Prognostic Role ofa Multigene ReverseTranscriptase-PCRAssay in Patients with Node-Negative Breast Cancer Not Receiving Adjuvant SystemicTherapy FranciscoJ. Esteva, 1 Aysegul A. Sahin, 2 Massimo Cristofanilli, 1 Kevin Coombes, 3 Sang-Joon Lee, 3 Joffre Baker, 4 Maureen Cronin, 4 Michael Walker, 4 Drew Watson, 4 Steven Shak, 4 and Gabriel N. Hortobagyi 1 Abstract Purpose: To test the ability of a reverse transcriptase-PCR (RT-PCR) assay, based on gene ex- pression profiles, to accurately determine the risk of recurrence in patients with node-negative breast cancer who did not receive systemic therapy using formalin-fixed, paraffin-embedded tis- sue. A secondary objective was to determine whether the quantitative RT-PCR data correlated with immunohistochemistry assay data regarding estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 status. Patients and Methods: We obtained archival paraffin-embedded tissue from patients with in- vasive breast cancer but no axillary lymph node involvement who had received no adjuvant sys- temic therapy and been followed for at least 5 years. RNA was extracted from three 10-Am-thick sections.The expression of 16 cancer-related genes and 5 reference genes was quantified using RT-PCR. A gene expression algorithm was used to calculate a recurrence score for each patient. We then assessed the abilityof the test to accurately predict distant recurrence-free survivalin this population. Results: We identified 149 eligible patients. Median age at diagnosis was 59 years; mean tumor diameter was 2 cm; and 69% of tumors were estrogen receptor positive. Median follow-up was 18 years. The 5-year disease-free survival rate for the group was 80%. The 21gene-based recurrence score was not predictive of distant disease recurrence. However, a high concordance between RT-PCR and immunohistochemical assays for estrogen receptor, progesterone recep- tor, and human epidermal growth factor receptor 2 status was noted. Conclusions: RT-PCR can be done on paraffin-embedded tissue to validate the large numbers of genes associated with breast cancer recurrence. However, further work needs to be done to de- velop an assay to identify the likelihood of recurrent disease in patients with node-negative breast cancer who do not receive adjuvant tamoxifen or chemotherapy. A great need exists for better molecular characterization of tumor tissue. This would have several benefits. It would help in the oncology decision-making process, facilitate treatment selection, and ultimately improve patient outcomes. There is a particular need for such information in women with early- stage breast cancer, as evidenced by the fact that there is currently great variety in the treatment prescribed for these women. The serious consequence of this is that some women are likely being undertreated whereas others are being over- treated. The few assays that currently exist to characterize the molecular properties of breast tumors include those that determine estrogen receptor (ER) status, which are routinely done as a standard assessment for hormonal treatment of breast cancer (1, 2). In addition, assays to determine the expression of the human epidermal growth factor receptor 2 (HER-2) were recently developed as prognostic tools and to aid in the selection of certain therapy options, such as treatment with the humanized monoclonal antibody trastuzumab (2 – 5). Clearly, additional molecular markers are needed to more accurately characterize breast tumors, determine prognosis, and predict the response to various treatments (6). In the past several years, great advances have been made in the development of sensitive and quantitative gene expression assays using reverse transcriptase-PCR (RT-PCR) and microarray technologies (7, 8). Initial studies have shown that it is possible for these assays to generate a molecular portrait of gene expression in tumor tissue (9, 10). However, fresh frozen tissue, which is not routinely available in standard clinical practice, www.aacrjournals.org ClinCancerRes2005;11(9)May1,2005 3315 Authors’Affiliations: 1 Departments of Breast Medical Oncology, 2 Pathology, and 3 Biostatistics, The University ofTexas M.D. Anderson Cancer Center, Houston,Texas and 4 Genomic Health, Inc., Redwood City, California Received8/23/04;revised1/11/05;accepted1/28/05. Grantsupport: GenomicHealth,Inc.(RedwoodCity,CA)andCareerDevelopment AwardfromtheNationalCancerInstitute(K23CA82119;F.J.Esteva). The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertisement in accordance with18U.S.C.Section1734solely toindicatethisfact. Note:Thisstudywaspresentedinpartatthe26thAnnualSanAntonioBreastCancer Symposium,December4,2003,SanAntonio,Texas. Requests for reprints: Francisco J. Esteva, Department of Breast Medical Oncology, Unit 424,The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston,TX 77030. Phone: 713-792-2817; Fax: 713-745- 5768; E-mail: festeva@mdanderson.org. F 2005 American Association for Cancer Research. Imaging, Diagnosis, Prognosis Research. on June 9, 2016. © 2005 American Association for Cancer clincancerres.aacrjournals.org Downloaded from