Analysis of the Mammaprint Breast Cancer Assay in a Predominantly Postmenopausal Cohort Ben S
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Imaging, Diagnosis, Prognosis Analysis of the MammaPrint Breast Cancer Assay in a Predominantly Postmenopausal Cohort Ben S. Wittner,1, 2 Dennis C. Sgroi,1, 3 Paula D. Ryan,1, 2 Ta ko J. B r uin s m a , 4 Annuska M. Glas,4 Anitha Male,1, 3 Sonika Dahiya,1, 3 Karleen Habin,1Rene Bernards,4,5 Daniel A. Haber,1, 2 LauraJ. Van’t Veer,4,6 and Sridhar Ramaswamy1, 2 Abstract Purpose: Most node-negative breast cancer patients are older and postmenopausal and are increasingly being offered adjuvant chemotherapy despite their low overall risk of distant relapse. A molecular diagnostic test with high negative predictive value (NPV) for distant metastasis in this subgroup would spare many older breast cancer patients adjuvant treatment. Experimental Design:We determined the NPVand positive predictive value of the MammaPrint assay in breast cancer patients who were consecutively diagnosed and treated at the Massachu- setts General Hospital between 1985 and 1997. Primary tumors from 100 patients with node- negative, invasive breast cancer (median age, 62.5 years; median follow-up, 11.3 years) were subjected to MammaPrint analysis and classified as being at either low or high risk for distant metastasis. Results: The MammaPrint 70-gene signature displayed excellent NPV as in previous studies, correctly identifying 100% of women at low risk for distant metastases at 5 years. However, this assay had a lower positive predictive value (12% at 5 years) than previously observed. Conclusions: The MammaPrint assay was originally designed to identify younger breast cancer patients at low risk for distant metastasis, who might consequently be spared systemic treatment. We show here that the same signature has a very high NPV for distant recurrence after adjuvant treatment in older breast cancer patients. Breast cancer is clinically heterogeneous. Patients differ widely are imperfect (3, 4). Alarge number of additional factors have with respect to natural history and response to treatment (1). been evaluated for determining likely prognosis or treatment This clinical heterogeneity is probably due to the varying response, but most have limited power and few reflect the mutational spectrum or cell type of origin of tumors and genetics of the disease (5). complex genetic differences among individuals (2). These DNAmicroarrays have recently been used to obtain genome- factors in combination influence the expression of many genes wide views of human tumor gene expression, and these studies involved in tumor growth, invasion, metastasis, and survival. have identified cancer biomarkers with diagnostic, prognostic, Although consensus criteria, based on clinical and histopath- and predictive potential in a wide variety of solid tumors (6). ologic features [age, tumor size, histologic tumor type, Breast cancer has proven particularly fertile ground for micro- pathologic grade, estrogen receptor (ER) status, and axillary array-based biomarker discovery, and recent studies have lymph node status], are currently used to assess risk of distant suggested that the clinical behavior of a patient’s cancer is relapse in patients with breast cancer, these clinical measures encoded in the gene expression profile of their primary tumor (7–9). Members of our group initially reported a 70-gene prognostic Authors’ Affiliations: 1Massachusetts General Hospital Cancer Center; microarray expression signature in primary tumors from Departments of 2Medicine and 3Pathology, Harvard Medical School, Boston, patients with breast cancer who were diagnosed and treated 4 5 Massachusetts and Agendia BV; Divisions of Molecular Carcinogenesis and between 1983 and 1996 at the Netherlands Cancer Institute 6Diagnostic Oncology, Netherlands Cancer Institute, Amsterdam, the Netherlands Received 10/24/07; revised 12/24/07; accepted 2/1/08. (NKI; ref. 9). Subsequently, retrospective validation studies Grant support: Avon Foundation (Massachusetts General Hospital Cancer showed that this 70-gene expression signature indeed can be Center) and Dutch National Genomics Initiative ‘‘Cancer Genomics Center’’ used to classify younger patients, with either node-positive or (Netherlands Cancer Institute). node-negative disease, into groups with significantly different The costs of publication of this article were defrayed in part by the payment of page probabilities of remaining metastasis-free (10, 11). Moreover, charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. the prognostic value of the 70-gene signature is additive to Note: Supplementary data for this article are available at Clinical Cancer Research currently used St. Gallen (3) or NIH (4) consensus criteria for Online (http://clincancerres.aacrjournals.org/). assessing risk of distant relapse. These initial observations Requests for reprints: Sridhar Ramaswamy, Massachusetts General Hospital suggest that the 70-gene signature is a powerful predictor of Cancer Center, 185 Cambridge Street, Boston, MA 02114. Phone: 617-643-3140; Fax: 617-643-3170; E-mail: [email protected]. clinical outcome in younger women with early-stage breast F 2008 American Association for Cancer Research. cancer and that clinically defined high-risk patients with doi:10.1158/1078-0432.CCR-07-4723 microarray-defined ‘‘good prognosis’’ disease might actually Clin Cancer Res 2008;14(10) May 15, 2008 2988 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on October 3, 2021. © 2008 American Association for Cancer Research. MammaPrint Breast CancerAssay be at low risk for developing distant metastases. In theory, assessed as absent or present. ER expression was estimated using ER these patients may be spared adjuvant chemotherapy with transcript levels on the microarray for each tumor. Fifteen to thirty 30- A its associated toxicity risk. These issues are currently being m sections were used for RNAisolation. Total RNAwas isolated with addressed in a prospective, multicenter, clinical trial in RNAzol B and dissolved in RNase-free water. Twenty-five micrograms of total RNAwere treated with DNase using the Qiagen RNase-free DNase Europe (12). kit and RNeasy spin columns for cleanup. RNAwas then dissolved in Most patients with breast cancer, however, are older than the RNase-free water to a final concentration of 0.2 Ag/AL. cohorts used to define and evaluate the 70-gene signature and Microarray expression profiling. RNAlabeling, microarray hybrid- + usually present with smaller, early-stage, ER tumors that tend ization, and scanning were done at Agendia using standardized to metastasize less frequently. In addition, such patients often methods and protocols (14). Briefly, cRNAwas generated by in vitro have comorbid disease that further complicates decisions about transcription with T7 RNApolymerase (Low Input Fluorescent Labeling the use of adjuvant chemotherapy. Despite this concern, many kit, Agilent Technologies) and labeled with Cy3 or Cy5 (Cy Dye, Perkin- older patients are increasingly being offered adjuvant chemo- Elmer). Cy-labeled cRNAfrom one breast cancer tumor was mixed with therapy. Amolecular test that can accurately identify older the same amount of reverse-color Cy-labeled product from a standard patients at low risk of distant metastasis, who could then be ‘‘mamma-reference’’ pooled RNAcontrol. Labeled cRNAswere hybrid- ized to an eight-pack Agendia MammaPrint microarray using standard spared chemotherapy toxicity, might therefore have clinical protocols (Agilent Oligo Microarray kit, Agilent Technologies). This value in selected circumstances. To explore these issues, we did microarray contains eight identical subarrays, each containing 60-mer a retrospective evaluation of the 70-gene MammaPrint assay in probes for the 70 prognosis genes in triplicate. Each sample was 100 older patients with node-negative breast cancer who were hybridized twice to do dye swaps. Fluorescence intensities on scanned initially diagnosed and treated at the Massachusetts General images were quantified, and the values were corrected for the Hospital (MGH) between 1985 and 1997. background level and normalized. Standard quality control measures were applied to determine technically acceptable hybridizations and poor-quality hybridizations were repeated. Materials and Methods MammaPrint tumor classification. Tumor classification was done using the previously reported 70-gene classification model (9). Briefly, Study design. Study design, patient selection, histopathologic for each of the 100 tumors, we calculated the cosine correlation analysis of tumors, clinical annotation, clinical interpretation, RNA coefficient of the level of expression of the 70 prognosis genes with the isolation, microarray profiling, and statistical analysis were carried out previously determined average profile of these genes in breast tumors jointly at the MGH, the NKI, and Agendia. from patients with a good prognosis (9, 14). Apatient with a Patient selection. We first identified those patients for whom frozen correlation coefficient >0.4 (the threshold in the original NKI study primary tumor material was available in the MGH Department of of 78 tumors that resulted in a 10% rate of false-negative results) was Pathology Breast Tumor Bank. We next identified patients who had then assigned to the ‘‘good’’ signature/low-risk group, and all others been consecutively diagnosed and treated for lymph node–negative were assigned to the ‘‘poor’’ signature/high-risk group. (pN0), invasive breast cancer at the