AFive-GeneMolecular Grade Index and HOXB 13 : IL 17 BR Are Complementary Prognostic Factors in Early Stage Breast Cancer

Purpose: Histologic tumor grade is a well-established prognostic factor for breast cancer, and tumor grade ^ associated genes are the common denominator of many prognostic gene signatures. The objectives of this study are as follows: (a) to develop a simple gene expression index for tumor grade (molecular grade index or MGI), and (b) to determine whether MGI and our previously described HOXB13:IL17BR index together provide improved prognostic information. Experimental Design: From our previously published list of genes whose expression correlates with both tumor grade and tumor stage progression, we selected five cell cycle ^ related genes to build MGI and evaluated MGI in two publicly available microarray data sets totaling 410 patients. Using two additional cohorts (n = 323), we developed a real-time reverse transcription PCR assay for MGI, validated its prognostic utility, and examined its interaction with HOXB13:IL17BR. Results:MGI performed consistently as a strong prognostic factor and was comparable with a more complex 97-gene genomic grade index in multiple data sets. In patients treated with endocrine therapy, MGI and HOXB13:IL17BR modified each other’s prognostic performance. High MGI was associated with significantly worse outcome only in combination with high HOXB13:IL17BR, and likewise, high HOXB13:IL17BR was significantly associated with poor outcome only in combination with high MGI. Conclusions:We developed and validated a five-gene reverse transcription PCR assay for MGI suitable for analyzing routine formalin-fixed paraffin-embedded clinical samples. The combination of MGI and HOXB13:IL17BR outperforms either alone and identifies a subgroup (f30%) of early stage estrogen receptor ^ positive breast cancer patients with very poor outcome despite endocrine therapy. The most recent (2005) St. Gallen consensus guidelines for treatment selection for early stage breast cancer consider both risk of recurrence and endocrine responsiveness to better balance risk and benefit of systemic adjuvant therapies (1). To better define risk stratification, genome-wide expression profiling studies have created multiple prognostic gene signatures for breast cancer (2, 3). An important issue is whether these signatures overlap in their prognostic information and whether combining several of these signatures would provide more accurate prognosis. In one comparative study, four signatures (the intrinsic subtypes, 70-gene signature, wound response signature, and Recurrence Score) were found to be highly concordant in classifying patients into low and high-risk groups (4). Notably, combining these signatures did not yield significant improvement in predictive accuracy, suggesting that the prognostic information provided by these signatures is largely overlapping (4). Sotiriou et al. (5) showed that a 97-gene tumor grade signature was comparable with the 70-gene signature and the Recurrence Score algorithm (6) in independent cohorts, and they hypothesized that most of the prognostic power of these Imaging, Diagnosis, Prognosis Authors’ Affiliations: AviaraDx, Inc., San Diego, California; Department of Pathology, Harvard Medical School, Molecular Pathology Research Unit, Massachusetts General Hospital and Division of Hematology and Oncology, Massachusetts General Hospital Cancer Center, Boston, Massachusetts ; Translational Research Unit, Jules Bordet Institute, Brussels, Belgium; and John Radcliffe Hospital, Oxford, United Kingdom Received11/30/07; revised1/17/08; accepted1/17/08. Grant support: National Cancer Institute grant RO1-1CA112021-01 (D.C. Sgroi), the Department of Defense grantW81XWH-04-1-0606 (D.C. Sgroi), the Susan G. Komen Breast Cancer Foundation grant BCTR0402932 (D.C. Sgroi), a grant from the Avon Foundation (D.C. Sgroi) and the NCI SPORE in breast cancer at Massachusetts General Hospital (D.C. Sgroi). 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 with18 U.S.C. Section1734 solely to indicate this fact. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Requests for reprints: Dennis Sgroi, Department of Pathology, Harvard Medical School, Molecular Pathology Research Unit, Massachusetts General Hospital, Boston, MA 02129. Phone: 617-726-5697; Fax: 617-726-5697; E-mail: dsgroi@ partners.org or Mark Erlander, 11025 Roselle St., Suite 200, San Diego, CA 92121. E-mail: merlander@aviaradx.com. F2008 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-07-5026 www.aacrjournals. org Clin Cancer Res 2008;14(9)May1, 2008 2601 Research. on June 15, 2017. © 2008 American Association for Cancer clincancerres.aacrjournals. org Downloaded from signatures comes from genes associated with cellular proliferation (7). Previously, we conducted a microarray analysis of 60 patients with hormone receptor–positive breast cancer treated with standard 5 years of adjuvant tamoxifen therapy (8). To facilitate discovery of novel biomarkers predictive of clinical outcome beyond standard prognostic factors, patients who developed recurrences were matched to those who did not with respect to tumor stage and grade. We identified three genes associated with clinical outcome, HOXB13, IL17BR , and CHDH , none of which had been previously implicated in breast cancer. Because high HOXB13 and low IL17BR expression levels are associated with recurrence, we proposed that a simple HOXB13:IL17BR two-gene ratio could serve as a novel biomarker for predicting recurrence in breast cancer patients receiving adjuvant tamoxifen therapy. Subsequent studies by us (9, 10) and others (11, 12) have shown that HOXB13:IL17BR is both prognostic (i.e., predicts the risk of breast cancer recurrence) and predictive of tamoxifen benefit (i.e., tamoxifen response/resistance). Given the well-established prognostic importance of histologic tumor grade (13) and tumor grade–associated genes (7) in breast cancer, a simple robust gene expression assay for tumor grade would be a valuable clinical tool. Herein, we developed a simple 5-gene tumor grade signature (MGI for molecular grade index) that recapitulates tumor grade and show that it predicts clinical outcome with comparable performance to the wellcharacterized 97-gene tumor grade index. We implemented a robust real-time reverse transcription-PCR (RT-PCR) assay for MGI and show that MGI together with HOXB13:IL17BR provides more accurate prognosis than either biomarker alone.