Kutteh, Leila A.

539 Background: An increased baseline sHER-2 concentration is an indicator of poor prognosis in the metastatic and adjuvant settings of HER-2+BC. This study evaluated the levels of sHER-2 during treatment and at time of recurrence in N9831. Methods: Aims were to describe sHER-2 levels during treatment and at time of recurrence in patients (pts) randomized to Arms A (standard chemotherapy) and C (standard chemotherapy with concurrent trastuzumab). Baseline serum samples of 1506 pts from both arms along with 86 recurrence serum samples were analyzed for sHER-2 (ng/mL). In addition, 556 serial samples were obtained in 148 patients over the treatment period. Median follow up was 4.5 years. Statistical methods included repeated measures linear models, Wilcoxon rank sum tests, and Cox regression models. Results: Analyzing serial sHER-2 levels during treatment, mean log sHER-2 remained constant in Arm C (estimated slope = 0.004, p = 0.44) while the estimated increase per month was 0.026 (on log e scale) for Arm A (p = 0.0003). Based on the linear model at 18 months, the mean log sHER-2 was 2.98 (95% CI: 2.87–3.09) and 2.55 (95% CI: 2.40–2.70) for Arms A and C, respectively. Among pts with disease recurrence, sHER-2 levels increased in Arm A from baseline to time of recurrence (mean = 72.9, median = 1.7, p =0.005) while sHER-2 levels remained unchanged in Arm C (mean = 9.2, median = -0.9, p = 0.65). While all 86 recurrence patients had baseline sHER2 levels <50, 24% (15/63) of Arm A patients had very high recurrence sHER-2 levels (≥50) as compared to 9% (2/23) on Arm C (p=0.10). Patients with recurrence sHER-2 levels ≥15 had shorter survival time following recurrence with 3 year overall survival of 51% compared to 76% for the <15 sHER-2 group (HR = 2.77; 95% CI: 1.20–6.43, p = 0.02). Conclusions: Based on serial specimens during treatment, the concurrent trastuzumab arm (C) had constant sHER-2 levels, whereas the standard chemotherapy arm (A) had increasing sHER-2 levels. In arm A, recurrence sHER-2 levels increased significantly from baseline while recurrence sHER-2 levels did not change in arm C. Additionally, pts with high sHER-2 levels at recurrence had shorter overall survival following recurrence. Partial support: CA25224, CA114740 , Genentech, Siemens, and the BCRF. [Table: see text]

579 Background: N9831 and other clinical trials have demonstrated the additive survival benefit of trastuzumab (H) therapy with chemotherapy for patients with resected HER-2 positive breast cancer. Cardiac toxicity (CTox) is increased by this strategy. Prediction of or early detection of CTox is important. Methods: 95 patients provided informed consent. Brain natriuretic peptide (BNP), C-reactive protein (CRP), troponin T (TnT) and I (TnI), TNF-alpha, IL-1b, and IL-6 were to be drawn at baseline, after initiation of doxorubicin/cyclophosphamide (AC), after initiation of paclitaxel (T), and after the initiation of H (may have been given concurrently with T). 67 patients with at least a baseline sample, a post-treatment sample, and a baseline and subsequent evaluation of LVEF were studied. No patient in this group had a grade 3 decline in LVEF. Only 3 of these patients did not receive H. Values above the 99th percentile were considered abnormal for troponin. 40 ng/ml was considered elevated for BNP. Results: Values of CRP, TNF-alpha, IL- 6 and IL1b changed in very few patients and without a discernible pattern. For further evaluation of BNP, TnI, and TnT, 2 definitions of CTox were tested: 1. a >10% drop in LVEF from baseline (n=27) and 2. a drop in LVEF of 10–15% to less than the institutional lower limit of normal (LLN) OR a >15% decline in LVEF (n=14). No pre-treatment marker appeared to predict a > 10% decline in LVEF (definition 1). For patients fulfilling definition 2, pre-treatment BNP > 40 was seen in 21% of patients vs 8% of normals (nls) (p= 0.18) and an abnormal TnI (>0.04) was seen in 21% vs 6% of nls (p=0.10). Only doubling of BNP appeared promising as a serial marker of CTox using definition 2 (27% vs 7% for nls (p = 0.09). Conclusions: Baseline BNP and TnI and serial measures of BNP may be promising for further investigation for the prediction and detection of treatment-related CTox. Due to limitations of the study including the small numbers of patients analyzed, the difficulty in defining CTox, and the variation in the timing of sample procurement and assessment, these data are considered exploratory. Supported by CA-25224, CA-2115, CA 38926, CA31946. No significant financial relationships to disclose.

Abstract 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 &lt; 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.

BACKGROUNDIncreased soluble human epidermal growth factor receptor 2 (sHER2) is an indicator of a poor prognosis in HER2‐positive metastatic breast cancer. In this study, the authors evaluated levels of sHER2 during treatment and at the time of disease recurrence in the adjuvant North Central Cancer Treatment Group N9831 clinical trial.METHODSThe objectives were to describe sHER2 levels during treatment and at the time of recurrence in patients who were randomized to treatment arms A (standard chemotherapy), B (standard chemotherapy with sequential trastuzumab), and C (standard chemotherapy with concurrent trastuzumab). Baseline samples were available from 2318 patients, serial samples were available from 105 patients, and recurrence samples were available from 124 patients. The cutoff sHER2 value for the assay was 15 ng/mL. Statistical methods included repeated measures linear models, Wilcoxon rank‐sum tests, and Cox regression models.RESULTSThere were differences between groups in terms of age, menopausal status, and hormone receptor status. Within treatment arms A, B, and C, patients who had baseline sHER2 levels ≥15 ng/mL had worse disease‐free survival than patients who had baseline sHER2 levels <15 ng/mL (arm A: hazard ratio, 1.81; P = .0014; arm B: hazard ratio, 2.08; P = .0015; arm C: hazard ratio, 1.96; P = .01). Among the 124 patients who experienced disease recurrence, sHER2 levels increased from baseline to the time of recurrence in arms A and B but remained unchanged in arm C. Patients who had recurrence sHER2 levels ≥15 ng/mL had a shorter survival after recurrence with a 3‐year overall survival rate of 51% compared with 77% for those who had recurrence sHER2 levels <15 ng/mL (hazard ratio, 2.36; 95% confidence interval, 1.19‐4.70; P = .01).CONCLUSIONSIn patients with early stage, HER2‐positive breast cancer, a high baseline sHER2 level was identified as a prognostic marker associated with shorter disease‐free survival, and a high sHER2 level at recurrence was predictive of shorter survival. Cancer 2013;119:2675–2682. © 2013 American Cancer Society.

Purpose It has been suggested that PTEN, a negative regulator of PI3K/AKT signaling, is involved in tumor sensitivity to trastuzumab. We investigated the association between tumor PTEN protein expression and disease-free survival (DFS) of patients randomly assigned to receive chemotherapy alone (arm A) or chemotherapy with sequential (arm B) or concurrent trastuzumab (arm C) in the phase III early-stage human epidermal growth factor receptor 2 (HER2) –positive trial—North Central Cancer Treatment Group (NCCTG) N9831. Patients and Methods The intensity and percentage of invasive cells with cytoplasmic PTEN staining were determined in tissue microarray sections containing three cores per block (n = 1,286) or in whole tissue sections (WS; n = 516) by using standard immunohistochemistry (138G6 monoclonal antibody). Tumors were considered positive for PTEN (PTEN-positive) if any core or WS had any invasive cells with ≥ 1+ staining. Median follow-up was 6.0 years. Results Of 1,802 patients included in this analysis (of 3,505 patients registered to N9831), 1,342 (74%) had PTEN-positive tumors. PTEN positivity was associated with hormone receptor negativity (χ2 P < .001) and nodal positivity (χ2 P = .04). PTEN did not have an impact on DFS within the various arms. Comparing DFS of arm C to arm A, patients with PTEN-positive and PTEN-negative tumors had hazard ratios (HRs) of 0.65 (P = .003) and 0.47 (P = .005), respectively (interaction P = .16). For arm B versus arm A, patients with PTEN-positive and PTEN-negative tumors had HRs of 0.70 (P = .009) and 0.85 (P = .44), respectively (interaction P = .47). Conclusion In contrast to selected preclinical and limited clinical studies suggesting a decrease in trastuzumab sensitivity in patients with PTEN-negative tumors, our data show benefit of adjuvant trastuzumab for patients with HER2-positive breast cancer, independent of tumor PTEN status.

Purpose Findings from the human epidermal growth factor receptor 2 (HER2) –positive National Surgical Adjuvant Breast and Bowel Project (NSABP) B31 trial suggested that MYC/HER2 coamplification (> 5.0 copies/nucleus) was associated with additional benefit from adjuvant trastuzumab in patients with early-stage breast cancer. To further explore this relationship, we investigated associations between MYC amplification and disease-free survival (DFS) in a similar adjuvant trastuzumab HER2-positive breast cancer trial—North Central Cancer Treatment Group (NCCTG) N9831. Patients and Methods This analysis included 799 patients randomly assigned to receive chemotherapy alone or with concurrent trastuzumab on N9831. Fluorescence in situ hybridization (FISH) was performed by using a dual-probe mixture for MYC and centromere 8 (MYC:CEP8) on tissue microarrays. MYC amplification was prespecified as MYC:CEP8 ratio > 2.2 or average MYC copies/nucleus > 5.0. Exploratory variables included polysomy 8. Results In comparing DFS (median follow-up, 4.0 years) between treatments, patients with MYC:CEP8 ratio ≤ 2.2 (n = 618; 77%) and > 2.2 (n = 181; 23%) had hazard ratios (HRs) of 0.46 (P < .001) and 0.67 (P = .33), respectively (interaction P = .38). Patients with MYC copies/nucleus ≤ 5.0 (n = 534; 67%) and > 5.0 (n = 265; 33%) had HRs of 0.52 (P = .002) and 0.48 (P = .02), respectively (interaction P = .94). Patients with MYC:CEP8 ratio < 1.3 with normal chromosome 8 copy number (n = 141; 18%) and ≥ 1.3 or < 1.3 with polysomy 8 (n = 658; 82%) had HRs of 0.66 (P = .28) and 0.44 (P < .001), respectively (interaction P = .23). Patients with MYC copies/nucleus < 2.5 (n = 130; 16%) and ≥ 2.5 (n = 669; 84%) had HRs of 1.07 (P = .87) and 0.42 (P < .001), respectively (interaction P = .05). Conclusion We did not confirm the B31 association between MYC amplification and additional trastuzumab benefit. Exploratory analyses revealed potential associations between alternative MYC/chromosome 8 copy number alterations and differential benefit of adjuvant trastuzumab.

Abstract Background: In 2007, the American Society of Clinical Oncology and College of American Pathologists (ASCO/CAP) recommended new guidelines to define HER2 positivity by immunohistochemistry (3+ IHC: uniform intense membrane staining of &gt;30% of invasive tumor cells) or fluorescent in situ hybridization (FISH+: HER2/CEP17 ratio &gt;2.2). The original criteria used for enrollment in the pivotal N9831 trial conducted from 2000-2005 were IHC&gt;10% (3+) or FISH ≥2.0.Purpose: To investigate the impact of the ASCO/CAP guidelines on patient (pt) eligibility and disease free survival (DFS) compared to the originally used FDA-approved definitions.Patients/Methods: This analysis included 2268 patients from the HER2+ N9831 adjuvant trastuzumab phase III trial. IHC was centrally performed at the Mayo Clinic using the DAKO HercepTest™ and was re-analyzed to determine the percent of tumor cells with 0, 1+, 2+, and 3+ staining intensities. DFS was compared between pts randomized to standard chemotherapy +/- concurrent trastuzumab (Arms A and C of the trial) within IHC/FISH subgroups using Cox proportional hazards regression stratified by hormone receptor and nodal status.Results: Based on 2268 pts, 83 pts (3.7%) were 3+ by FDA-approved guidelines but not 3+ by the ASCO/CAP guidelines. Among these pts, 77% (64/83) were FISH amplified centrally by ASCO/CAP guidelines. Only 1.5% (34/2268) of pts eligible under FDA-approved definitions from N9831 in this sample set would not meet the ASCO/CAP guidelines. In 1441 patients randomized to standard chemotherapy +/- concurrent trastuzumab, the exploratory DFS hazard ratio (HR) associated with trastuzumab in IHC/FISH subgroups were as follows: ASCO/CAP HER2+ HR=0.59 (95% CI: 0.47-0.75; p&lt;0.001); FDA-approved HER2+ HR=0.60 (95% CI: 0.47-0.75; p&lt;0.001); ASCO/CAP HER2 normal(not positive) HR=0.74 (95% CI: 0.35-1.57; p=0.43); and FDA-approved HER2 normal HR=0.53 (95% CI: 0.22-1.26; p=0.15).Conclusions: A small percentage (1.5%) of N9831 patients did not meet ASCO/CAP 2007 guidelines when applied retrospectively. Trastuzumab effect appeared similar for HER2+ patients regardless of ASCO/CAP or FDA-approved guidelines. Additional DFS analyses by IHC/FISH subgroups will be presented. We gratefully acknowledge support from the Breast Cancer Research Foundation, Genentech, and NIH CA25224 for this study. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 701.