated genes, suggesting that they may benefit the most
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from anti-HER2 therapies.19 A retrospective exploratory analysis of 114 patients with HER2þ tumors enrolled in the NOAH (Neoadjuvant Herceptin) trial showed that HER2-E tumors with a high proliferation score had the highest benefit from trastuzumabbased chemotherapy.20 Patients who achieved a pCR had significantly fewer recurrences and higher OS. However, when we compared the impact of pCR on prognosis after neoadjuvant therapy in the histologic subgroups of HER2þ BC, no significant difference in DFS and OS was detected: there was no significant difference in DFS and OS between patients with Ki-67 50% and Ki-67 < 50% tumors. This may result from the small case number in subgroups and short follow-up duration that could mask the potential associations, precluding the ability to reach a definitive conclusion. Another retrospective study including 102 cases of HER2þ BC reported that HR tumors and high Ki-67 expression were predictors for pCR but not for DFS and OS, although different neoadjuvant chemotherapy regimens combined with trastuzumab were administered, and ROC curve methodology was not used to find the optimal Ki-67 cutoff point.21 Notably, Von Minckwitz et al, in a large cohort of 6377 patients,22 demonstrated that a pCR after neoadjuvant chemotherapy was associated with an improved DFS only in patients with HER2þ (nonluminal) and triple-negative tumors, but not for luminal B/HER2þ and luminal A tumors. Our study does have certain limitations. First, it was a retrospective study with a relatively small number of patients and short follow-up. Second, it may be difficult to reproduce the centralized Ki-67 expression analysis rate in daily practice. However, we believe that our data can provide useful information for clinical practice because of the homogeneity of the treatment received by our patients in the neoadjuvant setting. An ROC curve methodology was used to obtain the Ki-67 cutoff that best identified patients who responded to trastuzumab-based chemotherapy. In conclusion, HER2þ BC is a biologically heterogeneous disease, and not all patients benefit from trastuzumab-based chemotherapy to the same extent. Although next-generation tumor sequencing may identify determinants of exquisite sensitivity to anti-HER2 therapy with or without chemotherapy, the proliferation measured by the Ki-67 marker could be a useful tool to better discriminate which patients will achieve a pCR.
Clinical Practice Points It is still unknown whether clinical and pathologic parameters
can be used to stratify HER2þ BC into subgroups with a different pathologic response. If we find a pretreatment predictive factor, it would have an important role in patient selection and management. Ki-67 is a well-established cell proliferation marker. Previous data underscore the importance of proliferation and support its incorporation into therapy decisions. Several meta-analyses suggested that a high Ki-67 index confers a higher risk of relapse and worse survival in patients with early BC. Furthermore, proliferation has been identified as the main component and common denominator for predicting clinical outcome in several gene signatures. Tumors with a high expression of proliferation-related genes are associated with high pCR rates after neoadjuvant chemotherapy, lending value to the relationship between proliferation and
Alfonso Sánchez-Muñoz et al chemosensitivity. However, the value of the Ki-67 proliferation biomarker as a predictive factor in HER2þ BC is less clear and has not been adequately investigated. We analyzed if proliferation measured by the Ki-67 index can be used for classification of HER2þ BC in subtypes with different pathologic responses. This study assessed the role of proliferation measured by the Ki-67 index as a predictive factor for pCR to trastuzumab-based chemotherapy in HER2þ BC. In our study, the occurrence of pCR was significantly different between histologic subgroups, being higher in HR/Ki-67 50% and HRþ/Ki-67 50% tumors compared with HRþ/ Ki-67 < 50% and HRþ/Ki-67 < 50% tumors. These results suggest that high proliferation measured by Ki-67 50% is an independent predictive factor for pCR in patients with HER2þ BC treated with trastuzumab-based chemotherapy. The impact on clinical practice could be to use the proliferation measured by the Ki-67 marker as a useful tool to better discriminate which patients will achieve a pCR.
Disclosure The authors have stated that they have no conflicts of interest.
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