Ballman, KV

Abstract Background: Unstained recuts from formalin-fixed paraffin-embedded tissues are commonly collected for cooperative group studies. There is concern among pathologists that improper storage conditions can lead to antigen degradation. In an effort to quantify this effect, we compared the expression of HER1 and HER2 on two sets of identical cohort tissue microarrays (TMAs) from the N9831 HER2+ adjuvant phase III trial (NCT00005970; www.clinicaltrials.gov); one freshly cut set (cut April 18, 2011) and a second set stored at 4 degrees for over two years (cut between Nov, 2007 and Jan, 2008). Methods: The two sets of TMA slides containing 1580 tumor samples from the N9831 cohort were treated identically using the AQUA method of quantitative immunofluorescence. HER1 was tested with D38B1 (rabbit monoclonal, Cell Signaling Technology, Inc.) and HER2 with CB11 (mouse monoclonal, Biocare, Inc.) on tumors from 695 patients (712 specimens) in the fresh TMAs and 779 patients (800 specimens) in the old TMAs in up to three-fold redundancy per specimen. Results: Frequency distributions of the expression of HER2 revealed bimodality in the fresh TMAs compared to an attenuated distribution of the old cases. The average score of the entire cohort was significantly lower in old TMAs compared to fresh cuts (paired t-test, p<0.0001). Linear regression of the average HER2 scores from new TMAs versus the average scores from old TMAs showed a slope term of 0.52, which is statistically significantly different from the hypothetical value of 1 (p<0.0001). Regressions between any two fresh slides showed slopes close to 1.0. Similar results were seen for HER1, but fewer positive cases made the changes less dramatic. Conclusions: The storage condition of tissue slides is a critical pre-analytical variable that can dramatically lower the score of HER1 and HER2, artificially. Thus, studies done on inadequately stored slides, either whole sections or TMAs, must be interpreted with caution. Tissue collection and analysis of biomarkers for cooperative group studies should not include unstained recuts, but rather, entire blocks or large cores from tissue blocks. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD05-04.

Abstract Background: Prediction of benefit from trastuzumab in patients (pts) with HER2+ breast cancer remains an important goal. We sought to investigate the predictive value of quantitative measurement of HER2, HER3, HER4, EGFR, ER and PTEN protein expression on the benefit of trastuzumab in the phase III HER2+ adjuvant N9831 study for pts randomized to chemotherapy alone (Arm A) or chemotherapy with sequential (Arm B) or concurrent trastuzumab (Arm C). Methods: For each marker, we evaluated quantitative expression, relationship with demographic data, and association with disease-free survival (DFS) of pts. Freshly cut tissue microarray slides with up to three-fold redundancy per specimen from the N9831 cohort were treated identically using the AQUA (Camp, et al; Nat Med 2002, JCO 2008) method of quantitative immunofluorescence for each marker. HER2 was tested with CB11 (mouse monoclonal, Biocare, Inc.) and preliminary results were available for 698 of nearly 1400 pt specimens to be tested. The minimum value per pt was used in statistical analysis. Specimens were classified with high versus low expression based on a median value cutpoint for each marker. Median follow-up was 7.0 yrs. Results: Quantitative HER2 was compared with centrally performed HER2 testing by IHC and FISH. Median quantitative HER2 via AQUA was 10,017 units for the HER2 IHC 3+ group (n=607) versus 1058, 831, and 970 for the HER2 IHC 2+ (n=68), 1+ (n=11), and 0 (n=11) groups, respectively. The Spearman correlation between quantitative HER2 and FISH HER2/CEP17 ratio was 0.32 (p<0.001). High quantitative HER2 was associated with lower percentage of hormone receptor positivity (48% vs 59%, chi-sq p=0.003) but not associated with age, race, nodal positivity, tumor histology, grade, or size. High HER2 did not impact DFS in any arm of the study (See Table). Data for additional HER2 testing, HER3, HER4, EGFR, ER and PTEN are in process and will be ready by September, 2011. Conclusions: Similar to results based on standard HER2 testing by IHC and FISH in N9831, quantitative HER2 did not impact benefit from adjuvant trastuzumab. Results for additional markers will be presented. Our complete quantitative results for a second epitope on HER2, HER3, HER4, ER and EGFR will be the first report of these markers in a large patient cohort in the adjuvant setting. Disease Free Survival Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD05-03.

Abstract Background Obesity, as defined by body mass index (BMI), has been associated with increased recurrence rate, shorter DFS and increased death rates due to breast cancer (BC). Most of the studies to date have examined the relationship of BMI and DFS in patients with hormone receptor positive disease. To our knowledge, BMI and its relationship with outcome in early stage HER2 positive breast cancer has not previously been examined. The N9831 is a large phase III trial testing the role of trastuzumab in the adjuvant setting of high risk patients with early stage HER2+ BC. We hypothesized that the occurrence of overweight and obesity may correlate with outcome. Methods: This analysis presents BMI and its relation to tumor characteristics and DFS in patients (pts) enrolled in the N9831 clinical trial. Pts were categorized as normal weight, overweight or obese using the WHO BMI classification parameters of < 25%, 25–29% and ≥ 30% respectively. For patient characteristics, patients were grouped into non-obese (BMI< 30) and obese (≥ 30) cohorts. DFS was estimated by the Kaplan-Meier method. Comparisons between arms A (chemotherapy alone), B (chemotherapy plus sequential trastuzumab) and C (chemotherapy plus concurrent trastuzumab) were performed using the Cox proportional hazards model, stratified by BMI. Results: Analysis was completed on 3,017 eligible pts. Obese pts were more likely to be older and postmenopausal (p<0.0001 for both). There was no significant association between BMI and ER/PR status (p=0.07) or histologic tumor grade (p=0.33). Obese pts were found to have significantly larger tumors ≥ 2 cm (p=0.002) and more positive lymph nodes (p=0.02). There was no significant difference in DFS within each intrinsic arm (A, B and C) between the obese and non-obese pts at 3, 5 or 7 yrs of follow up. However, pts in the non-obese group had significantly improved DFS in arm B and C compared to arm A (p=0.001 and p<0.0001 respectively). Also obese pts in arm C had significantly improved DFS compared to obese pts in arm A (p=0.008). There was a trend of improved DFS in the obese group in arm B compared to arm A, but this was not statistically significant (p=0.09). Pts in the normal weight and overweight groups did significantly better in arm B (p=0.02 for both) and arm C (p=0.01 and p=0.002 respectively) compared to arm A. Conclusions: This analysis of data from the N9831 study confirms that obese pts with early stage HER2+ tumors have worse clinical outcome than pts with BMI < 30%. Adjuvant trastuzumab improved clinical outcome regardless of BMI. This study supports weight loss intervention for obese women with early stage HER2+ BC. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-12-02.