Association of Stromal Tumor-Infiltrating Lymphocytes With Recurrence-Free Survival in the N9831 Adjuvant Trial in Patients With Early-Stage HER2-Positive Breast Cancer | Breast Cancer | JAMA Network
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Figure 1.  Cohort Patient Flow
Cohort Patient Flow

An indication of the patients from the parent study who were included in the analysis.

Figure 2.  Recurrence-Free Survival by Treatment Arm for Patients Stratified by Stromal Tumor-Infiltrating Lymphocytes (STILs) Status
Recurrence-Free Survival by Treatment Arm for Patients Stratified by Stromal Tumor-Infiltrating Lymphocytes (STILs) Status

Patients in arm A received doxorubicin-cyclophosphamide followed by weekly paclitaxel; patients in arm C received doxorubicin-cyclophosphamide followed by weekly paclitaxel plus trastuzumab followed by trastuzumab alone.

Table 1.  Distribution of Stromal Tumor-Infiltrating Lymphocyte (STIL) Classification for Each Arm Separately and for Entire Group
Distribution of Stromal Tumor-Infiltrating Lymphocyte (STIL) Classification for Each Arm Separately and for Entire Group
Table 2.  Comparison of Baseline Patient and Disease Characteristics Between Patients With Lymphocyte-Predominant Breast Cancer (LPBC) and Non-LPBC
Comparison of Baseline Patient and Disease Characteristics Between Patients With Lymphocyte-Predominant Breast Cancer (LPBC) and Non-LPBC
Table 3.  Multivariable Models for Recurrence-Free Survival (RFS) by Study Group
Multivariable Models for Recurrence-Free Survival (RFS) by Study Group
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Original Investigation
January 2016

Association of Stromal Tumor-Infiltrating Lymphocytes With Recurrence-Free Survival in the N9831 Adjuvant Trial in Patients With Early-Stage HER2-Positive Breast Cancer

Author Affiliations
  • 1Division of Hematology/Oncology, Department of Medicine, Mayo Clinic, Jacksonville, Florida
  • 2Division of Biostatistics and Bioinformatics, Department of Health Science Research, Mayo Clinic, Rochester, Minnesota
  • 3Department of Cancer Biology, Mayo Clinic, Jacksonville, Florida
  • 4Department of Pathology, Department of Pathology and Laboratory Medicine, Indiana University, Indianapolis, Indiana
  • 5Genomic Health Inc, Redwood City, California
  • 6Department of Pathology, University of California, San Francisco
JAMA Oncol. 2016;2(1):56-64. doi:10.1001/jamaoncol.2015.3239
Abstract

Importance  The presence of tumor-infiltrating lymphocytes at diagnosis is reported to be prognostic in triple-negative breast cancer.

Objective  To evaluate the association of stromal tumor-infiltrating lymphocytes (STILs) with recurrence-free survival (RFS) in women with human epidermal growth factor receptor 2 (HER2)–positive breast cancer treated with chemotherapy or chemotherapy plus trastuzumab in the N9831 trial.

Design, Setting, and Participants  Hematoxylin-eosin–stained tumor slides from patients with early-stage HER2-positive breast cancer in 2 of the 3 arms of the N9831 trial were assessed for STILs at an academic medical center. The amounts of STILs were quantitated in deciles, and a level of at least 60% STILs was used for the prespecified categorical cutoff. The association between STILs and RFS was evaluated with Cox models.

Exposure  Standard chemotherapy consisting of doxorubicin-cyclophosphamide followed by weekly paclitaxel (arm A) or doxorubicin-cyclophosphamide followed by weekly paclitaxel plus trastuzumab followed by trastuzumab alone (arm C).

Main Outcomes and Measures  Stromal tumor-infiltrating lymphocytes and their association with RFS.

Results  A total of 489 patients from arm A and 456 patients from arm C were assessed with a median (range) follow-up of 4.4 (0-13.6) years. The 10-year Kaplan-Meier estimates for RFS in arm A were 90.9% and 64.5% for patients with high and low levels of STILs, respectively (hazard ratio [HR], 0.23 [95% CI, 0.07-0.73]; P = .01). The 10-year estimates for RFS in arm C were 80.0% and 80.1% for patients with high and low levels of STILs, respectively (HR, 1.26 [95% CI, 0.50-3.17]; P = .63). The test for interaction between trastuzumab treatment and STIL status was statistically significant (P = .03). In a multivariable analysis, STIL status remained significantly associated with RFS in arm A and not significantly associated in arm C (HR, 1.01 [95% CI, 0.89-1.15]; interaction P = .04).

Conclusions and Relevance  This analysis of participants in the N9831 trial found that the presence of STILs was prognostically associated with RFS in patients treated with chemotherapy alone but not in patients treated with chemotherapy plus trastuzumab. High levels of STILs were associated with lack of trastuzumab therapy benefit, in contrast to a previously reported association between increased levels of STILs and increased trastuzumab benefit in HER2-positive patients.

Trial Registration  clinicaltrials.gov Identifier: NCT00005970

Introduction

The presence of dense lymphocytic infiltrates in breast carcinoma has long been recognized by breast histopathologists.1 The term medullary carcinoma was first used in 1949 to describe a high-grade breast carcinoma growing in anastomosing sheets composed of large cells with numerous mitoses and an “intimate” stromal lymphoid infiltrate that was associated with a better-than-average prognosis.1 The association of dense stromal lymphocytic infiltrates characteristic of medullary carcinoma and a good prognosis continued to be documented throughout the 20th century; however, the etiology of this better prognosis remained uncertain.2-4 Medullary carcinomas are by definition estrogen receptor negative. Microarray-based comparative genomic hybridization studies examining the enriched tumor DNA of medullary carcinoma show that medullary breast carcinomas share common genomic alterations with basal-like carcinomas, the most frequent being 1q and 8q gains and X losses. However, medullary breast carcinomas appear to be a distinct entity within the basal-like spectrum characterized by higher proportions of genome copy number aberrations than basal carcinomas and recurrent 10p, 9p, and 16q gains, 4p losses, and 1q, 8p, 10p, and 12p amplicons and, most importantly, are associated with better prognosis.5,6

Today, the role of the immune system in breast cancer development and outcome is undergoing substantial study, especially in the setting of triple-negative breast cancer (TNBC) and human epidermal growth factor receptor 2 (HER2)–positive breast cancer. Recent retrospective analyses have demonstrated a prognostic association of stromal tumor-infiltrating lymphocytes (STILs) with outcome in patients receiving adjuvant or neoadjuvant chemotherapy for TNBC.7-13 These studies have confirmed that STILs are most frequently found in highly proliferative TNBC and to a slightly lesser degree in HER2-positive breast cancer. Their presence at diagnosis is associated with pathologic response to neoadjuvant therapy, and with improved disease-free and overall survival after adjuvant chemotherapy.8-10,14,15 Subset analysis of HER2-positive breast cancers from the BIG 02-98 adjuvant study has documented that higher levels of STILs were significantly associated with improved survival in patients who did not receive taxane.10 Furthermore, analysis of HER2-positive cancers from patients enrolled in the FinHER adjuvant study has suggested that the levels of STILs are predictive of benefit from adjuvant trastuzumab therapy.14 However, these data from the FinHER trial are based on only 209 patients randomized to chemotherapy with or without trastuzumab and are associated with a small number of events (49 events) between the 2 treatment groups. The goal of the present study was to determine whether the data from FinHER could be validated in a larger adjuvant trial with the standard 1 year of trastuzumab therapy such as N9831.

Herein we describe a prospective-retrospective exploratory analysis of the association between the presence of STILs and recurrence-free survival (RFS) in patients enrolled in the N9831 adjuvant trial, which evaluated chemotherapy alone or chemotherapy with trastuzumab in patients with early-stage HER2-positive breast cancer. Guidelines followed for our analysis included those recommended by Simon et al16 and the REMARK (Reporting Recommendations for Tumor Marker Prognostic Studies).17 Archived specimens collected at baseline for patients with centrally tested HER2-positive breast cancer enrolled in arms A and C of N9831 (chemotherapy alone [arm A] vs chemotherapy plus concurrent trastuzumab [arm C]) were evaluated. Arm C represents the current standard of care, and this treatment group exhibited maximum difference in RFS after trastuzumab, compared with RFS after chemotherapy alone (arm A 10-year RFS, 67.1%; arm C 10-year RFS, 79.7%).18 The purpose of our analyses was to determine whether the presence of STILs is predictive of RFS for HER2-positive patients treated with trastuzumab.

Box Section Ref ID

At a Glance

  • The presence of stromal tumor-infiltrating lymphocytes (STILs) at diagnosis is reported to be prognostic in triple-negative and HER2-positive breast cancer and predictive of trastuzumab therapy benefit in HER2-positive breast cancer.

  • In HER2-positive patients who had been treated with chemotherapy or chemotherapy plus trastuzumab in the N9831 trial, we evaluated the association of STILs and recurrence-free survival (RFS) using a cutoff of at least 60% STILs by hematoxylin-eosin.

  • Stromal tumor-infiltrating lymphocytes were prognostically associated with RFS in patients treated with chemotherapy alone (hazard ratio, 0.23 [95% CI, 0.07-0.73]; P = .01) but not in patients treated with chemotherapy plus trastuzumab (hazard ratio, 1.26 [95% CI, 0.50-3.17]; P = .63).

  • High levels of STILs were associated with lack of trastuzumab therapy benefit.

Methods
Study Patients

The N9831 phase 3 randomized trial included 3505 women with histologically confirmed node-positive or high-risk node-negative HER2-positive invasive breast cancer. It was approved by the Mayo Clinic Institutional Review Board. Eligible patients were randomly assigned to doxorubicin-cyclophosphamide followed by weekly paclitaxel (control arm, arm A); doxorubicin-cyclophosphamide followed by weekly paclitaxel followed by trastuzumab (sequential arm, arm B); or doxorubicin-cyclophosphamide followed by weekly paclitaxel plus trastuzumab followed by trastuzumab alone (concurrent arm, arm C). Results of the different arms of N9831 were published in 2011, demonstrating that although each arm that included trastuzumab treatment led to statistically significant better disease-free survival compared with chemotherapy alone, the largest difference was observed in the arm C vs arm A comparisons. The present analyses were approved by the Mayo Clinic Institutional Review Board and included only patients randomly assigned to arm A or C, enrolled from May 25, 2000, through April 25, 2005. Radiation and/or hormonal therapy was administered after the completion of chemotherapy, as indicated. Patient accrual occurred from 2000 through 2005; follow-up is ongoing, although the primary and secondary clinical objectives have been published.18,19 Baseline estrogen receptor, progesterone receptor, and HER2-positive status was assessed according to protocol guidelines as described previously.18

Pathologic Analysis of STILs

Histopathologic analysis of the percentage of STILs was prespecified and performed using a single hematoxylin-eosin–stained section from each tumor using the criteria by Loi et al,10 Denkert et al,9 Adams et al,11 and Salgado et al.20 Levels of STILs were defined as the percentage of tumor stroma containing infiltrating lymphocytes that were not in direct contact with tumor cells from an assessment of the entire tumor-containing area of the section. Areas of noninvasive cancer or crush artifacts were not included in the analyses. The STIL data were collected as deciles. Specifically, each specimen was determined to consist of 0% to 9%, 10% to 19%, 20% to 29%, 30% to 29%, 40% to 49%, 50% to 59%, 60% to 69%, 70% to 79%, 80% to 89%, or 90% to 100% STILs. A priori, tumors were classified as lymphocyte-predominant breast cancer (LPBC) if they consisted of at least 60% STILs, consistent with Denkert et al.15 This histopathological review was conducted in tandem by 2 pathologists for the first 100 cases (H.B., F.L.B.) followed by a single pathologist (H.B.). Twelve samples were randomly selected from each of the 10 STIL bins (N = 120), and an independent pathologist (S.S.B.) reviewed them using the same published criteria but without any teaching set.15,20 All were blinded to the patient’s treatment assignment, tumor staging, and clinical outcome.

Statistical Analyses

Differences in continuous variables between groups were evaluated with a t test or a Wilcoxon rank sum test if the distribution was skewed. Differences in categorical variables were evaluated with a χ2 test with a level of significance of .05. The interrater agreement was assessed with a weighted κ statistic where 0 indicates no agreement and 1 indicates perfect agreement. Recurrence-free survival was defined as time from randomization until recurrent disease (local, regional, or distant recurrence of breast cancer). Patients who had not experienced a disease recurrence at the time of last follow-up or death were censored at the date of last follow-up or death. Kaplan-Meier curves were used to summarize the RFS experience, and the curves were compared with a log-rank test. A Cox proportional model was used to determine whether there was an interaction between LPBC status and treatment arm in terms of an association with RFS. The model contained the main effects (treatment arm and LPBC status), as well as the interaction term of LPBC status*treatment arm. Because the interaction term was significant, separate analyses were done for each treatment arm. Univariable and multivariable Cox models were used to generate hazard ratios (HRs) and corresponding 95% confidence intervals (CIs) for determining associations between variables of interest and RFS. A secondary analysis was performed using the decile levels as continuous measurements in place of LPBC status within the Cox models; the coding of the deciles was 0% to 9% coded as 1, 10% to 19% coded as 2, 20% to 29% as 3, and so forth. This recoded variable was treated as continuous.

This was an unplanned ad hoc analysis with a predetermined sample size that resulted from the number of patients enrolled in the trial who had provided written consent to the use of their specimen for analysis and who had sufficient tumor tissue for analysis. Given this fact, a power calculation was not performed. Instead, we provide 95% CIs for all the results so that the reader can determine whether the intervals contain values that would be considered clinically significant in the cases in which the results were not found to be significant.

Results
Patient Characteristics

A total of 2027 eligible patients were enrolled in arms A and C of N9831: 1081 in arm A and 946 in arm C. A subset of 945 patients was included in the STILs analysis. These patients were eligible and had provided sufficient tissue for analysis (see Figure 1). The patients assessed had a median (range) follow-up of 4.4 (0-13.6) years. A comparison between those patients included in the STILs analysis and those not included revealed that the 2 groups differed significantly with respect to race, with a greater percentage of white patients included in the STILs analysis (81% in the cohort not included and 88% in the cohort included; P < .001). The 2 groups did not differ significantly on other baseline variables (eTable 1 in the Supplement). In the parent study (N = 2027; total number of events = 352), the HR comparing the RFS of arm C to arm A was 0.51 (95% CI, 0.41-0.64; P < .001; 240 events in 1081 patients for arm A; 112 events in 946 patients for arm C). The HR for RFS in the cohort of patients in this study was similar (HR, 0.55 [95% CI, 0.40-0.77]; P < .001).

STILs Data

A comparison was made of the STILs evaluations between the 2 independent pathologists (S.S.B., H.B.). There was good concordance of the STILs assessment by deciles, with 55% agreement and a weighted κ statistic of 0.66 (95% CI, 0.57-0.74). The concordance of the dichotomous variable of LPBC status (LPBC if STILs ≥60%) classification between the 2 pathologists was excellent, with 98% agreement (κ = 0.85 [95% CI, 0.64-1.00]). The analysis only used the STILs data from the pathologist (H.B.) who reviewed all the specimens.

The distribution of STILs overall and by study arm is presented in Table 1. A majority of the samples were classified as having between 0 and 19% STILs. There were 94 samples (9.9%) that were classified as LPBC, and this was balanced between the arms: 48 of 489 (9.8%) in arm A and 46 of 456 (10.1%) in arm C (P = .89).

Characteristics Associated With LPBC

Women with LPBC (approximately 10% of all patients) were less likely to have hormone receptor–positive (estrogen receptor positive or progesterone receptor positive or both) disease compared with women with non-LPBC (31% vs 57%, respectively; P <.001) (Table 2). In addition, women with LPBC disease were more likely to have undergone breast-conserving surgery (47% vs 38%) and to have poor tumor grade (80% vs 70%) and were more likely to have stage N0 disease (21% vs 13%), although these differences were not statistically significant. There were no other differences in baseline characteristics between the 2 groups. Importantly, the 2 groups were balanced with respect to treatment arm assignment.

RFS and STILs

There were 162 disease recurrence events: 8 events in the LPBC group and 154 in the non-LPBC group. Patients without recurrent disease were observed for a median (range) of 6.9 (0.0-13.6) years. There was a significant interaction between treatment arm and LPBC status (P = .03). In particular, patients with LPBC tumors did not appear to derive any additional benefit from the addition of trastuzumab therapy (HR, 2.43 [95% CI, 0.58-10.22]; P = .22). This is in contrast to patients with non-LPBC tumors, who appeared to derive benefit from the addition of trastuzumab (HR, 0.49 [95% CI, 0.35-0.69]; P < .001) to chemotherapy. Figure 2 contains the Kaplan-Meier curves comparing the RFS by treatment group for each patient group. In arm A, the 10-year Kaplan-Meier estimates for patients with LPBC tumors and patients with non-LPBC tumors are 90.9% and 64.3%, respectively (P = .004). The corresponding Kaplan-Meier estimates of 10-year RFS for the 2 groups were 80.0% and 79.6%, respectively, for patients in arm C (P = .79). We performed an exploratory analysis of the at least 50% cut point, and the results were the same. There was limited power in the analysis of LPBC status due to the fact that there were only 8 recurrence events in the LPBC set. Exploratory splitting of the LPBC group into hormone receptor–positive and hormone receptor–negative groups results in even less power. The number of patients and number of events for the 4 different groups as defined by LPBC status and hormone receptor status are as follows: LPBC and hormone receptor positive: N = 29, events = 0; LPBC and hormone receptor negative: N = 65, events = 8; non-LPBC and hormone receptor positive: N = 482, events = 84; non-LPBC and hormone receptor negative: N = 369, events = 70. In the LPBC and hormone receptor–positive group, there were no recurrence events among 29 patients. This means that in the model it is not possible to get an estimate for the hormone receptor–positive subset in this group (eTable 2 in the Supplement). However, the relationship in this group appears to be similar to that in the hormone receptor–negative group (ie, both HRs are <1).

When we adjusted the Cox model for important prognostic variables (age, nodal status, hormone receptor status, tumor grade, and tumor size), the interaction term for treatment arm and LPBC status remained significant (P = .04), so we performed a separate multivariable analysis for each arm (Table 3 and eFigure 1 in the Supplement). Lymphocyte-predominant breast cancer status was significantly associated with RFS in arm A (HR, 0.19 [95% CI, 0.06-0.61]; P = .005) but not in arm C (HR, 1.01 [95% CI, 0.39-2.60]; P = .98). Hormone receptor status was associated with RFS in arm A (HR, 0.63 [95% CI, 0.42-0.94]; P = .02) but not in arm C (HR, 0.75 [95% CI, 0.43-1.32]; P = .32).

When we performed the analysis that treated the STILs decile levels as a continuous variable, the relationships did not change. In particular, STILs analyzed as deciles was associated with RFS in the multivariable model for arm A (P < .001) and was not associated with RFS in the multivariable model for arm C (P = .84); the arm by STIL decile interaction was significant (P = .008) (eFigure 2 in the Supplement).

Discussion

In this prospectively defined, retrospective study, we report that STILs assessed dichotomously (LPBC; ≥60% STILs) were significantly associated with outcome in HER2-positive patients treated with chemotherapy alone; that is, patients with LPBC had a better prognosis following treatment with doxorubicin-cyclophosphamide followed by weekly paclitaxel without trastuzumab. This association was not observed following treatment with doxorubicin-cyclophosphamide followed by weekly paclitaxel with trastuzumab. Notably, we did not confirm that increased STILs, either assessed in deciles or dichotomously (LPBC; ≥60% STILs), were predictive of increased benefit from adjuvant trastuzumab. To the contrary, in exploratory analyses from this landmark adjuvant trastuzumab trial, we showed that patients with high levels of STILs (LPBC; ≥60% STILs) did not benefit from the addition of trastuzumab therapy. However, it should be noted that only 94 patients were classified as having LPBC and there was a total of only 8 disease recurrence events. This means that this study was likely underpowered to detect a treatment effect in this group. On the other hand, the interaction P value was significant, which indicates that the trastuzumab treatment effect does appear to differ by LPBC status; at the very least, the trastuzumab effect in the patients with LPBC appears to be less than that in those without LPBC.

Previous reports have suggested that increasing level of STILs, either as a semicontinuous variable (deciles) or as a dichotomous variable (LPBC; ≥60% STILs), is prognostic of decreased residual risk following chemotherapy in estrogen receptor–negative breast cancer.10,14 We confirmed this semicontinuous association of decreasing residual risk as a function of STILs by decile in patients treated with adjuvant chemotherapy alone, and the interobserver concordance was good. Using the dichotomous cutoff of at least 60% STILs, the interobserver concordance was excellent, and the previously prognostic association between those with LPBC and a decreased residual risk was confirmed in patients treated with chemotherapy alone.

These data have implications with respect to cancer pathogenesis and metastasis. For more than 75 years, STILs have been a known prognostic factor, and we have confirmed this finding in patients with HER2-positive breast cancer treated with chemotherapy without trastuzumab in the adjuvant setting.1 The presence of a tumor-specific immune response may stimulate immune surveillance in these antigenic primary cancers for primary tumor control, possibly as a function of the composition of the T-cell receptor repertoire of STILs.21,22 These STILs data may seem counterintuitive in light of other data from whole-transcriptome analyses, which identified a cohort of genes that can be assigned to immune function gene ontology terms and which is predictive of long-term RFS in trastuzumab-treated patients.23

Specifically, our model23 was derived from HER2-positive samples from a large randomized adjuvant trial of chemotherapy with or without trastuzumab in which a large number of unselected genes were assessed and we systematically excluded any genes that were prognostic following chemotherapy. Thus, our model includes genes that strictly are predictive of trastuzumab response. There are also other important differences between our data and those reported by others. The recently reported study by Denkert et al15 was a neoadjuvant study in which STILs were assessed in smaller needle core biopsy hematoxylin-eosin–stained tissue sections; the study included triple-negative breast carcinomas, which may have higher levels of STILs; the end point was different (pathologic complete response); and the HER2-targeted treatment was different: the inclusion of a tyrosine kinase inhibitor in the trial may substantially alter the association between the immune system and the tumor.15 Importantly, with respect to their immune signature, Denkert et al used a selected candidate genes list, with little or no information provided on how the genes were selected. If the genes were preselected on the basis of association with increased lymphocyte infiltration, then their reported association is predictable. Finally, some immune function genes may be expressed in epithelial cells, and the expression of many immune function genes reflects cellular activity rather than cell number; thus, the relationship between gene expression profiles, derived from mixed cell populations, and number of lymphocytes is complex.

How do these results and other studies of STILs affect clinical treatment decisions, in particular the decision of whether to administer adjuvant trastuzumab therapy? These results do not confirm those of Loi et al.14 Our findings do not show that increased levels of STILs, either assessed in deciles or dichotomously (LPBC; ≥60% STILs), were predictive of increased benefit from adjuvant trastuzumab but rather that patients with high levels of STILs (LPBC; ≥60% STILs) did not benefit from the addition of trastuzumab. The observed lack of trastuzumab therapy benefit in the small population of patients with LPBC treated with chemotherapy plus trastuzumab is limited by the small numbers of patients, limited number of events, and the exploratory nature of the study; thus, these results should only be considered hypothesis generating and will require further study in the other landmark adjuvant trastuzumab trials.24,25

The strengths of this study include the predefined methods and cut points for STILs assessment using consensus guidelines, tandem STILs assessment and adjudication of challenging cases, low degree of interobserver variability in STILs assessment, and study in a landmark adjuvant trastuzumab randomized clinical trial.20 The major limitations of the study were the fact that this analysis was not prespecified in the N9831 trial and that only a subset of the enrolled patients in the N9831 trial were included in this analysis, although there did not appear to be a substantially meaningful difference between patients who provided tissue for analysis and had sufficient tissue for analysis and those who did not.

Conclusions

This was an exploratory analysis of the association between STILs and RFS from a subset of N9831 trial participants with HER2-positive disease treated with chemotherapy alone or treated with concurrent chemotherapy and trastuzumab followed by trastuzumab. These results show that patients with tumors classified as LPBC had better RFS when treated with chemotherapy alone than patients with tumors not classified as LPBC. Importantly, LPBC status was not associated with RFS in patients treated concurrently with chemotherapy and trastuzumab. A significant treatment interaction between LPBC status and trastuzumab therapy benefit was observed, which raises the question whether women with HER2-positive, LPBC require treatment with trastuzumab; however, this finding, contradictory to previously published results, will require further study.

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Article Information

Corresponding Author: Edith A. Perez, MD, Departments of Hematology/Oncology and Cancer Biology, Mayo Clinic, 4500 San Pablo Rd, S, Jacksonville, FL 32224 (perez.edith@mayo.edu).

Published Online: October 15, 2015. doi:10.1001/jamaoncol.2015.3239.

Author Contributions: Dr Perez had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study concept and design: Perez, Ballman, Thompson, Baehner.

Acquisition, analysis, or interpretation of data: All authors.

Drafting of the manuscript: Perez, Ballman, Tenner, Badve, Baehner.

Critical revision of the manuscript for important intellectual content: Perez, Ballman, Thompson, Badve, Bailey, Baehner.

Statistical analysis: Ballman, Tenner, Baehner.

Administrative, technical, or material support: Thompson, Badve, Bailey.

Study supervision: Perez, Thompson.

Conflict of Interest Disclosures: Dr. Perez is an employee of Genentech and owner of Roche stock (employment began August 2015, after completion of manuscript). Drs Bailey and Baehner are employees of and own stock in Genomic Health. No other disclosures are reported.

Funding/Support: The N9831 trial was coordinated by North Central Cancer Treatment Group (now part of the Alliance for Clinical Trials in Oncology) and supported in part by grant CA129949 from the National Cancer Institute. Correlative analysis of N9831 was partially funded by the National Institutes of Health/National Cancer Institute grant CA152045 (principal investigator, E.A.P.) and grant CA15083 to the Mayo Clinic Comprehensive Cancer Center. Additional funding was provided by the Donna Foundation. Biospecimens were provided by the Mayo Clinic Biospecimen Accession Pathology Laboratory.

Role of the Funder/Sponsor: The funders had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.

Disclaimer: The content of this article is solely the responsibility of the authors and does not necessarily reflect the official views of the National Cancer Institute or any of the other funding groups.

Additional Contributions: We thank Natasha Calhoun, BS, Department of Cancer Biology, Mayo Clinic, Jacksonville, Florida, for secretarial assistance and all patients who participated in N9831 and provided consent for these studies.

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