Obesity at diagnosis is associated with inferior outcomes in hormone receptor-positive operable breast cancer

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1 Obesity at diagnosis is associated with inferior outcomes in hormone receptor-positive operable breast cancer Joseph A. Sparano, Albert Einstein College of Medicine Molin Wang, Harvard University Fengmin Zhao, Harvard University Vered Stearns, Johns Hopkins University Silvana Martino, The Angeles Clinic and Research Institute Jennifer A. Ligibel, Harvard University Edith A. Perez, Mayo Clinic Tom Saphner, Green Bay Oncology Antonio C. Wolff, Johns Hopkins University George W. Sledge, Indiana University Only first 10 authors above; see publication for full author list. Journal Title: Cancer Volume: Volume 118, Number 23 Publisher: Wiley: 12 months , Pages Type of Work: Article Post-print: After Peer Review Publisher DOI: /cncr Permanent URL: Final published version: Copyright information: 2012 American Cancer Society. Accessed June 19, :09 PM EDT

2 NIH Public Access Author Manuscript Published in final edited form as: Cancer December 1; 118(23): doi: /cncr Obesity at Diagnosis Is Associated With Inferior Outcomes in Hormone Receptor-Positive Operable Breast Cancer Joseph A. Sparano, MD 1, Molin Wang, PhD 2, Fengmin Zhao, PhD 2, Vered Stearns, MD 3, Silvana Martino, DO 4, Jennifer A. Ligibel, MD 2, Edith A. Perez, MD 5, Tom Saphner, MD 6, Antonio C. Wolff, MD 3, George W. Sledge Jr, MD 7, William C. Wood, MD 8, John Fetting, MD 3, and Nancy E. Davidson, MD 9 1 Albert Einstein College of Medicine, Montefiore Medical Center, Bronx, New York 2 Dana Farber Cancer Institute, Harvard University, Boston, Massachusetts 3 Kimmel Cancer Center, Johns Hopkins University, Baltimore, Maryland 4 The Angeles Clinic and Research Institute, Santa Monica, California 5 Mayo Clinic Jacksonville, Jacksonville, Florida 6 Green Bay Oncology, Green Bay, Wisconsin 7 Simon Cancer Center, Indiana University School of Medicine, Indianapolis, Indiana 8 Winship Cancer Center, Emory University, Atlanta, Georgia 9 University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania. Abstract BACKGROUND Obesity has been associated with inferior outcomes in operable breast cancer, but the relation between body mass index (BMI) and outcomes by breast cancer subtype has not been previously evaluated. METHODS The authors evaluated the relation between BMI and outcomes in 3 adjuvant trials coordinated by the Eastern Cooperative Oncology Group that included chemotherapy regimens with doxorubicin and cyclophosphamide, including E1199, E5188, and E3189. Results are expressed as hazard ratios (HRs) from Cox proportional hazards models (HR >1 indicates a worse outcome). All P values are 2-sided. RESULTS When evaluated as a continuous variable in trial E1199, increasing BMI within the obese (BMI, 30 kg/m 2 ) and overweight (BMI, kg/m 2 ) ranges was associated with inferior outcomes in hormone receptor-positive, human epidermal growth receptor 2 (HER-2)/neunegative disease for disease-free survival (DFS; P =.0006) and overall survival (OS; P =.0007), but not in HER-2/neu overexpressing or triple-negative disease. When evaluated as a categorical variable, obesity was associated with inferior DFS (HR, 1.24; 95% confidence interval [CI], ; P =.0008) and OS (HR, 1.37; 95% CI, ; P =.002) in hormone receptorpositive disease, but not other subtypes. In a model including obesity, disease subtype, and their interaction, the interaction term was significant for OS (P =.02) and showed a strong trend for DFS (P =.07). Similar results were found in 2 other trials (E5188, E3189) American Cancer Society. Corresponding author: Joseph A. Sparano, MD, Montefiore Medical Center-Weiler Division, 1825 Eastchester Road, Bronx, NY 10461; Fax: (718) ; jsparano@montefiore.org. CONFLICT OF INTEREST DISCLOSURES The authors made no disclosures. The first author, as the principal investigator, participated in all phases of this analysis, interpretation, and article preparation. All coauthors participated in data interpretation. The study biostatisticians (second and third authors) conducted all analyses. Coauthors reviewed the article contents and approved the submission version.

3 Sparano et al. Page 2 CONCLUSIONS In a clinical trial population that excluded patients with significant comorbidities, obesity was associated with inferior outcomes specifically in patients with hormone receptor-positive operable breast cancer treated with standard chemohormonal therapy. Keywords obesity; breast cancer; prognosis; hormone receptor positive; disparity INTRODUCTION Obesity is a major public health problem in North America, Europe, and other developed regions of the world. 1 A body mass index (BMI) of 30 kg/m 2 or higher has been used to define obesity in most reports, including those showing an association between obesity and increased risk of cancers, cancer mortality, and mortality from all causes. 2,3 Obesity rates have increased 2-fold in adults and 3-fold in children in the past 30 years in the United States, and are also rising in other developed regions worldwide. 1 Obesity has been associated with an increased risk of breast cancer, which is the most common cancer in women and the second leading cause of cancer death in the United States and other developed nations worldwide. 4,5 Obesity at breast cancer diagnosis has also been associated with inferior survival, including breast cancer-specific survival. 6 Other analyses have indicated that for a clinical trial population of patients with lymph node-negative, estrogenreceptor positive breast cancer treated with tamoxifen who have a relatively low risk of cancer recurrence, obesity is associated with an increased rate of contralateral breast cancer, second primary cancers, and other noncancer-related deaths. 7,8 Gene expression profiling studies have shown that breast cancer is a heterogeneous disease characterized by distinct subtypes that have differing clinical behaviors and response to therapy Management decisions are based upon predictive factors for response to targeted therapies, such as estrogen receptor (ER) and/or progesterone receptor (PR) protein expression for selecting hormonal therapy, 12 and human epidermal growth receptor 2 (HER-2)/neu protein overexpression and/or gene amplification for selecting anti HER-2- directed therapy. 13 Patterns of ER, PR, and HER-2 expression have been shown to correspond to genotypically defined subsets. 14 Understanding the relation between hostrelated factors, such as obesity, and tumor-related factors, such as breast cancer subtype, may have important implications for identifying factors contributing to recurrence, and thus may impact the management of localized and advanced disease. To disentangle the influence of obesity from other factors influencing recurrence and survival, we compared the outcomes of obese patients with others in a large cohort of women with stage I to III breast cancer who participated in 3 National Cancer Institute (NCI)-sponsored trials evaluating the role of chemotherapy or endocrine therapy regimens These trials were completed before trastuzumab was approved as adjuvant therapy for HER-2 positive disease. 18,19 All of the trials required normal cardiac, renal, hepatic, and bone marrow function and good performance status, thereby excluding patients with significant comorbidities, potentially minimizing this as a confounding factor. In addition, patients received standardized care as stipulated by the trial. We found that obesity was independently associated with inferior outcomes specifically in patients with hormone receptor-positive, HER-2/neu negative disease, but not other breast cancer subtypes.

4 Sparano et al. Page 3 MATERIALS AND METHODS Patient Selection and Treatment Statistical Analysis The analysis included patients enrolled on 3 NCI-sponsored clinical trials coordinated by the Eastern Cooperative Oncology Group (ECOG) for whom BMI data were available. Briefly, patients with operable adenocarcinoma of the female breast with axillary lymph node metastases (T1-3; N1 or 2) or high-risk lymph node-negative disease (T2-3, N0 for E1199 only) without distant metastases were eligible. Other details regarding eligibility, treatment administered, and results have been previously reported and are summarized in Table Chemotherapy was prescribed by actual body weight and was not adjusted for obese patients in all trials. The primary analysis was initially performed for E1199, which was the most recent trial and included the most contemporary chemotherapy regimens (doxorubicin, cyclophosphamide, and paclitaxel or docetaxel) and endocrine therapy (tamoxifen or aromatase inhibitors). After a relation between obesity and breast cancer was defined in the E1199 study population, similar analyses were performed in the 2 older studies (E5188, E3189) that provided confirmatory findings. The primary trial endpoints were disease-free survival (DFS) and overall survival (OS). DFS was defined to be time from randomization to a first event, including disease recurrence, contralateral breast cancer, or death from any cause. OS was defined as time from randomization to death from any cause. To determine whether inferior outcomes were attributable to breast cancer recurrence rather than comorbidities, we also evaluated breast cancer-specific survival (BCSS), which was defined as death attributed to breast cancer or preceded by a breast cancer recurrence, with follow-up censored at the time of death from other causes. Event-time distributions for DFS and OS were estimated using Kaplan-Meier analysis. Cox proportional hazards methods were used to estimate hazard ratios (HRs) and test for significance for event times. The relation between BMI as a continuous variable and DFS/OS was evaluated using a flexible family of curves (a natural spline with 3 degrees of freedom) to model the effect of BMI in a proportional hazards model. 20 Patient characteristics were compared using Fisher exact test if they were categorical and Wilcoxon 2-sample test if they were continuous. The adverse events were compared using Fisher exact test. All P values are 2-sided; confidence intervals (CIs) are at the 95% level. The analysis was based on a dataset downloaded in April 2011 for trial E1199, in which patients are still being followed for recurrence and survival; the median follow-up for surviving patients was 95 months (7.9 years; range, months), at which time there were 1234 DFS events and 891 deaths (including 695 BCSS events). The BCSS events included 568 patients who were coded by the treating institutions as dying from breast cancer (64% of all deaths and 82% of all BCSS events) and 127 patients who had a breast recurrence before death whose death was coded by the treating site as from an unknown cause (14% of all deaths and 18% of all BCSS events). Other deaths included 118 patients who were coded as dying from other causes (13% of all deaths), and 78 patients who were coded as dying from an unknown cause and who did not have breast cancer recurrence before death (9% of all deaths). The median time from recurrence to death for those coded as dying from breast cancer was 15.2 months; for those coded as dying from an unknown cause who had a breast cancer recurrence before death it was 12.4 months. Data Management and Regulatory Issues The studies were sponsored by the NCI, reviewed and approved by the Cancer Therapy Evaluation Program at NCI, and developed and coordinated by the ECOG (ClinicalTrials.gov identifier, NCT ). The protocol was reviewed and approved by

5 Sparano et al. Page 4 the institutional review board at each participating institution, and all patients provided written informed consent. RESULTS Patient Characteristics The characteristics of obese and nonobese patients for trial E1199 are shown in Table 2. Of the 4770 patients with BMI data, 1745 (36.6%) were obese, 1540 (32.3%) were overweight, 1447 (30.3%) had a normal BMI, and 38 (0.8%) were underweight at the time they were enrolled on the trial after surgery and before initiation of chemotherapy. Obese and overweight patients were older and more likely to be postmenopausal and black. Obese patients and overweight patients had somewhat larger primary tumors and were more likely to have breast-conserving surgery, and exhibited somewhat different distribution of nodal metastases, although the median number of nodes involved was similar. There were no significant differences in the biologic characteristics of the tumor, as reflected by ER, PR, or HER-2 expression, nor in the type of endocrine therapy or chemotherapy given. Delivery of Adjuvant Therapy and Adverse Events The administration of adjuvant chemotherapy and grade 3 to 4 adverse events in obese compared with nonobese patients for the E1199 trial are shown in Table 3. There were generally no significant differences in the proportion who received at least 90% of cycle 1 dosing for cyclophosphamide, doxorubicin, paclitaxel, or docetaxel, indicating that clinicians adhered to the protocol and prescribed chemotherapy dosing by actual body weight. When evaluating all cycles, drug administration was similar for doxorubicin, cyclophosphamide, and docetaxel, although obese patients were less likely to receive at least 90% relative dose intensity for every 3-week paclitaxel (80% vs 87%; P =.007) and weekly paclitaxel administration (78% vs 85%; P =.008). During doxorubicin and cyclophosphamide chemotherapy, obese patients exhibited less grade 4 neutropenia (32% vs 40%; P <.0001) and more grade 3 to 4 cardiac toxicity (0.2% vs 0%; P =.05), but had comparable rates of grade 3-4 febrile neutropenia, infection, and overall cardiac toxicity. During taxane therapy, obese patients exhibited less grade 4 neutropenia (12% vs 15%; P =. 027) and febrile neutropenia (3% vs 5%; P =.022), but more grade 2 to 4 neuropathy (21% vs 19%; P =.042) and grade 3 to 4 neuropathy (7% vs 5%; P =.0041). Relation Between Obesity and Clinical Outcomes Univariate analyses were performed evaluating the relation between obesity and clinical outcomes in the E1199 trial. When the entire study population was included, compared with nonobese women, obesity was associated with significantly inferior DFS (HR, 1.17; 95% CI, ; P =.0077) and OS (HR, 1.23; 95% CI, ; P =.0025). For patients with hormone receptor-positive/her-2 negative/unknown disease, obesity was likewise associated with inferior outcomes, including DFS (HR, 1.31; 95% CI, ; P =.0009) and OS (HR, 1.46; 95% CI, ; P =.0001; = Fig. 1A, B), an effect that was more pronounced than in the overall population. This relation was not observed, however, in patients with triple-negative disease (Fig. 1C, D) or HER-2 positive disease (Fig. 1E, F). Only 0.8% (n = 38) of patients were underweight (BMI, <18.5 kg/m 2 ), and the results were not altered if these patients were excluded (DFS: HR, 1.31; 95% CI, ; P =.001; OS: HR, 1.45; 95% CI, ; P =.0001 for hormone receptor-positive, HER-2 negative disease in obese compared with nonobese women). The results were also similar if outcomes for obese women (BMI, 30 kg/m 2 ) were compared with those for women with a normal BMI ( kg/m 2 ), including DFS (HR, 1.40; 95% CI, ; P =.0007) and OS (HR, 1.69; 95% CI, ; P <.0001) for women with hormone receptorpositive, HER-2 negative disease, but not other subtypes (data not shown).

6 Sparano et al. Page 5 Analysis for Interaction Between Obesity and Breast Cancer Subtype To further evaluate the interaction between obesity and breast cancer subtype (hormone receptor-positive/her-2 negative or unknown vs other) in the E1199 trial, we constructed models including obesity, the breast cancer subtype, and their interaction. For OS, the interaction term of P =.021 was statistically significant (HR for the obese over nonobese for hormone receptor-positive/her-2 negative or unknown was 1.45, P = vs HR 1.05 for other subtypes, P =.62). For DFS, the action term of P =.070 indicated a strong trend (HR for hormone receptor-positive/her-2 negative or unknown was 1.30, P =.0012 vs HR 1.05, P =.60). For BCSS, the interaction term of P =.0087 was statistically significant (HR for hormone receptor-positive/her-2 negative or unknown was 1.47, P =.0008 vs HR 0.96, P =.74). Relation Between BMI as a Continuous Variable and Clinical Outcomes The relation between BMI as a continuous variable after adjustment for other covariates was also performed for the E1199 trial. There was a significant relation between increasing BMI and inferior DFS (overall P =.00063, nonlinearity P =.83) and OS (P =.00067, nonlinearity P =.31) in patients with hormone receptor-positive/her-2 negative or unknown disease (Fig. 2). A similar relation was not observed for patients with triple-negative disease (P =. 89 for DFS [nonlinearity, P =.13] and P =.45 for OS [nonlinearity, P =.22]), nor in HER-2 positive disease (P =.51 for DFS [nonlinearity, P =.34] and P =.61 for OS [nonlinearity, P =.55]). Evaluation of Obesity in Multivariate Models and Validation in Other Trials After identifying a relation between obesity and inferior outcomes in trial E1199 specifically in patients with hormone receptor-positive, HER-2 negative/unknown disease, we next constructed multivariate models (Table 4) adjusted for other covariates (including age, race, menopausal status, tumor size, number of positive axillary lymph nodes, and type of surgery), and also performed similar analyses in 2 other clinical trial populations that included only patients with hormone receptor-positive disease (E5188) or hormone receptornegative disease (E3189). When adjusted for other covariates, obesity remained strongly associated in the E1199 trial population with inferior DFS (HR, 1.24; 95% CI, ; P =.0079) and OS (HR, 1.37; 95% CI, ; P =.0015) only for patients with hormone receptor-positive, HER-2 negative/unknown disease in trial E1199. For the E5188 trial, which included only premenopausal women with ER-positive, axillary lymph node-positive breast cancer, obesity was likewise associated with inferior DFS (HR, 1.41; 95% CI, ; P <.0001) and OS (HR, 1.51; 95% CI, ; P <.0001). For the E3189 trial, which included only patients with ER/PR-negative disease, there was no relation between obesity and outcomes. Although trials E5188 and E3189 were performed before routine HER-2/neu testing in clinical practice and the information was not available for this analysis, the findings are nonetheless consistent with the relation observed in the E1199 trial. To provide additional confirmation that breast cancer recurrence was contributing to the inferior DFS and OS observed only in specific breast cancer subtypes, we also evaluated BCCS. For trial E1199, BCSS was likewise inferior for those with hormone receptorpositive, HER-2 negative disease (HR, 1.40; 95% CI, ; P =.0042), but not for other subtypes (Table 4). Inferior BCSS was also observed in trial E5188, which included only patients with ER-positive disease (HR, 1.54; 95% CI, ; P <.0001), but not in trial E3189, which included only patients with ER-negative disease (HR, 0.85; 95% CI, ; P =.29). In addition to the results using cause-specific hazard analysis based on the Cox proportional hazard methods described above, we also conducted the

7 Sparano et al. Page 6 subdistribution hazards analysis for competing risks, 21 which produced consistent results (data not shown). DISCUSSION We evaluated the relation between BMI and clinical outcomes in a cohort of 6885 women with stage I to III breast cancer enrolled in 3 clinical trials that included adjuvant doxorubicin-containing chemotherapy. Similar to previous individual reports and a metaanalysis of these other reports, 6 we found that obese patients, defined as having a BMI of 30 kg/m 2 or higher, exhibited a significantly higher risk of recurrence and death. This is likewise consistent with a population-based study that demonstrated inferior outcomes for obese patients. 22 However, analysis of the E1199 dataset is the first report to demonstrate that worse outcomes are observed specifically in women with hormone receptor-positive/ HER-2 negative disease who were treated with contemporary chemoendocrine therapy (including anthracyclines, taxanes, and aromatase inhibitors) but not other breast cancer subtypes. Furthermore, there was a continuous relation between increasing BMI and inferior outcomes in this specific subtype, indicating that excessive weight may also be a surrogate for other factors contributing to recurrence not only in obese but also in overweight patients. Although numerous previous studies have shown an association between obesity and inferior breast cancer outcomes, this report addresses critical limitations of previous analyses, including uniform adjuvant therapy administration, adjustment for other potentially confounding covariates, evaluation of outcomes in specific breast cancer subtypes, and sufficient maturity of follow-up data to detect late relapses typically associated with hormone receptor-positive disease. 23 Few previous studies have evaluated the effect of obesity stratified by hormone receptor expression, and none also included information regarding HER-2/neu overexpression. Majed et al reported that in a cohort of 14,709 patients treated at the Curie Institute over a 19-year period, there was a statistically significant interaction between obesity and distant recurrence for ER-positive compared with ER-negative disease (P =.05), although only about 30% of the population received adjuvant chemotherapy, and not all patients with ER-positive disease received endocrine therapy. 24 In contrast, de Azambuja et al reported worse outcomes for ER-positive compared with ER-negative disease, but the interaction P values were not significant. 25 Sestak et al reported that in postmenopausal women with ER-positive disease enrolled in the ATAC (Arimidex, Tamoxifen Alone or in Combination) trial, not only was high baseline BMI associated with more distant recurrence, but also better outcomes were observed for adjuvant anastrozole compared with tamoxifen primarily in women who were not obese. 26 Obesity is known to be associated with more comorbidities. 27 However, the studies included in this analysis required an excellent performance status and normal cardiac, renal, hepatic, and bone marrow function, thereby excluding those with cardiovascular disease or other comorbidities compromising normal organ function or functional status. Moreover, by evaluating BCSS, we confirmed that the inferior DFS and OS was attributable to higher risk of breast cancer recurrence and not solely to comorbidities. An important consideration in interpreting this work is that the relation between increasing BMI and increased recurrence is biologically plausible for several reasons. First, ERpositive breast cancer exhibits significantly higher gene expression of the insulin growth factor pathway (eg, IRS1, IGFR1, IGFB2) and downstream elements, such as Ras pathway (RhoB, RhoC, RAB27B, GGPS1) and mitogen-activated protein (MAP) kinase (MAPK3) pathway, 28 and there is significant cross-talk between the estrogen and insulin signaling pathways. 29 Second, higher insulin levels, even within a normal physiologic range, have been associated with increased recurrence in operable breast cancer 30,31 ; in addition,

8 Sparano et al. Page 7 Acknowledgments REFERENCES elevated C-peptide levels are associated with increased risk of cancer-related death specifically in ER-positive breast cancer. 31 Binding of insulin to the insulin receptor activates the MAP kinase and PI3 kinase pathways, and also actives ER-alpha mediated transcription. Finally, obesity is known to be associated with hyperinsulinemia, and metabolic syndrome (characterized by central obesity and hyperinsulinemia) is known to be associated with increased breast cancer recurrence. 32 Although increased production of endogenous estrogens in obese women has been postulated as a contributing factor, this has not been confirmed. 33,34 The results of this analysis clearly establish a relation between higher BMI at the time of breast cancer diagnosis and higher risk of recurrence and death, specifically in hormone receptor-positive, HER-2 negative disease, which accounts for about 2/3 of all breast cancers. What remains uncertain, however, is whether dietary and lifestyle interventions resulting in weight loss after a breast cancer diagnosis could substantially reduce the risk of recurrence, and also perhaps provide secondary benefits in reducing cardiovascular morbidity and mortality. The effect of dietary fat reduction in early stage breast cancer has already been evaluated in 2 clinical trials, including the WINS (Women s Intervention Nutrition Study) 35 and WHEL (Women s Healthy Eating and Living) trials. 36 Dietary fat reduction associated with modest average weight loss of 6 pounds was associated with reduced recurrence in the WINS trial (HR, 0.76; P =.034 for adjusted Cox model analysis), but was not observed in the WHEL trial, in which the dietary intervention did not produce weight loss. The 25% reduction in the risk of recurrence observed in the WINS trial is comparable to the treatment effect associated with adjuvant chemotherapy. 37 Other studies have shown that significant weight loss may be achieved with other dietary strategies, and was also associated with comparable reduction in insulin levels, 38 suggesting that patients may have several dietary options that may be effective in reducing recurrence risk. Further evaluation of dietary and lifestyle modification is warranted. FUNDING SOURCES Supported in part by grants from the Department of Health and Human Services and the National Institutes of Health (NIH): CA14958 to the Albert Einstein College of Medicine, CA23318 to the ECOG statistical center, CA66636 to the ECOG data management center, CA21115 to the ECOG coordinating center and chairman s office, CA32012 to the Southwest Oncology Group, CA11789 to the Cancer and Leukemia Group B, CA25224 to the North Central Cancer Treatment Group, CA49883 to the Indiana University School of Medicine, and CA16116 to Johns Hopkins Oncology Center. The contents of the article are solely the responsibility of the authors and do not necessary represent the official view of the National Center for Research Resources, NIH, or American Society of Clinical Oncology. 1. Flegal KM, Carroll MD, Ogden CL, Curtin LR. Prevalence and trends in obesity among US adults, JAMA. 2010; 303: [PubMed: ] 2. Ryan DH, Kushner R. The state of obesity and obesity research. JAMA. 2010; 304: [PubMed: ] 3. Renehan AG, Tyson M, Egger M, Heller RF, Zwahlen M. Body-mass index and incidence of cancer: a systematic review and meta-analysis of prospective observational studies. Lancet. 2008; 371: [PubMed: ] 4. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ. Cancer statistics, CA Cancer J Clin. 2009; 59: [PubMed: ] 5. Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, CA Cancer J Clin. 2005; 55: [PubMed: ]

9 Sparano et al. Page 8 6. Protani M, Coory M, Martin JH. Effect of obesity on survival of women with breast cancer: systematic review and meta-analysis. Breast Cancer Res Treat. 2010; 123: [PubMed: ] 7. Dignam JJ, Wieand K, Johnson KA, Fisher B, Xu L, Mamounas EP. Obesity, tamoxifen use, and outcomes in women with estrogen receptor-positive early-stage breast cancer. J Natl Cancer Inst. 2003; 95: [PubMed: ] 8. Dignam JJ, Wieand K, Johnson KA, et al. Effects of obesity and race on prognosis in lymph nodenegative, estrogen receptor-negative breast cancer. Breast Cancer Res Treat. 2006; 97: [PubMed: ] 9. Sparano JA, Fazzari M, Kenny PA. Clinical application of gene expression profiling in breast cancer. Surg Oncol Clin N Am. 2010; 19: [PubMed: ] 10. Sorlie T, Tibshirani R, Parker J, et al. Repeated observation of breast tumor subtypes in independent gene expression data sets. Proc Natl Acad Sci U S A. 2003; 100: [PubMed: ] 11. Sorlie T, Perou CM, Tibshirani R, et al. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci U S A. 2001; 98: [PubMed: ] 12. Hammond ME, Hayes DF, Dowsett M, et al. American Society of Clinical Oncology/College Of American Pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer. J Clin Oncol. 2010; 28: [PubMed: ] 13. Wolff AC, Hammond ME, Schwartz JN, et al. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. J Clin Oncol. 2007; 25: [PubMed: ] 14. Nielsen TO, Hsu FD, Jensen K, et al. Immunohistochemical and clinical characterization of the basal-like subtype of invasive breast carcinoma. Clin Cancer Res. 2004; 10: [PubMed: ] 15. Sparano JA, Wang M, Martino S, et al. Weekly paclitaxel in the adjuvant treatment of breast cancer. N Engl J Med. 2008; 358: [PubMed: ] 16. Davidson NE, O Neill AM, Vukov AM, et al. Chemoendocrine therapy for premenopausal women with axillary lymph node-positive, steroid hormone receptor-positive breast cancer: results from INT 0101 (E5188). J Clin Oncol. 2005; 23: [PubMed: ] 17. Fetting JH, Gray R, Fairclough DL, et al. Sixteen-week multidrug regimen versus cyclophosphamide, doxorubicin, and fluorouracil as adjuvant therapy for node-positive, receptornegative breast cancer: an Intergroup study. J Clin Oncol. 1998; 16: [PubMed: ] 18. Romond EH, Perez EA, Bryant J, et al. Trastuzumab plus adjuvant chemotherapy for operable HER2-positive breast cancer. N Engl J Med. 2005; 353: [PubMed: ] 19. Piccart-Gebhart MJ, Procter M, Leyland-Jones B, et al. Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer. N Engl J Med. 2005; 353: [PubMed: ] 20. Gray RJ. Spline-based tests in survival analysis. Biometrics. 1994; 50: [PubMed: ] 21. Fine JP, Gray RJ. A proportional hazards model for the subdistribution of a competing risk. J Am Stat Assoc. 1999; 94: Ewertz M, Jensen MB, Gunnarsdottir KA, et al. Effect of obesity on prognosis after early-stage breast cancer. J Clin Oncol. 2011; 29: [PubMed: ] 23. Saphner T, Tormey DC, Gray R. Annual hazard rates of recurrence for breast cancer after primary therapy. J Clin Oncol. 1996; 14: [PubMed: ] 24. Majed B, Moreau T, Senouci K, Salmon RJ, Fourquet A, Asselain B. Is obesity an independent prognosis factor in woman breast cancer? Breast Cancer Res Treat. 2008; 111: [PubMed: ] 25. de Azambuja E, McCaskill-Stevens W, Francis P, et al. The effect of body mass index on overall and disease-free survival in node-positive breast cancer patients treated with docetaxel and

10 Sparano et al. Page 9 doxorubicin-containing adjuvant chemotherapy: the experience of the BIG trial. Breast Cancer Res Treat. 2010; 119: [PubMed: ] 26. Sestak I, Distler W, Forbes JF, Dowsett M, Howell A, Cuzick J. Effect of body mass index on recurrences in tamoxifen and anastrozole treated women: an exploratory analysis from the ATAC trial. J Clin Oncol. 2010; 28: [PubMed: ] 27. Schelbert KB. Comorbidities of obesity. Prim Care. 2009; 36: [PubMed: ] 28. Sparano JA, Gray R, Goldstein LJ, et al. Gene expression profiling of phenotypically-defined hormone-receptor positive breast cancer: evidence for increased transcriptional activity of the insulin growth factor receptor pathway and other pathways [abstract]. Breast Cancer Res Treat Abstract Yee D, Lee AV. Crosstalk between the insulin-like growth factors and estrogens in breast cancer. J Mammary Gland Biol Neoplasia. 2000; 5: [PubMed: ] 30. Goodwin PJ, Ennis M, Bahl M, et al. High insulin levels in newly diagnosed breast cancer patients reflect underlying insulin resistance and are associated with components of the insulin resistance syndrome. Breast Cancer Res Treat. 2009; 114: [PubMed: ] 31. Irwin ML, Duggan C, Wang CY, et al. Fasting C-peptide levels and death resulting from all causes and breast cancer: the health, eating, activity, and lifestyle study. J Clin Oncol. 2011; 29: [PubMed: ] 32. Pasanisi P, Berrino F, De Petris M, Venturelli E, Mastroianni A, Panico S. Metabolic syndrome as a prognostic factor for breast cancer recurrences. Int J Cancer. 2006; 119: [PubMed: ] 33. Lunardi G, Del Mastro L, Bighin C, et al. Gruppo Italiano Mammella (GIM). Effects of body mass index (BMI) on plasma levels of estrone sulfate (ES) in postmenopausal women with breast cancer (BC) during letrozole (L) treatment [abstract]. ASCO Meeting Abstracts. May Vol. 29(No 15_suppl) Supplement): Abstract Egle D, Oberguggenberger A, Achleitner R, et al. Do obese women benefit less from adjuvant endocrine therapy with aromatase inhibitors? Preliminary analysis on the association of BMI and aromatase inhibitor plasma levels [abstract]. ASCO Meeting Abstracts. May Vol 29(No 15_suppl):e Supplement), Chlebowski RT, Blackburn GL, Thomson CA, et al. Dietary fat reduction and breast cancer outcome: interim efficacy results from the Women s Intervention Nutrition Study. J Natl Cancer Inst. 2006; 98: [PubMed: ] 36. Pierce JP, Natarajan L, Caan BJ, et al. Influence of a diet very high in vegetables, fruit, and fiber and low in fat on prognosis following treatment for breast cancer: the Women s Healthy Eating and Living (WHEL) randomized trial. JAMA. 2007; 298: [PubMed: ] 37. Early Breast Cancer Trialists Collaborative Group (EBCTG). Effects of chemotherapy and hormonal therapy for early breast cancer on recurrence and 15-year survival: an overview of the randomised trials. Lancet. 2005; 365: [PubMed: ] 38. Shai I, Schwarzfuchs D, Henkin Y, et al. Weight loss with a low-carbohydrate, Mediterranean, or low-fat diet. N Engl J Med. 2008; 359: [PubMed: ]

11 Sparano et al. Page 10 Figure 1. Disease-free survival (DFS) and overall survival (OS), respectively, are shown for patients enrolled in the E1199 trial with (A, B) hormone receptor-positive (HR positive), human epidermal growth receptor 2 (HER-2)-negative/unknown disease, (C, D) triple-negative disease, and (E, F) HER-2 positive disease. HR, hazard ratio.

12 Sparano et al. Page 11 Figure 2. Relation between body mass index (BMI) and disease-free survival (DFS) and overall survival (OS), respectively, is shown for patients enrolled in the E1199 trial with hormone receptor-positive (HR+), human epidermal growth receptor 2-negative(HER2 )/unknown disease. HR, hazard ratio.

13 Sparano et al. Page 12 Studies Included in Analysis Table 1 Characteristic Trial E1199 Trial E5188 Trial E3189 Analysis Primary analysis Confirmatory analysis Confirmatory analysis No. with BMI data included in analysisa Time period study performed October 1999-January 2002 July 1989-February 1994 September 1989-April 1993 Population Node positive and high risk node negative ER positive, node positive, premenopausal Median follow-up for living patients 7.9 years 14.0 years 14.4 years ER negative, node positive Chemotherapy AC taxane CAF CAF vs 16-week regimen Endocrine therapy BMI Tamoxifen or tamoxifen followed by an AI No therapy vs goserelin vs goserelin plus tamoxifen 30 kg/m 2 37% 25% 31% kg/m 2 32% 29% 29% <25 kg/m 2 31% 45% 40% Age at diagnosis, median y (range) 51 (22-84) 43 (24-57) 47 (25-78) 45 years 30% 61% 41% years 60% 39% 51% 66 years 10% 0% 8% Tumor size, cm % 38% 29% % 54% 57% % 9% 14% Axillary nodal status Negative 12% positive 56% 59% 54% 4 positive 32% 41% 46% Abbreviations: AC, doxorubicin and cyclophosphamide; AI, aromatase inhibitor; BMI, body mass index; CAF, cyclophosphamide, doxorubicin, and 5-flourouracil; ER, estrogen receptor. a BMI data were available in 4770 of 4950 patients (96%) enrolled in E1199, and all patients enrolled in trials E5188 and E3189. None

14 Sparano et al. Page 13 Table 2 Comparison of Patient Characteristics for Obese, Overweight, and Normal Weight Patients Enrolled in Trial E1199 Characteristic Normal or Underweight, BMI <25 kg/m 2a Overweight, BMI kg/m 2 Obese, BMI 30 kg/m 2 No (31%) 1540 (32%) 1745 (37%) P, Obese vs Other Age < Median y [range] 49 [22-79] 51 [24-81] 52 [25-84] 45 years 568 (38.3%) 435 (28.3%) 434 (24.9%) years 801 (53.9%) 931 (60.5%) 1130 (64.8%) 66 years 116 (7.8%) 174 (11.3%) 181 (10.4%) Premenopausal, No. b 811 (54.6%) 684 (44.4%) 724 (41.5%) < Race/ethnicity < White 1314 (88.5%) 1310 (85.1%) 1407 (80.6%) Hispanic 41 (2.8%) 67 (4.4%) 69 (4.0%) Black 57 (3.8%) 109 (7.1%) 234 (13.4%) Other 65 (4.4%) 49 (3.2%) 31 (1.8%) Unknown 8 (0.5%) 5 (0.3%) 4 (0.2%) Tumor size Median cm [range] 2.5 [0.1-17] 2.5 [0.1-14] 2.5 [0.1-15] 2 cm 593 (39.9%) 532 (34.6%) 601 (34.4%) >2 and 5 cm 768 (51.7%) 838 (54.4%) 926 (53.1%) >5 cm 111 (7.5%) 156 (10.1%) 201 (11.5%) Unknown 13 (0.9%) 14 (0.9%) 17 (1.0%) Positive nodes Median [range] 2 [0-40] 2 [0-30] 2 [0-41] (10.2%) 178 (11.6%) 224 (12.8%) (58.5%) 859 (55.8%) 917 (52.6%) (22.4%) 352 (22.9%) 400 (22.9%) (8.6%) 143 (9.3%) 193 (11.1%) Unknown 6 (0.4%) 8 (0.5%) 11 (0.6%) Hormone receptor positivec Positive 1084 (73.0%) 1091 (70.8%) 1236 (70.8%) Negative 381 (25.7%) 426 (27.7%) 486 (27.9%) Unknown 20 (1.4%) 23 (1.5%) 23 (1.3%) HER-2/neuexpression Positive 302 (20.3%) 295 (19.2%) 343 (19.7%) Negative 1043 (70.2%) 1086 (70.5%) 1215 (69.6%) Unknown 140 (9.4%) 159 (10.3%) 187 (10.7%) Triple-negative diseased 262 (17.6%) 289 (18.8%) 327 (18.7%) Most extensive surgery P, Obese vs Overweight vs Normal

15 Sparano et al. Page 14 Characteristic Normal or Underweight, BMI <25 kg/m 2a Overweight, BMI kg/m 2 Obese, BMI 30 kg/m 2 Breast-sparing surgery 541 (36.4%) 610 (39.6%) 728 (41.7%) Mastectomy 935 (63.0%) 922 (59.9%) 1010 (57.9%) Unknown 9 (0.6%) 8 (0.5%) 7 (0.4%) P, Obese vs Other Radiation therapy Given 814 (54.8%) 885 (57.5%) 995 (57.0%) Not given 671 (45.2%) 655 (42.5%) 750 (43.0%) Treatment arms Paclitaxel every 3 weeks 369 (24.9%) 396 (25.7%) 449 (25.7%) Paclitaxel weekly 371 (25.0%) 394 (25.6%) 417 (23.9%) Docetaxel every 3 weeks 389 (26.2%) 363 (23.6%) 438 (25.1%) Docetaxel weekly 356 (24.0%) 387 (25.1%) 441 (25.3%) Endocrine therapy givene No. with hormone receptor-positive disease Tamoxifen alone 412 (38.0%) 388 (35.6%) 446 (36.1%) Tamoxifen, then AI 598 (55.2%) 631 (57.8%) 693 (56.1%) AI alone 43 (4.0%) 33 (3.0%) 54 (4.4%) None 22 (2.0%) 23 (2.1%) 31 (2.5%) Unknown 9 (0.8%) 16 (1.5%) 12 (1.0%) Abbreviations: AI, aromatase inhibitor; BMI, body mass index; HER-2, human epidermal growth receptor 2. P, Obese vs Overweight vs Normal Wilcoxon 2-sample test was used for comparisons for age, tumor size, and number of positive nodes, and Fisher exact test was used for other variables. a Normal BMI group includes 38 patients who were underweight (BMI, <18.5 kg/m 2 ). b Premenopausal or <50 years of age if menopausal status was unknown. c Hormone receptor positive is defined as estrogen receptor- and/or progesterone receptor-positive disease. d The reported percentages are percentage of patients with triple-negative disease. The P values are for all patients. e In the test, the variable was treated as a 4-level categorical variable: tamoxifen alone, sequential tamoxifen followed by an AI, AI alone, and none.

16 Sparano et al. Page 15 Table 3 Chemotherapy Drug Delivery and Most Common Grade 3 to 4 AEs During Chemotherapy in Trial E1199 Drug Administration/AEs BMI <30 kg/m 2 Drug administration, at least 90% of intended dose given Doxorubicin BMI 30 kg/m 2 P a Cycle 1 99% 99% NS All cycles 95% 94% NS Cyclophosphamide Cycle 1 99% 99% NS All cycles 94% 94% NS Paclitaxel every 3 weeks Cycle 1 99% 98% NS All cycles 87% 80%.007 Paclitaxel weekly Cycle 1 100% 99% NS All cycles 85% 78%.008 Docetaxel every 3 weeks Cycle 1 98% 98% NS All cycles 66% 67% NS Docetaxel weekly Cycle 1 99% 98% NS All cycles 69% 66% NS AC chemotherapy AEs Neutropenia, grade 4 only 40% 32% < Febrile neutropenia 6% 6% NS Infection 9% 10% NS Cardiac toxicity 0% 0.2%.050 Taxane therapy AEs Neutropenia, grade 4 only 15% 12%.027 Febrile neutropenia 5% 3%.022 Infection 5% 6% NS Cardiac toxicity 0.1% 0.2% NS Neuropathy Grade 3-4 5% 7%.0041 Grade % 21%.042 Abbreviations: AC, doxorubicin and cyclophosphamide; AE, adverse event per National Cancer Institute Common Toxicity Criteria, version 2.0; BMI, body mass index; NS, not significant. a Fisher exact test.

17 Sparano et al. Page 16 Table 4 Estimated HRs and 95% CIs for Obese Patients Compared With Others Derived From Multivariate Models for Disease-Free Survival and Overall Survival Trial Population HR for Disease-Free Survival (95% CI) E1199 Hormone receptor-positive, HER-2-negative/unknown disease HR for Overall Survival (95% CI) HR for BCSS (95% CI) 1.24 ( ), P = ( ), P = ( ), P =.0042 Triple-negative disease 1.02 ( ), P = ( ), P = ( ), P =.996 HER-2-positive disease 1.06 ( ), P = ( ), P = ( ), P =.98 E5188 ER-positive disease 1.41 ( ), P < ( ), P < ( ), P <.0001 E3189 ER/PR-negative disease 0.90 ( ), P = ( ), P = ( ), P =.29 Abbreviations: BCSS, breast cancer-specific survival; CI, confidence interval; ER, estrogen receptor; HER-2, human epidermal growth receptor 2; HR, hazard ratio; PR, progesterone receptor. Other covariates for all trials included 1) age ( 45 vs >45 years), 2) race (black vs other), 3) premenopausal vs other (for E1199 and E3189 only), 4) tumor size ( 2 cm vs >2 cm), 5) axillary nodal status (1-3 vs 0, and 4 vs 0 positive for E1199; 4 positive nodes vs 1-3 positive nodes for E5188 and E3189), 6) surgery (breast sparing vs mastectomy), 7) use of radiation therapy (yes vs no; E5188 and E3189), and 8) use of systemic therapy (E5188 and E3189). For the E3189, other covariates included chemotherapy treatment arm (cyclophosphamide, doxorubicin, and 5- flourouracil vs 16-week regimen). For the E5188 trial, 5 adherence variables representing treatment duration of endocrine therapy were used.