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Originally published as JCO Early Release 10.1200/JCO.2007.11.7762 on October 22 2007 © 2007 American Society of Clinical Oncology.
Different Prognostic Value of Cytokeratin-19 mRNA–Positive Circulating Tumor Cells According to Estrogen Receptor and HER2 Status in Early-Stage Breast Cancer
From the Department of Medical Oncology and Pathology, University General Hospital of Heraklion; Laboratory of Tumor Cell Biology and Department of Biostatistics, School of Medicine, University of Crete, Crete; Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, Greece; and the Functional Genomics and Translational Research Unit, Jules Bordet Institut, Brussels, Belgium Address reprint requests to Dimitris Mavroudis, MD, PhD, Department of Medical Oncology, University General Hospital of Heraklion, PO Box 1352, Heraklion 711 10, Crete, Greece; e-mail: mavrudis{at}med.uoc.gr
Purpose To examine the prognostic value of cytokeratin-19 (CK-19) mRNA–positive circulating tumor cells (CTCs) in early-stage breast cancer patients focusing on clinically relevant subgroups based on estrogen receptor (ER) and HER2 expression. Patients and Methods CK-19 mRNA–positive CTCs were detected by real-time reverse transcriptase polymerase chain reaction in the blood of 444 consecutive, stage I-III, breast cancer patients before initiation of adjuvant chemotherapy. The association between detection of CK-19 mRNA–positive CTCs and clinical outcome was analyzed for patients with ER-positive, ER-negative, triple-negative, HER2-positive, and ER-positive/HER2-negative tumors. Results CK-19 mRNA–positive CTCs were detected in 181 (40.8%) of 444 patients; 109 (41.9%) of 260 patients with ER-positive tumors; 71 (40.6%) of 175 patients with ER-negative tumors; 27 (35%) of 77 patients with triple-negative tumors; 35 (39.8%) of 88 patients with HER2-positive tumors; and 82 (44.1%) of 186 patients with ER-positive/HER2-negative tumors. After a median follow-up of 53.5 months, patients with CK-19 mRNA–positive CTCs experienced reduced disease-free survival (DFS; P < .001) and overall survival (OS; P < .001); this was mainly observed in patients with ER-negative (P < .001 and P < .001, respectively) but not ER-positive tumors (P = .172 and P = .425, respectively) and in patients with triple-negative (P = .008 and P = .001, respectively) and HER2-positive (P = .023 and P = .040, respectively) but not ER-positive/HER2-negative tumors (P = .210 and P = .578, respectively). In multivariate analysis, the interaction between CK-19 mRNA–positive CTCs and ER status was the strongest independent prognostic factor for reduced DFS (hazard ratio [HR], 3.808; 95% CI, 2.415 to 6.003; P < .001) and OS (HR, 4.172; 95% CI, 2.477 to 9.161; P < .001). Conclusion Detection of CK-19 mRNA–positive CTCs before adjuvant chemotherapy predicts poor clinical outcome mainly in patients with ER-negative, triple-negative, and HER2-positive early-stage breast cancer.
Different markers have been used for the molecular detection of circulating tumor cells (CTCs). Among them, cytokeratin-19 (CK-19), a cytoskeletal protein expressed on epithelial but not on mesenchymal cells, has been the most extensively studied.1 Although studies using reverse transcriptase polymerase chain reaction (RT-PCR) of CK-19 mRNA as a biomarker for CTCs, have been flawed by problems of illegitimate expression and pseudogene amplification, our group has optimized the assay to improve its sensitivity and specificity.2,3 Thus, using a highly sensitive and specific real-time RT-PCR assay,3 we demonstrated that the detection of CK-19 mRNA–positive CTCs before the initiation of adjuvant chemotherapy was an independent prognostic factor for disease recurrence and decreased survival in patients with node-negative breast cancer.4 Until now the prognostic value of micrometastatic disease has been studied without considering the heterogeneity of breast cancer.4-9 Primary tumor gene expression profiling studies using unsupervised hierarchical clustering analysis have shown that breast tumors are grouped into two main clusters: predominately estrogen receptor (ER) negative and ER positive.10-15 Moreover, from these studies, at least three stable molecular subtypes have been consistently identified, namely the ER-negative/HER2-negative or basal-like, the HER2-positive, and the ER-positive/HER2-negative or luminal subtypes.16 Apart from differences in gene expression profiles, ER-negative and ER-positive tumors differ in their response to treatment and clinical course.17 The last Early Breast Cancer Trialists’ Collaborative Group meta-analysis demonstrated that women with ER-negative early-stage breast cancer experienced relapse more often than those with ER-positive tumors during the first 5 years, while the opposite was true for the period of 5 to 15 years after diagnosis.17 Similarly, the breast cancer molecular subtypes have been reported to differ in their response to treatment and clinical outcome.11,15,16,18 In this study we sought to validate our previous results regarding the prognostic value of peripheral blood CK-19 mRNA–positive cells in an extended cohort of 444 patients with stage I-III breast cancer. Furthermore, considering the heterogeneity of the disease, we investigated the prognostic value of CK-19 mRNA–positive cells, in the ER-negative and ER-positive subgroups and in the three molecular subtypes—namely the ER-negative/HER2–negative/progesterone receptor (PR)-negative or triple-negative, the HER2-positive, and the ER-positive/HER2-negative subtypes.
Patients From 1997 until 2004, a total of 444 consecutive patients who had all received adjuvant chemotherapy for stage I-III breast cancer at the Department of Medical Oncology of the University Hospital of Heraklion (Crete, Greece) and who had sufficient follow-up (at least 10 months) were included in this study. For every patient enrolled, a complete diagnostic evaluation to exclude the presence of distant metastasis was performed consisting of chest x-rays, mammography, ultrasound of the liver, and a whole-body bone scan. Computed tomography scans and/or magnetic resonance imaging studies were performed if clinically indicated (patients with symptoms, physical signs, or abnormal findings on chest x-ray, ultrasound of the liver, and bone scan). The administration of adjuvant chemotherapy and hormone treatment was decided independently of the CK-19 mRNA detection. All patients included in this study received adjuvant chemotherapy and most of them were treated in the context of research protocols of the Hellenic Oncology Research Group. Adjuvant chemotherapy regimens consisted of either FEC (fluorouracil 700 mg/m2 day 1 plus epirubicin 75 mg/m2 day 1 plus cyclophosphamide 700 mg/m2 day 1 every 3 weeks for six cycles) or EC-T (epirubicin 75 mg/m2 day 1 plus cyclophosphamide 700 mg/m2 day 1 every 3 weeks for four cycles followed by docetaxel 100 mg/m2 day 1 every 3 weeks for four additional cycles) or classical CMF (cyclophosphamide 100 mg/m2 orally days 1 through 14, methotrexate 40 mg/m2 days 1 and 8, and fluorouracil 600 mg/m2 days 1 and 8 every 4 weeks for six cycles). All patients with ER- and/or PR-positive tumors received tamoxifen 20 mg daily for 5 years; premenopausal women also received luteinizing hormone-releasing hormone (LHRH) analogs for 2 years. There were no subgroups of patients who received hormone therapy only or no systemic therapy at all. Patients with HER2-positive tumors did not receive adjuvant trastuzumab because all patients were enrolled before the positive results from the adjuvant trastuzumab trials were reported.19,20 Patient follow-up consisted of clinical examination with laboratory and imaging studies every 3 months for the first 2 years, every 6 months for the next 3 years, and yearly thereafter. All patients signed an informed consent to participate in the study which was approved by the ethics and scientific committees of our institution.
Clinical Samples, Real-Time RT-PCR Assay for CK-19 mRNA
The procedures of RNA extraction and cDNA synthesis have already been described elsewhere.3,4 The real-time RT-PCR assay for CK-19 mRNA–positive CTCs and the primers used, have been previously described in detail and were used in this study without any modification.3,4 According to the analytic detection limit of our assay, the presence of
Immunohistochemistry for HER2, ER, and PR ER and PR expression of the primary tumors was detected by IHC with monoclonal antibodies to ER and PR (DakoCytomation, Denmark, A/S), respectively, using the same automated system as above. All carcinoma cells in three hot spots (areas with the highest density of ER-positive or PR-positive carcinoma cell nuclei) per slide were evaluated independently by two pathologists (M.K., E.N.S.), and the mean of the two independent counts was considered the final value for each field and hot spot. The final immunoreactivity index (score) was calculated as the mean percentage of ER-positive or PR-positive carcinoma cell nuclei in the three hot spots. Specimens were interpreted as positive for ER or PR if at least 10% of the cells demonstrated nuclear staining of any intensity of reactivity, from 1+ to 3+. Staining intensity was graded as negative (0), weak (1+), intermediate (2+), or strong (3+), and reported separately.
Molecular Subtypes
Statistical Analysis
Patient Characteristics The characteristics of the 444 patients are presented in Table 1. The patients’ median age was 54 years (range, 26 to 78 years). Primary tumor size was less than or equal to 2 cm, absence of axillary lymph node infiltration and ER-positive disease were observed in 35.4%, 36.7%, and 58.6% of the patients, respectively. Overall, CK-19 mRNA–positive CTCs were detected in 181 patients (40.8%) and detection of CTCs was not significantly associated with any of the known clinicopathological characteristics (Table 1). There was no significant difference in the proportion of patients with detectable CK-19 mRNA–positive CTCs in the ER-negative and ER-positive subgroups (40.6% and 41.9%, respectively; P = .779) as well as in the three molecular subtypes namely the triple negative, the HER2 positive, and the ER positive/HER2 negative (35%, 39.8%, and 44.1%, respectively; P = .385). Similarly, there was no significant difference in the distribution (median, range) of CK-19 mRNA values between the ER-negative and ER-positive subgroups (P = .559) and between the three molecular subtypes (P = .185; Appendix Table A1, online only).
Detection of CK-19 mRNA–Positive CTCs and Clinical Outcome We first investigated the association between detection of CK-19 mRNA–positive CTCs and clinical outcome of the whole population. The median follow-up was 53.5 months (range, 10 to 106 months). During this period, 94 (21%) of 444 patients relapsed (18 local and 76 distant) and 42 patients (9%) died due to disease progression. Median follow-up for alive patients was 56.4 months (range, 10 to 96 months) and 45% of alive patients had follow-up for longer than 5 years. Relapse and death were more frequent in patients with CK-19 mRNA–positive CTCs (P < .001 and P = .001; Table 2). The median DFS and OS of patients with and without CK-19 mRNA–positive CTCs cannot, as of yet, be estimated; however, patients with CK-19 mRNA–positive CTCs had significantly shorter DFS (P < .001; Fig 1A) and OS (P < .001; Fig 2A).
Because axillary nodal status is very important in tumor staging, we sought to examine the prognostic value of the detection of CK-19 mRNA–positive CTCs in patients’ groups based on nodal infiltration. The detection of CK-19 mRNA–positive CTCs was associated with worse DFS and OS both in the node-negative (log-rank test, P = .003 and P = .001, respectively) and in the node-positive subgroup (log-rank test, P = .001 and P = .046, respectively).
Subgroups Based on ER Status ER-positive patients. In the ER-positive subgroup (n = 260), relapses and deaths did not differ significantly for patients with detectable CK-19 mRNA–positive CTCs and those without (P = .315 and P = .499, respectively; Table 2). Although no significant differences in DFS and OS were observed, with longer follow-up there was a nonsignificant trend for worse DFS in ER-positive patients with detectable CK-19 mRNA–positive CTCs, (P = .172; Fig 1C and P = .425; Fig 2C, respectively). DFS and OS of ER-positive/CK-19–negative, ER-positive/CK-19–positive, ER-negative/CK-19–negative, ER-negative/CK-19–positive patients is depicted in Appendix Figure A1 (online only).
Subgroups Based on Molecular Subtypes HER2-positive patients. In the HER2-positive subgroup (n = 88), relapses and deaths were observed with higher frequency in patients with CK-19 mRNA–positive CTCs (P = .033 and P = .038, respectively; Table 2). Patients with CK-19 mRNA–positive CTCs had also significantly shorter DFS and OS (P = .023, Fig 1E; and P = .040, Fig 2E, respectively). ER-positive/HER2-negative patients. Among the ER-positive/HER2-negative subgroup (n = 186), disease recurrences and deaths were not significantly different in patients with detectable CK-19 mRNA–positive CTCs versus those without (P = .392 and P = .731, respectively; Table 2). Although no significant differences in DFS and OS were observed, with longer follow-up there was a nonsignificant trend for worse DFS in ER-positive/HER2-negative patients with detectable CK-19 mRNA–positive CTCs (P = .210; Fig 1F and P = .578; Fig 2F, respectively). The 5-year DFS and OS for patients with or without CK-19 mRNA–positive CTCs in the entire population, the subgroups based on ER status, and the three molecular subtypes are depicted in Appendix Table A2 (online only).
Univariate and Multivariate Analysis
In this study of an extended cohort of 444 node-negative and node-positive breast cancer patients, we confirmed our previous results4 on the adverse, independent prognostic value of CK-19 mRNA–positive CTCs in early-stage breast cancer. The node-negative patients included in this analysis were to a large extent different from the node-negative patients of our previous report.4 In this study, we report for the first time (to our knowledge) that the presence of CK-19 mRNA–positive CTCs predicted poor clinical outcome (relapse and death) in patients with ER-negative but not ER-positive early-stage breast cancer, despite the similar proportion of patients with detectable CK-19 mRNA–positive CTCs in both subgroups. Similarly, the presence of CK-19 mRNA–positive CTCs was associated with shorter DFS and OS in the triple-negative and HER2-positive, but not in the ER-positive/HER2-negative subgroups. In multivariate analysis, the interaction between CK-19 mRNA–positive CTCs and ER status was the strongest independent prognostic factor for DFS and OS. During the 53.5-month median follow-up, patients with ER-negative tumors relapsed more frequently than patients with ER-positive tumors (28% v 16.5%; P = .004). This is in accordance with the Oxford meta-analysis, where ER-negative patients relapsed more frequently than ER-positive patients during the first 5 years after diagnosis.17 Therefore, CK-19 mRNA–positive CTCs are correlated with the development of early metastasis within the first 5 years in patients with ER-negative but not in patients with ER-positive disease. A possible explanation for this is that in the ER-negative group, micrometastatic cells could be targeted only by adjuvant chemotherapy, whereas in the ER-positive subgroup they could be controlled by both adjuvant chemotherapy as well as hormone treatment. Nevertheless, it appears that the curves in the ER-positive group tend to separate after 5 years (Figs 1C and 2C), which would be consistent with the 5 years of tamoxifen received by ER-positive patients. Therefore, with longer follow-up, initial CK-19 mRNA levels might also predict long-term outcome of ER-positive patients and thus might help identifying those who could benefit from extended adjuvant hormone therapy. Therefore, the 53.5-month median follow-up of this study is relatively short to draw definite conclusions for patients with ER-positive tumors. Furthermore, since all patients received adjuvant chemotherapy with or without hormone therapy, the information we derived from the detection of CK-19 mRNA–positive cells is not purely prognostic. Validation of our results in a well-designed, prospective, multicenter trial, where therapeutic decision will be based on CK-19 mRNA detection, is needed. Further development of this biomarker should take into consideration the problems related to tumor marker prognostic studies described by McShane et al.23 Based on our results, we could also hypothesize that CK-19 mRNA–positive CTCs have different biologic behavior in patients with ER-negative (basal-like CTCs) and ER-positive (luminal-like CTCs) tumors. However, molecular and immunophenotypic characterization of CK-19 mRNA–positive CTCs in patients with ER-negative and ER-positive disease is required to further validate this hypothesis. Furthermore, it could be argued that the presence of CK-19 mRNA–positive CTCs in a given patient may reflect the biology of the primary tumor as determined by the ER status. According to Klein et al, 24 occult micrometastatic cells are heterogeneous in a given patient with early-stage breast cancer. After the identification of tumorigenic breast cancer cells,25 it would be interesting to examine whether there are different subpopulations of micrometastatic cells with stem-cell/progenitor properties responsible for the development of metastasis in ER-negative and ER-positive patients. Another important question is whether the study of CK-19 mRNA–positive CTCs could provide additional prognostic information to currently developed gene expression signatures.26-30 Interestingly, according to these signatures, the majority of ER-negative tumors are assigned to the poor outcome group, whereas ER-positive tumors comprise a mixture of poor and good prognosis tumors.30-33 Therefore, gene expression signatures are more useful for predicting clinical outcome in ER-positive disease.15,34 On the contrary, our results indicate that the detection of CK-19 mRNA–positive CTCs could subdivide ER-negative and triple-negative as well as HER2-positive patients into better and worse prognosis groups. Therefore, it would be interesting to prospectively assess the hypothesis that by combining information from primary tumor gene expression profiling and the detection of micrometastatic cells, we could further improve prognosis in early-stage breast cancer. Furthermore, the monitoring of CK-19 mRNA–positive CTCs could be used to investigate the potential value of secondary adjuvant strategies. We have previously reported that a short course of trastuzumab could eliminate chemotherapy-resistant CK-19 mRNA–and HER2 mRNA–positive CTCs and bone marrow disseminated tumor cells for patients with breast cancer.35 Thus, the identification of suitable targets for individualized adjuvant breast cancer treatment may need to take into account, not only the characteristics of the primary tumor, but also those of micrometastatic cells.
The author(s) indicated no potential conflicts of interest.
Conception and design: Michail Ignatiadis, Christos Sotiriou, Vassilis Georgoulias, Dimitris Mavroudis Financial support: Vassilis Georgoulias, Dimitris Mavroudis Administrative support: Michail Ignatiadis, Nikos Xenidis, Maria Perraki, Stella Apostolaki, Eleni Politaki, Aliki Stathopoulou, Evi Lianidou, Grigorios Chlouverakis Provision of study materials or patients: Vassilis Georgoulias, Dimitris Mavroudis Collection and assembly of data: Michail Ignatiadis, Nikos Xenidis, Maria Perraki, Stella Apostolaki, Eleni Politaki, Maria Kafousi, Efstathios Stathopoulos, Aliki Stathopoulou, Evi Lianidou Data analysis and interpretation: Michail Ignatiadis, Nikos Xenidis, Maria Perraki, Stella Apostolaki, Eleni Politaki, Maria Kafousi, Efstathios Stathopoulos, Aliki Stathopoulou, Evi Lianidou, Grigorios Chlouverakis, Christos Sotiriou, Vassilis Georgoulias, Dimitris Mavroudis Manuscript writing: Michail Ignatiadis, Maria Kafousi, Efstathios Stathopoulos, Grigorios Chlouverakis, Christos Sotiriou, Vassilis Georgoulias, Dimitris Mavroudis Final approval of manuscript: Michail Ignatiadis, Nikos Xenidis, Maria Perraki, Stella Apostolaki, Eleni Politaki, Maria Kafousi, Efstathios Stathopoulos, Aliki Stathopoulou, Evi Lianidou, Grigorios Chlouverakis, Christos Sotiriou, Vassilis Georgoulias, Dimitris Mavroudis
We thank Marc Buyse, PhD, for reviewing the manuscript.
published online ahead of print at www.jco.org on October 22, 2007. M.I. and N.X. have contributed equally to this article. Authors’ disclosures of potential conflicts of interest and author contributions are found at the end of this article.
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Collett D: Modeling Survival Data in Medical Research. London, United Kingdom, Chapman and Hall/CRC, 1999 23. McShane LM, Altman DG, Sauerbrei W: Reporting recommendations for tumor marker prognostic studies. J Clin Oncol 23:9067-9072, 2005 24. Klein CA, Blankenstein TJ, Schmidt-Kittler O, et al: Genetic heterogeneity of single disseminated tumour cells in minimal residual cancer. Lancet 360:683-689, 2002[CrossRef][Medline] 25. Al-Hajj M, Wicha MS, Ito-Hernandez A: Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A 100:3983-3988, 2003 26. van't Veer LJ, Dai H, van de Viljer MJ, et al: Gene expression profiling predicts clinical outcome of breast cancer. Nature 415:530-536, 2002[CrossRef][Medline] 27. Wang Y, Klijn JG, Zhang Y, et al: Gene-expression profiles to predict distant metastasis of lymph-node-negative primary breast cancer. Lancet 365:671-679, 2005[Medline] 28. Chang HY, Nuyten DS, Sneddon JB, et al: Robustness, scalability, and integration of a wound-response gene expression signature in predicting breast cancer survival. Proc Natl Acad Sci U S A 102:3738-3743, 2005 29. Sotiriou C, Wirapati P, Loi S, et al: Gene expression profiling in breast cancer: Understanding the molecular basis of histologic grade to improve prognosis. J Natl Cancer Inst 98:262-272, 2006 30. Liu R, Wang X, Chen GY, et al: The prognostic role of a gene signature from tumorigenic breast-cancer cells. N Engl J Med 356:217-226, 2007 31. van de Vijver, He YD, van't Veer LJ, et al: A gene-expression signature as a predictor of survival in breast cancer. N Engl J Med 347:1999-2009, 2002 32. Buyse M, Loi S, van't VL, et al: Validation and clinical utility of a 70-gene prognostic signature for women with node-negative breast cancer. J Natl Cancer Inst 98:1183-1192, 2006 33. Desmedt C, Piette F, Loi S, et al: Strong time dependence of the 76-gene prognostic signature for node-negative breast cancer patients in the TRANSBIG multicenter independent validation series. Clin Cancer Res 13:3207-3214, 2007 34. Desmedt C, Sotiriou C. Proliferation: The most prominent predictor of clinical outcome in breast cancer. Cell Cycle 5:2198-2202, 2006[Medline] 35. Bozionellou V, Mavroudis D, Perraki M, et al: Trastuzumab administration can effectively target chemotherapy-resistant cytokeratin-19 messenger RNA-positive tumor cells in the peripheral blood and bone marrow of patients with breast cancer. Clin Cancer Res 10:8185-8194, 2004 Submitted March 19, 2007; accepted August 20, 2007.
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Copyright © 2007 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
0.6 MCF-7 equivalents/5 µg of total RNA was a positive result. Using the above cutoff, only two of 89 healthy female donors were positive (2.2%).
