Purpose: This study investigated the association between tumor MYC protein expression and disease-free survival (DFS) of patients randomized to receive chemotherapy alone (Arm A) or chemotherapy with sequential (Arm B) or concurrent trastuzumab (Arm C) in the N9831 (Alliance) adjuvant HER2+ trastuzumab breast cancer trial.

Experimental Design: This analysis included 1,736 patients randomized to Arms A, B, and C on N9831. Nuclear MYC protein expression was determined in tissue microarray sections containing three biopsies per patient or whole tissue sections using standard immunohistochemistry (clone 9E10). A tumor was considered positive for MYC protein overexpression (MYC+) if the nuclear 3+ staining percentage was more than 30%.

Results: Five hundred and seventy-four (33%) tumors were MYC+. MYC+ was associated with hormone receptor positivity (χ2, P = 0.006), tumors 2 cm or more (χ2, P = 0.02), and a higher rate of nodal positivity (χ2, P < 0.001). HRs for DFS (median follow-up: 6.1 years) for Arm C versus A were 0.52 (P = 0.006) and 0.65 (P = 0.006) for patients with MYC+ and MYC tumors, respectively (Pinteraction = 0.40). For Arm B versus A, HRs for patients with MYC+ and MYC tumors were 0.79 (P = 0.21) and 0.74 (P = 0.04), respectively (Pinteraction = 0.71). For Arm C versus B, HRs for patients with MYC+ and MYC tumors were 0.56 (P = 0.02) and 0.89 (P = 0.49), respectively (Pinteraction = 0.17).

Conclusions: Our data do not support an impact of tumor MYC protein expression on differential benefit from adjuvant trastuzumab. Clin Cancer Res; 19(20); 5798–807. ©2013 AACR.

Translational Relevance

Despite therapy, up to a quarter of women diagnosed with HER2+ early-stage breast cancer develop tumor relapse within 3 years. Identifying markers that could help predict trastuzumab benefit is therefore an important clinical goal. Previous evidence from B31 and N9831 suggested that MYC gene copy number anomalies may be associated with additional benefit to adjuvant trastuzumab. We then investigated the association between MYC protein overexpression (MYC+; nuclear 3+ staining in >30% tumor cells) and disease-free survival of patients in N9831. Patients with MYC+ and MYC tumors both significantly benefited from concurrent trastuzumab compared with standard chemotherapy alone, and the level of benefit was not significantly different. Patients with MYC+ but not with MYC tumors significantly benefited from concurrent trastuzumab compared with sequential trastuzumab. Our N9831 data indicate that MYC protein expression is not significantly associated with differential benefit to concurrent adjuvant trastuzumab with chemotherapy.

Patients with human EGF receptor-2–positive (HER2+) breast tumors have greatly benefited from the development of trastuzumab, a humanized monoclonal antibody directed against HER2 (1). Trastuzumab has proven to prolong the survival of women with metastatic breast cancer and significantly increase disease-free survival (DFS) of patients with HER2+ early breast cancer (2, 3). However, many women who receive trastuzumab for advanced disease develop tumor progression within 1 year, and 15% to 25% of women diagnosed with HER2+ early disease develop tumor relapse within 3 years, despite therapy (4). Thus, identifying molecular markers that could predict the patients who are most likely to benefit from trastuzumab is an important research and clinical goal.

MYC is one of several markers reported to be involved in trastuzumab sensitivity and resistance. The MYC oncoprotein is a pleiotropic transcription factor and key regulator of cell growth, proliferation, metabolism, differentiation, apoptosis, and pathways that regulate genome stability and cell death (5, 6). MYC has been shown to be overexpressed in breast cancer with reported frequencies between 12% to 100% depending on antibody/technique and cutpoint used, patient heterogeneity, and molecular subtype of the breast tumor (5–7). MYC acts as a downstream target of HER2-driven proliferative signals in breast cancer cells in vitro (8), and deregulation of MYC contributes to breast cancer tumorigenesis and progression and is typically associated with poor outcomes (7).

In addition, MYC gene amplification has been reported to predict additional trastuzumab benefit in a retrospective analysis of the National Surgical Adjuvant Breast and Bowel Project Cooperative Group (NSABP) B31 adjuvant trial. NSABP B31 showed that patients with MYC/HER2 coamplification (defined as average copies/nucleus >5.0) in their primary breast tumors, benefited significantly more (Pinteraction = 0.007) from trastuzumab than patients with only HER2 amplification, although a significant benefit of trastuzumab was observed in both MYC amplified and nonamplified patients (9).

Conversely, however, our results from the North Central Cancer Treatment Group (NCCTG) N9831 (10) did not support the link between MYC gene amplification and benefit from trastuzumab strictly on the basis of MYC amplification defined as more than 5.0 average copies/nucleus. In the N9831 Intergroup adjuvant trastuzumab phase III trial, we observed differential benefit of trastuzumab in groups of patients with HER2+ tumors with less than 2.5 average MYC copies/nucleus and patients with alternative MYC and chromosome 8 copy number alterations (10).

Considering that protein overexpression may be independent of gene amplification (5), we designed the translational component of the N9831 trial to also include an analysis of the role of MYC protein overexpression in trastuzumab sensitivity. We therefore evaluated the association between MYC protein expression and DFS of patients randomized to receive chemotherapy alone (Arm A) or chemotherapy with sequential (Arm B) or concurrent trastuzumab (Arm C) on N9831.

Patients

The N9831 trial (NCT00005970) was a phase III trial in which patients were randomized to three arms: Arm A, doxorubicin and cyclophosphamide followed by weekly paclitaxel; Arm B, same as Arm A but followed by 1 year of sequential trastuzumab; and Arm C, same as Arm A but with 1-year concurrent trastuzumab, started the same day as weekly paclitaxel (Supplementary Fig. S1). Patients randomly assigned to the concurrent trastuzumab arm had a significantly increased DFS [P < 0.001; stratified HR, 0.52; 95% confidence interval (CI), 0.45–0.60] and overall survival (OS; P < 0.001; stratified HR, 0.61; 95% CI, 0.50–0.75) compared with patients assigned to the control arm (2). In the N9831 comparison of sequential versus concurrent trastuzumab chemotherapy, there was an increase in DFS with concurrent trastuzumab (P = 0.02; HR, 0.77; 99.9% CI, 0.53–1.11; ref. 11). The 5-year OS rate for the sequential and concurrent arms was estimated at 89.7% (95% CI, 87.7%–91.8%) and 91.9% (95% CI, 90.0%–93.7%), respectively.

All patients' tumors included in these analyses were tested for HER2 protein overexpression or gene amplification at a central laboratory (Mayo Clinic). Patients were considered positive for HER2 according to the U.S. Food and Drug Administration-approved guidelines (immunohistochemistry, IHC: complete 3+ membrane staining ≥ 10% invasive cells; FISH: HER2:CEP17 ratio ≥ 2.0; refs. 12, 13). N9831 was approved by all treating sites' Institutional Review Boards, and all patients signed informed consent. The Mayo Institutional Review Board and the Correlative Science Committee of the North American Breast Cancer Group approved this translational study.

This study included 1,736 eligible/consented patients with sufficient tissue for analyses. Six hundred and eighty-two were excluded (failed central review: 283, ineligible: 61, canceled: 28, no consent: 187, lost to follow-up: 123) and 1,087 had insufficient tissue for analyses (Supplementary Fig. S2). The number of patients represented on tissue microarrays (TMA) with evaluable tissue cores were 1,216 and the number of different patients with evaluable whole sections were 520 (Supplementary Fig. S2).

Tissue microarrays and whole tissue sections

TMAs were constructed as part of the translational study component of N9831 using an ATA-27 automated TMA construction system (Beecher Instruments) as described previously (10). Each TMA contained control biopsies from non-neoplastic human liver, placenta, and tonsil tissues. Whole tissue sections from tumors not represented on TMAs were also examined. We evaluated the concordance between TMA and whole section protein analyses of 86 independent breast tumors and observed a concordance of 90% and 92% using the minimum and maximum TMA scores, respectively, of nuclear 3+ staining in more than 30% invasive cells.

MYC testing methods

Standard laboratory protocols were followed for IHC and quality control measures. Antigen retrieval was conducted on deparaffinized whole or TMA sections (5 μm) using preheated citrate buffer (98°C; 40 minutes). The tissue sections were treated with Peroxidase Blocking Reagent (Dako) and serum-free Protein Block (Dako) before IHC staining for c-MYC (mouse monoclonal clone 9E10; Sigma-Aldrich, #5546; dilution 1:250; 60-minute incubation) using a Dako Autostainer Plus (Reference #S3800). The sections were incubated in secondary antibody (Dako Envision Plus Dual Link Horseradish Peroxidase Kit; Dako # K4061). The high-sensitivity 3,3′-diaminobenzidine (DAB+) chromogenic substrate system (Betazoid DAB, Biocare) was used for colorimetric visualization followed by counter staining with hematoxylin.

MYC protein overexpression (MYC+) was defined as more than 30% of invasive cells with 3+ nuclear staining, based on the criteria used for HER2 protein overexpression established by the 2007 American Society of Clinical Oncologists/College of American Pathologists guidelines, as well as the lack of any widely accepted other criteria for MYC positivity in the literature (14). We also evaluated cytoplasmic MYC protein expression because MYC has been observed in the cytoplasm of tumor cells (5), which has been shown to be correlated with increased survival of patients with breast cancer (15).

Statistical analysis

The primary endpoint of N9831 was DFS and was defined as local, regional, or distant recurrence, contralateral breast cancer, another primary cancer (except squamous or basal cell carcinoma of the skin, carcinoma in situ of the cervix, or lobular carcinoma in situ of the breast), or death from any cause. Duration of DFS was defined as the time from registration to the first DFS event. DFS was estimated by the Kaplan–Meier method. Comparisons between Arms A, B, and C within subgroups were conducted using Cox proportional hazards models stratified by nodal status (1–3 vs. 4–9 vs. ≥10 positive nodes vs. positive sentinel node only vs. negative sentinel node with no axillary nodal dissection vs. axillary nodal dissection with no positive nodes) and hormone receptor status (estrogen receptor positive and/or progesterone receptor positive vs. negative for both receptors). We tested MYC protein expression as a predictor for differential trastuzumab benefit between MYC subgroups using Cox proportional hazards models (also stratified by nodal status and hormone receptor status), which included a treatment arm by MYC subgroup interaction term. The maximum nuclear MYC protein expression of the whole section or of the replicate TMA biopsies was used for all analyses associated with patient outcome.

Study patients

The trial N9831 registered 3,505 patients into Arms A (1,232 patients), B (1,216 patients), and C (1,057 patients) of which 1,736 patients (A: 584, B: 624, and C: 528) were included in the statistical analysis of MYC protein expression (Supplementary Fig. S2). The 1,769 patients who were excluded from analysis were excluded for the following reasons: failed central HER2 pathology review (283 patients), ineligible (61 patients), canceled before treatment initiation (28 patients), withdrew consent (187 patients), lost to follow-up (123 patients), and no/inadequate tissue or a technical failure of the assay (1,087 patients). The median follow-up time was 6.1 years (September 21, 2010).

The clinicopathological characteristics of the 1,736 patients enrolled on Arms A, B, and C reported herein were similar to the 1,087 consented and eligible patients on Arms A, B, and C excluded from analysis because of a lack of MYC results (Supplementary Table S1), except that included patients tended to have larger tumors. The clinicopathological characteristics of the 1,736 patients whose tumors had nuclear 3+ MYC protein staining in >30% and ≤ 30% invasive tumor cells are shown in Table 1. Patients whose tumors had nuclear 3+ staining in more than 30% invasive cells had a higher rate of hormone receptor positivity, larger tumors, higher rate of mastectomy, and higher number of positive nodes than those patients whose tumors had nuclear 3+ MYC staining in 30% or less invasive tumor cells.

Table 1.

Patient characteristics by % 3+ nuclear staining

≤30% 3 + Nuclear staining N = 1,162 (67%)>30% 3 + Nuclear staining N = 574 (33%)
CharacteristicN (%)N (%)χ2P
Age (median) 50 (22–80) 49 (25–79)  
Age group 
 <40 200 (17) 106 (18) 0.49a 
 40–49 370 (32) 185 (32)  
 50–59 379 (33) 181 (32)  
 ≥ 60 213 (18) 102 (18)  
Race 
 White 993 (85) 495 (86) 0.66 
 Other 169 (15) 79 (14)  
Menopausal status 
 Pre 610 (52) 316 (55) 0.32 
 Post 552 (48) 258 (45)  
ER/PR Status 
 ER or PR Positive 584 (50) 329 (57) 0.006 
 Other 578 (50) 245 (43)  
Surgery 
 Breast conserving 482 (41) 202 (35) 0.01 
 Mastectomy 680 (58) 372 (65)  
Nodal statusb 
 Node positive (1–3+ nodes) 429 (37) 249 (43) <0.001 
 Node positive (4–9+ nodes) 298 (26) 150 (26)  
 Node positive (≥10+ nodes) 135 (12) 90 (16)  
 Node negative (no pos. nodes) 90 (8) 23 (4)  
 Positive sentinel node 94 (8) 38 (7)  
 Negative sentinel node 116 (10) 24 (4)  
Predominant tumor histology 
 Ductal 1,097 (94) 544 (95) 0.55 
 Lobular 33 (3) 19 (3)  
 Other 31 (3) 11 (2)  
 Missing  
Histologic tumor grade (Elston/Scarff-Bloom-Richardson) 
 Well/intermediate 327 (28) 159 (28) 0.85 
 Poor 835 (72) 415 (72)  
Pathologic tumor size 
 < 2 cm 384 (33) 159 (28) 0.02 
 ≥ 2 cm 778 (67) 415 (72)  
≤30% 3 + Nuclear staining N = 1,162 (67%)>30% 3 + Nuclear staining N = 574 (33%)
CharacteristicN (%)N (%)χ2P
Age (median) 50 (22–80) 49 (25–79)  
Age group 
 <40 200 (17) 106 (18) 0.49a 
 40–49 370 (32) 185 (32)  
 50–59 379 (33) 181 (32)  
 ≥ 60 213 (18) 102 (18)  
Race 
 White 993 (85) 495 (86) 0.66 
 Other 169 (15) 79 (14)  
Menopausal status 
 Pre 610 (52) 316 (55) 0.32 
 Post 552 (48) 258 (45)  
ER/PR Status 
 ER or PR Positive 584 (50) 329 (57) 0.006 
 Other 578 (50) 245 (43)  
Surgery 
 Breast conserving 482 (41) 202 (35) 0.01 
 Mastectomy 680 (58) 372 (65)  
Nodal statusb 
 Node positive (1–3+ nodes) 429 (37) 249 (43) <0.001 
 Node positive (4–9+ nodes) 298 (26) 150 (26)  
 Node positive (≥10+ nodes) 135 (12) 90 (16)  
 Node negative (no pos. nodes) 90 (8) 23 (4)  
 Positive sentinel node 94 (8) 38 (7)  
 Negative sentinel node 116 (10) 24 (4)  
Predominant tumor histology 
 Ductal 1,097 (94) 544 (95) 0.55 
 Lobular 33 (3) 19 (3)  
 Other 31 (3) 11 (2)  
 Missing  
Histologic tumor grade (Elston/Scarff-Bloom-Richardson) 
 Well/intermediate 327 (28) 159 (28) 0.85 
 Poor 835 (72) 415 (72)  
Pathologic tumor size 
 < 2 cm 384 (33) 159 (28) 0.02 
 ≥ 2 cm 778 (67) 415 (72)  

aMantel–Haenszel trend test.

bSentinel node findings are based on sentinel node dissection not followed by axillary dissection.

Abbreviations: ER, estrogen receptor; PR, progesterone receptor.

Distribution of MYC protein expression and relationship to HER2 protein expression

Of 1,736 patients with evaluable IHC analyses, 33% (n = 574) had more than 30% invasive cells, 28% (n = 494) had 10% to 30%, and 38% (n = 668) had less than 10% invasive cells with 3+ nuclear staining (Table 2). Nuclear and cytoplasmic 3+ staining had high agreement (81%; P < 0.001), and the correlation between 3+ nuclear and 3+ cytoplasmic staining was 0.66 (P < 0.001; Table 2). No significant association was observed between 3+ nuclear staining and HER2 IHC staining (P = 0.10; Table 3). Representative staining patterns of MYC protein expression are shown in Fig. 1.

Figure 1.

Representative IHC staining of MYC. A, representative staining of a specimen with more than 30% 3+ nuclear staining. B, representative staining of a specimen with less than 30% 3+ nuclear staining. C, representative staining of a specimen with 0% 3+ nuclear staining. ×10 magnification.

Figure 1.

Representative IHC staining of MYC. A, representative staining of a specimen with more than 30% 3+ nuclear staining. B, representative staining of a specimen with less than 30% 3+ nuclear staining. C, representative staining of a specimen with 0% 3+ nuclear staining. ×10 magnification.

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Table 2.

Correlation of MYC cytoplasmic staining with MYC nuclear staining

MYC% 3+ nuclear staining (maximum across cores)
0%–9%10%–30%31%–60%61%–100%Total
MYC% 3+ cytoplasmic staining (maximum across cores)a,b 0%–9% 536 (64%) 203 (24%) 78 (9%) 27 (3%) 844 
 10%–30% 118 (27%) 205 (46%) 75 (17%) 43 (10%) 441 
 31%–60% 11 (6%) 63 (32%) 74 (38%) 46 (24%) 194 
 61%–100% 3 (1%) 23 (9%) 54 (21%) 177 (69%) 257 
 Total 668 494 281 293 1,736 
MYC% 3+ nuclear staining (maximum across cores)
0%–9%10%–30%31%–60%61%–100%Total
MYC% 3+ cytoplasmic staining (maximum across cores)a,b 0%–9% 536 (64%) 203 (24%) 78 (9%) 27 (3%) 844 
 10%–30% 118 (27%) 205 (46%) 75 (17%) 43 (10%) 441 
 31%–60% 11 (6%) 63 (32%) 74 (38%) 46 (24%) 194 
 61%–100% 3 (1%) 23 (9%) 54 (21%) 177 (69%) 257 
 Total 668 494 281 293 1,736 

a3+ cytoplasmic staining versus 3+ nuclear staining Mantel–Haenszel χ2P ≤ 0.001.

bSpearman correlation between 3+ cytoplasmic staining and 3+ nuclear staining = 0.66 (P ≤ 0.001).

Table 3.

Correlation of MYC nuclear staining with HER2 status

MYC% 3+ nuclear staining (maximum across cores)
0%–9%10%–30%31%–60%61%–100%Total
HER2 IHCa 0, 1+ 10 (23%) 10 (23%) 9 (20%) 15 (34%) 44 
 2+ 70 (42%) 52 (31%) 16 (10%) 29 (17%) 167 
 3+ 582 (38%) 432 (28%) 256 (17%) 249 (16%) 1,519 
 Total 662 494 281 293 1,730 
MYC% 3+ nuclear staining (maximum across cores)
0%–9%10%–30%31%–60%61%–100%Total
HER2 IHCa 0, 1+ 10 (23%) 10 (23%) 9 (20%) 15 (34%) 44 
 2+ 70 (42%) 52 (31%) 16 (10%) 29 (17%) 167 
 3+ 582 (38%) 432 (28%) 256 (17%) 249 (16%) 1,519 
 Total 662 494 281 293 1,730 

aHER2 IHC staining versus 3+ nuclear staining Mantel–Haenszel χ2P = 0.10.

Associations between MYC protein expression and DFS

No significant differences in DFS were observed between MYC+ and MYC patients in any of the three arms (Table 4). Comparing DFS between arms C and A, patients with MYC+ (Fig. 2A) and MYC (Fig. 2B) tumors had HRs of 0.52 (P = 0.006) and 0.65 (P = 0.006), respectively (Pinteraction = 0.40). Comparing DFS between arms B and A, patients with MYC+ and MYC tumors had HRs of 0.79 (P = 0.21) and 0.74 (P = 0.04), respectively (Pinteraction = 0.71; Fig. 2A and B). Comparing DFS between arms C and B, patients with MYC+ and MYC tumors had HRs 0.56 (P = 0.02) and 0.89 (P = 0.49), respectively (Pinteraction = 0.17; Fig. 2A and B).

Figure 2.

DFS by MYC protein level and treatment arm. A, nuclear 3+ MYC staining in more than 30% of invasive tumor cells by treatment arm. B, nuclear 3+ MYC staining in 30% or less of invasive tumor cells by treatment arm. A, doxorubicin; C, cyclophosphamide; T, paclitaxel; H, trastuzumab. DFS stratified by receptor and nodal status; Arm A versus B Pinteraction = 0.71; Arm A versus C Pinteraction = 0.40; Arm B versus C Pinteraction = 0.17. C, forest plots of DFS by nuclear MYC protein expression (Arm C vs. A).

Figure 2.

DFS by MYC protein level and treatment arm. A, nuclear 3+ MYC staining in more than 30% of invasive tumor cells by treatment arm. B, nuclear 3+ MYC staining in 30% or less of invasive tumor cells by treatment arm. A, doxorubicin; C, cyclophosphamide; T, paclitaxel; H, trastuzumab. DFS stratified by receptor and nodal status; Arm A versus B Pinteraction = 0.71; Arm A versus C Pinteraction = 0.40; Arm B versus C Pinteraction = 0.17. C, forest plots of DFS by nuclear MYC protein expression (Arm C vs. A).

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Table 4.

DFS by MYC protein status within treatment arm

DFS
Arm3+ nuclear staining groupN# EventsHR (95% CI)P3 y5 y
A (N = 584) ≤ 30% 390 108  81.8 74.9 
 > 30% 194 58 1.04 (0.76–1.44) 0.77 79.3 71.4 
B (N = 624) ≤ 30% 411 86  86.3 81.6 
 > 30% 213 56 1.24 (0.87–1.76) 0.24 84.0 76.4 
C (N = 528) ≤ 30% 361 66  88.6 84.0 
 > 30% 167 28 0.80 (0.51–1.27) 0.34 91.0 88.0 
DFS
Arm3+ nuclear staining groupN# EventsHR (95% CI)P3 y5 y
A (N = 584) ≤ 30% 390 108  81.8 74.9 
 > 30% 194 58 1.04 (0.76–1.44) 0.77 79.3 71.4 
B (N = 624) ≤ 30% 411 86  86.3 81.6 
 > 30% 213 56 1.24 (0.87–1.76) 0.24 84.0 76.4 
C (N = 528) ≤ 30% 361 66  88.6 84.0 
 > 30% 167 28 0.80 (0.51–1.27) 0.34 91.0 88.0 

In addition, patients with nuclear MYC staining of 3+ in 0% to 9%, 10% to 30%, 31% to 60%, and 61% to 100% of cells had HRs (C vs. A) of 0.68 (95% CI, 0.45–1.02), 0.64 (95% CI, 0.39–1.04), 0.63 (95% CI, 0.31–1.24), and 0.44 (95% CI, 0.23–0.84), respectively (Mantel–Haenszel χ2 = 0.30; Fig. 2C).

MYC is a highly regulated and multifunctional transcription factor that regulates up to 15% of human genes and plays a central role in proliferation and malignant transformation of human and animal cells (16, 17). Previous evidence from B31 and N9831 suggested that MYC gene copy number anomalies may be associated with additional benefit to adjuvant trastuzumab (9, 10). Reports have been inconsistent with regard to the association of MYC protein expression and both clinicopathological characteristics and prognosis (5). To further explore these relationships, we designed the translational component of the N9831 trial to include an analysis of MYC protein overexpression in patients with HER2+ tumors.

Overall, we found that MYC protein expression was heterogeneous and characterized by both cytoplasmic and nuclear localization. We observed nuclear MYC protein overexpression (defined as nuclear 3+ staining in >30% invasive cells) in tumors from 33% of N9831 patients with HER2+ breast cancer. Early studies using IHC have shown that approximately 50% to 100% of breast cancer cases have increased levels of MYC protein (5, 15, 18–23). In addition, strong MYC protein positivity was found in more than 20% nuclei, in 45% of 440 primary breast tumors (24), and 70% (36/51) showed 1+ or higher intensity staining (25) using the 9E10 clone used in our present study. Our study observed a lower incidence of 33% most likely due to our more strict cutoff criteria of 3+ staining in more than 30% nuclei and the fact that our patient population was HER2+ and not a general population of patients with breast cancer.

We also observed strong (3+) cytoplasmic staining in malignant cells, which correlated with strong (3+) nuclear staining. Other groups have found predominant cytoplasmic localization of MYC (15, 19), and this cytoplasmic staining has been associated with better survival (15). These results support the idea of nuclear exclusion of MYC, which has been observed in high-grade tumors, and could serve to attenuate select functions of MYC in later stages of disease progression (25, 26).

In this analysis, MYC nuclear protein overexpression was associated with hormone receptor positivity, nodal positivity, and larger tumors with associated increase in mastectomy rates. In agreement with our findings, MYC protein expression has been correlated with positive nodes (20, 27) and with estrogen receptor positivity (19). MYC also has been shown to be an estrogen-responsive gene (28). Although a significant association has not been consistently observed between MYC protein expression and breast tumor size (5, 19), MYC DNA levels (as detected by Southern blot analysis) have been correlated with tumor size (5), and the Ki-67 proliferation marker has been shown to correlate with MYC protein level (20). The association between MYC overexpression and tumor size in our study is consistent with MYC being a transcriptional activator of the cell proliferation pathway, an additional marker for the assessment of tumor cell proliferation (5). In contrast, other studies have shown no significant associations with estrogen receptors (18, 20–22, 24, 29) or with lymph node status (15, 18, 20–22, 24, 29–32).

We did not observe a significant difference in outcome between patients with and without MYC protein overexpression within any treatment arm and specifically, the DFS of N9831 patients treated with chemotherapy only was similar, regardless of MYC protein overexpression. Although this suggests that MYC overexpression is not a marker for prognosis, the true prognostic significance of MYC protein overexpression cannot be addressed in this study as all patients were treated with chemotherapy, and subgroups of patients also received protocol-specified radiotherapy and/or hormonal therapy. We did observe that MYC overexpression was associated with larger tumors and nodal positivity, both of which are powerful prognostic indicators. In N9831, however, radiotherapy was directed on the basis of surgery type and number of positive nodes. MYC overexpression was also associated with hormone receptor positivity, another disease characteristic, which was used in directing additional therapy, namely hormonal therapy. Therefore, one possibility to explain similar DFS within arm regardless of MYC overexpression status is that MYC may have disparate prognostic impact in subtypes of breast cancer, which washes out when treated differently and considered together. Another possibility is that MYC overexpression confers a worse prognosis but perhaps a greater response to chemotherapy, making it an overall null biomarker in this chemotherapy-treated population.

Several investigations have found that higher expression of MYC protein correlated with poorer outcome (33–35), whereas other studies have shown positive associations between MYC mRNA levels and survival (36) and between MYC protein levels and survival, most notably for node-negative patients (15). Our findings are consistent with previous findings that showed that MYC protein expression alone was not related to recurrence (30) and are supported by a limited number of studies that did not find associations between MYC expression and prognosis (19, 22). Importantly, however, we observed a benefit of concurrent trastuzumab in patients with or without MYC protein overexpression.

We did observe a trend toward greater benefit from concurrent trastuzumab with increasing MYC protein expression. Trastuzumab has been shown to sensitize HER2-overexpressing cells to apoptosis (37, 38), possibly through induction of the proapoptotic function of MYC (39–41). Higher MYC protein expression could then result in an increased rate of apoptosis in tumors with HER2 protein overexpression. Alternatively, trastuzumab was recently shown to inhibit glycolysis in HER2+ cells (42), and downregulation of MYC protein has been shown to contribute to cancer cell survival under dual deficiency of oxygen and glucose (43). Conceivably, when MYC protein is overexpressed in HER2+ tumors that are treated with trastuzumab, the protective effect of MYC downregulation on cancer cell survival in low glucose settings would be lost. This may result in increased tumor cell death and improved outcome of patients with increasing MYC protein expression.

We also observed statistically significantly improved DFS from concurrent trastuzumab compared with sequential trastuzumab among patients with MYC protein overexpression, but not in patients without MYC protein overexpression. The interaction between MYC protein overexpression and timing of trastuzumab did not reach statistical significance, but the large improvement in the HR for patients with MYC overexpression relative to patients without MYC overexpression gives rise to the speculation that the timing/schedule of trastuzumab administration may be important in using MYC as an additional marker of trastuzumab sensitivity. As MYC induces cell proliferation and HER2+ breast tumors tend to have a high proliferation index (44–46), those tumors with overexpression for both HER2 and MYC may be more susceptible to the growth inhibitory synergistic effects observed with the combination of chemotherapy and trastuzumab (37, 38, 47).

Overall, our data indicate that MYC protein expression alone is not significantly associated with differential benefit to concurrent trastuzumab, but potentially could help differentiate benefit between concurrent and sequential trastuzumab treatment. Distinct mechanisms of regulation for MYC have been defined over the past decade, and several signal transduction pathways and regulatory mechanisms have evolved to keep MYC expression under tight control (17). Ongoing protein expression analyses of regulators and effectors of MYC (e.g., PTEN and insulin-like growth factor receptor-I; refs. 48, 49), and whole-genome expression profiling of N9831 tumors will provide important information about the interactions between MYC and other pertinent proteins and genes and the effects of these interactions on the sensitivity/resistance to adjuvant trastuzumab. Understanding the full extent of the oncogenic effects of these interactions is critical to the development of more effective, targeted therapies for patients with breast cancer that exhibit HER2+ disease.

P.A. Kaufman has a commercial research grant, has honoraria from speakers' bureau, and is a consultant/advisory board member of Genentech. J. Gralow has a commercial research grant from Amgen, Novartis, Genentech, and Roche. No potential conflicts of interest were disclosed by the other authors.

Conception and design: A.C. Dueck, M.M. Reinholz, W. Lingle, E.A. Perez

Development of methodology: A.C. Dueck, M.M. Reinholz, R.B. Jenkins, A.E. McCullough, W. Lingle, E.A. Perez

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): M.M. Reinholz, M.M. Reinholz, R.B. Jenkins, D. Riehle, N.E. Davidson, S. Martino, G.W. Sledge, J. Gralow, L. Harris, J.N. Ingle, W. Lingle, E.A. Perez

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): A.C. Dueck, M.M. Reinholz, K.S. Tenner, K.V. Ballman, R.B. Jenkins, G.W. Sledge, P.A. Kaufman, L. Harris, E.A. Perez

Writing, review, and/or revision of the manuscript: A.C. Dueck, M.M. Reinholz, M.M. Reinholz, K.S. Tenner, K.V. Ballman, R.B. Jenkins, B. Chen, A.E. McCullough, N.E. Davidson, S. Martino, G.W. Sledge, P.A. Kaufman, L.A. Kutteh, J. Gralow, L. Harris, J.N. Ingle, W. Lingle, E.A. Perez

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): A.C. Dueck, M.M. Reinholz, K.S. Tenner, R.B. Jenkins

Study supervision: M.M. Reinholz, R.B. Jenkins, P.A. Kaufman, E.A. Perez

This work was supported by NIH grants CA25224-31 and CA114740 (PI: J.C. Buckner) for the North Central Cancer Treatment Group and associated Biospecimen Resource, respectively, NIH grant CA129949 (PI: E.A. Perez/M.M. Reinholz), and the Breast Cancer Research Foundation (PI: E.A. Perez).

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 with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Perez
EA
,
Palmieri
FM
,
Brock
SM
. 
Trastuzumab
.
Cancer Treat Res
2009
;
151
:
181
96
.
2.
Perez
EA
,
Romond
EH
,
Suman
VJ
,
Jeong
JH
,
Davidson
NE
,
Geyer
CE
 Jr
, et al
Four-year follow-up of trastuzumab plus adjuvant chemotherapy for operable human epidermal growth factor receptor 2-positive breast cancer: joint analysis of data from NCCTG N9831 and NSABP B-31
.
J Clin Oncol
2011
;
29
:
3366
73
.
3.
Piccart-Gebhart
MJ
,
Procter
M
,
Leyland-Jones
B
,
Goldhirsch
A
,
Untch
M
,
Smith
I
, et al
Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer
.
N Engl J Med
2005
;
353
:
1659
72
.
4.
Bedard
PL
,
de Azambuja
E
,
Cardoso
F
. 
Beyond trastuzumab: overcoming resistance to targeted HER-2 therapy in breast cancer
.
Curr Cancer Drug Targets
2009
;
9
:
148
62
.
5.
Liao
DJ
,
Dickson
RB
. 
c-Myc in breast cancer
.
Endocr Relat Cancer
2000
;
7
:
143
64
.
6.
Lutz
W
,
Leon
J
,
Eilers
M
. 
Contributions of Myc to tumorigenesis
.
Biochim Biophys Acta
2002
;
1602
:
61
71
.
7.
Xu
J
,
Chen
Y
,
Olopade
OI
. 
MYC and Breast Cancer
.
Genes Cancer
2010
;
1
:
629
40
.
8.
Neve
RM
,
Sutterluty
H
,
Pullen
N
,
Lane
HA
,
Daly
JM
,
Krek
W
, et al
Effects of oncogenic ErbB2 on G1 cell cycle regulators in breast tumour cells
.
Oncogene
2000
;
19
:
1647
56
.
9.
Kim
C
,
Bryant
J
,
Horne
Z
,
Geyer
C
,
Wickerham
D
,
Wolmark
N
, et al
Trastuzumab sensitivity of breast cancer with co-amplification of HER2 and cMYC suggests pro-apoptotic function of dysregulated cMYC in vivo. [Abstract 46]
.
Breast Cancer Res Treat
2005
;
94
:
S6
.
10.
Perez
EA
,
Jenkins
RB
,
Dueck
AC
,
Wiktor
AE
,
Bedroske
PP
,
Anderson
SK
, et al
C-MYC alterations and association with patient outcome in early-stage HER2-positive breast cancer from the north central cancer treatment group N9831 adjuvant trastuzumab trial
.
J Clin Oncol
2011
;
29
:
651
9
.
11.
Perez
EA
,
Suman
VJ
,
Davidson
NE
,
Gralow
JR
,
Kaufman
PA
,
Visscher
DW
, et al
Sequential versus concurrent trastuzumab in adjuvant chemotherapy for breast cancer
.
J Clin Oncol
2011
;
29
:
4491
7
.
12.
Gianni
L
,
Zambetti
M
,
Clark
K
,
Baker
J
,
Cronin
M
,
Wu
J
, et al
Gene expression profiles in paraffin-embedded core biopsy tissue predict response to chemotherapy in women with locally advanced breast cancer
.
J Clin Oncol
2005
;
23
:
7265
77
.
13.
Perez
EA
,
Roche
PC
,
Jenkins
RB
,
Reynolds
CA
,
Halling
KC
,
Ingle
JN
, et al
HER2 testing in patients with breast cancer: poor correlation between weak positivity by immunohistochemistry and gene amplification by fluorescence in situ hybridization
.
Mayo Clin Proc
2002
;
77
:
148
54
.
14.
Wolff
AC
,
Hammond
ME
,
Schwartz
JN
,
Hagerty
KL
,
Allred
DC
,
Cote
RJ
, 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
:
118
45
.
15.
Pietilainen
T
,
Lipponen
P
,
Aaltomaa
S
,
Eskelinen
M
,
Kosma
VM
,
Syrjanen
K
. 
Expression of c-myc proteins in breast cancer as related to established prognostic factors and survival
.
Anticancer Res
1995
;
15
:
959
64
.
16.
Dang
CV
. 
Enigmatic MYC conducts an unfolding systems biology symphony
.
Genes Cancer
2010
;
1
:
526
31
.
17.
Meyer
N
,
Penn
LZ
. 
Reflecting on 25 years with MYC
.
Nat Rev Cancer
2008
;
8
:
976
90
.
18.
Locker
AP
,
Dowle
CS
,
Ellis
IO
,
Elston
CW
,
Blamey
RW
,
Sikora
K
, et al
c-myc oncogene product expression and prognosis in operable breast cancer
.
Br J Cancer
1989
;
60
:
669
72
.
19.
Mizukami
Y
,
Nonomura
A
,
Noguchi
M
,
Taniya
T
,
Koyasaki
N
,
Saito
Y
, et al
Immunohistochemical study of oncogene product ras p21, c-myc and growth factor EGF in breast carcinomas
.
Anticancer Res
1991
;
11
:
1485
94
.
20.
Pavelic
ZP
,
Pavelic
L
,
Lower
EE
,
Gapany
M
,
Gapany
S
,
Barker
EA
, et al
c-myc, c-erbB-2, and Ki-67 expression in normal breast tissue and in invasive and noninvasive breast carcinoma
.
Cancer Res
1992
;
52
:
2597
602
.
21.
Spandidos
DA
,
Yiagnisis
M
,
Papadimitriou
K
,
Field
JK
. 
ras, c-myc and c-erbB-2 oncoproteins in human breast cancer
.
Anticancer Res
1989
;
9
:
1385
93
.
22.
Spaventi
R
,
Kamenjicki
E
,
Pecina
N
,
Grazio
S
,
Pavelic
J
,
Kusic
B
, et al
Immunohistochemical detection of TGF-alpha, EGF-R, c-erbB-2, c-H-ras, c-myc, estrogen and progesterone in benign and malignant human breast lesions: a concomitant expression
.
In Vivo
1994
;
8
:
183
9
.
23.
Walker
RA
,
Senior
PV
,
Jones
JL
,
Critchley
DR
,
Varley
JM
. 
An immunohistochemical and in situ hybridization study of c-myc and c-erbB-2 expression in primary human breast carcinomas
.
J Pathol
1989
;
158
:
97
105
.
24.
Naidu
R
,
Wahab
NA
,
Yadav
M
,
Kutty
MK
. 
Protein expression and molecular analysis of c-myc gene in primary breast carcinomas using immunohistochemistry and differential polymerase chain reaction
.
Int J Mol Med
2002
;
9
:
189
96
.
25.
Blancato
J
,
Singh
B
,
Liu
A
,
Liao
DJ
,
Dickson
RB
. 
Correlation of amplification and overexpression of the c-myc oncogene in high-grade breast cancer: FISH, in situ hybridisation and immunohistochemical analyses
.
Br J Cancer
2004
;
90
:
1612
9
.
26.
Liao
DJ
,
Natarajan
G
,
Deming
SL
,
Jamerson
MH
,
Johnson
M
,
Chepko
G
, et al
Cell cycle basis for the onset and progression of c-Myc-induced, TGFalpha-enhanced mouse mammary gland carcinogenesis
.
Oncogene
2000
;
19
:
1307
17
.
27.
Pavelic
ZP
,
Steele
P
,
Preisler
HD
. 
Evaluation of c-myc proto-oncogene in primary human breast carcinomas
.
Anticancer Res
1991
;
11
:
1421
7
.
28.
Miller
TL
,
Huzel
NJ
,
Davie
JR
,
Murphy
LC
. 
C-myc gene chromatin of estrogen receptor positive and negative breast cancer cells
.
Mol Cell Endocrinol
1993
;
91
:
83
9
.
29.
Saccani Jotti
G
,
Fontanesi
M
,
Bombardieri
E
,
Gabrielli
M
,
Veronesi
P
,
Bianchi
M
, et al
Preliminary study on oncogene product immunohistochemistry (c-erbB-2, c-myc, ras p21, EGFR) in breast pathology
.
Int J Biol Markers
1992
;
7
:
35
42
.
30.
Bland
KI
,
Konstadoulakis
MM
,
Vezeridis
MP
,
Wanebo
HJ
. 
Oncogene protein co-expression. Value of Ha-ras, c-myc, c-fos, and p53 as prognostic discriminants for breast carcinoma
.
Ann Surg
1995
;
221
:
706
18
.
31.
Bolufer
P
,
Molina
R
,
Ruiz
A
,
Hernandez
M
,
Vazquez
C
,
Lluch
A
. 
Estradiol receptors in combination with neu or myc oncogene amplifications might define new subtypes of breast cancer
.
Clin Chim Acta
1994
;
229
:
107
22
.
32.
Sirotkovic-Skerlev
M
,
Krizanac
S
,
Kapitanovic
S
,
Husnjak
K
,
Unusic
J
,
Pavelic
K
. 
Expression of c-myc, erbB-2, p53 and nm23-H1 gene product in benign and malignant breast lesions: coexpression and correlation with clinicopathologic parameters
.
Exp Mol Pathol
2005
;
79
:
42
50
.
33.
Guerin
M
,
Barrois
M
,
Terrier
MJ
,
Spielmann
M
,
Riou
G
. 
Overexpression of either c-myc or c-erbB-2/neu proto-oncogenes in human breast carcinomas: correlation with poor prognosis
.
Oncogene Res
1988
;
3
:
21
31
.
34.
Pertschuk
LP
,
Feldman
JG
,
Kim
DS
,
Nayeri
K
,
Eisenberg
KB
,
Carter
AC
, et al
Steroid hormone receptor immunohistochemistry and amplification of c-myc protooncogene. Relationship to disease-free survival in breast cancer
.
Cancer
1993
;
71
:
162
71
.
35.
Scorilas
A
,
Trangas
T
,
Yotis
J
,
Pateras
C
,
Talieri
M
. 
Determination of c-myc amplification and overexpression in breast cancer patients: evaluation of its prognostic value against c-erbB-2, cathepsin-D and clinicopathological characteristics using univariate and multivariate analysis
.
Br J Cancer
1999
;
81
:
1385
91
.
36.
Bieche
I
,
Laurendeau
I
,
Tozlu
S
,
Olivi
M
,
Vidaud
D
,
Lidereau
R
, et al
Quantitation of MYC gene expression in sporadic breast tumors with a real-time reverse transcription-PCR assay
.
Cancer Res
1999
;
59
:
2759
65
.
37.
Arteaga
CL
. 
Can trastuzumab be effective against tumors with low HER2/Neu (ErbB2) receptors?
J Clin Oncol
2006
;
24
:
3722
5
.
38.
Henson
ES
,
Hu
X
,
Gibson
SB
. 
Herceptin sensitizes ErbB2-overexpressing cells to apoptosis by reducing antiapoptotic Mcl-1 expression
.
Clin Cancer Res
2006
;
12
:
845
53
.
39.
Adhikary
S
,
Eilers
M
. 
Transcriptional regulation and transformation by Myc proteins
.
Nat Rev Mol Cell Biol
2005
;
6
:
635
45
.
40.
Nilsson
JA
,
Cleveland
JL
. 
Myc pathways provoking cell suicide and cancer
.
Oncogene
2003
;
22
:
9007
21
.
41.
Cole
MD
,
McMahon
SB
. 
The Myc oncoprotein: a critical evaluation of transactivation and target gene regulation
.
Oncogene
1999
;
18
:
2916
24
.
42.
Zhao
Y
,
Zhou
LM
,
Chen
YY
,
Yang
SG
,
Tian
WM
. 
MYC genes with differential responses to tapping, mechanical wounding, ethrel and methyl jasmonate in laticifers of rubber tree (Hevea brasiliensis Muell. Arg.)
.
J Plant Physiol
2011
;
168
:
1649
58
.
43.
Okuyama
H
,
Endo
H
,
Akashika
T
,
Kato
K
,
Inoue
M
. 
Downregulation of c-MYC protein levels contributes to cancer cell survival under dual deficiency of oxygen and glucose
.
Cancer Res
2010
;
70
:
10213
23
.
44.
Rudolph
P
,
Olsson
H
,
Bonatz
G
,
Ratjen
V
,
Bolte
H
,
Baldetorp
B
, et al
Correlation between p53, c-erbB-2, and topoisomerase II alpha expression, DNA ploidy, hormonal receptor status and proliferation in 356 node-negative breast carcinomas: prognostic implications
.
J Pathol
1999
;
187
:
207
16
.
45.
Trihia
H
,
Murray
S
,
Price
K
,
Gelber
RD
,
Golouh
R
,
Goldhirsch
A
, et al
Ki-67 expression in breast carcinoma: its association with grading systems, clinical parameters, and other prognostic factors–a surrogate marker?
Cancer
2003
;
97
:
1321
31
.
46.
Yerushalmi
R
,
Woods
R
,
Ravdin
PM
,
Hayes
MM
,
Gelmon
KA
. 
Ki67 in breast cancer: prognostic and predictive potential
.
Lancet Oncol
2010
;
11
:
174
83
.
47.
Menendez
JA
,
Mehmi
I
,
Lupu
R
. 
Trastuzumab in combination with heregulin-activated Her-2 (erbB-2) triggers a receptor-enhanced chemosensitivity effect in the absence of Her-2 overexpression
.
J Clin Oncol
2006
;
24
:
3735
46
.
48.
Perez
EA
,
Dueck
AC
,
McCullough
AE
,
Chen
B
,
Geiger
XJ
,
Jenkins
RB
, et al
Impact of PTEN protein expression on benefit from adjuvant trastuzumab in early-stage human epidermal growth factor receptor 2-positive breast cancer in the North Central Cancer Treatment Group N9831 Trial
.
J Clin Oncol
2013
;
31
:
2115
22
.
49.
Reinholz
MM
,
Dueck
AC
,
Chen
B
,
Geiger
X
,
McCullough
AE
,
Jenkins
RB
, et al
Effect of IGF1R protein expression on benefit to adjuvant trastuzumab in early-stage HER2+ breast cancer in NCCTG adjuvant trial N9831
.
J Clin Oncol
2011
;
29
(
15_suppl
):
10503
.