McCullough, Ann E.

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 < 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.

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: Gene expression profiling is an important tool in prediction, prognosis, and cancer modeling. The DASL® assay (cDNA –mediated Annealing, extension, Selection, and Ligation; Illumina; San Diego, CA) is a gene expression profiling system designed for degraded RNAs such as those derived from formalin-fixed paraffin-embedded (FFPE) tumor samples. FFPE tumor samples are invaluable resources for cancer research, being the most widely available materials for which patient outcomes are known. Purpose: The objective of this study was to evaluate the performance of Illumina's Whole Genome DASL platform (24,526; 24K) at distinguishing HER2-positive (HER2+) from HER2-negative (HER2-) FFPE breast tumors. Methods: We measured the relative gene expression of biological and technical replicates using nine HER2+ and 11 HER2- primary breast tumors from patients that were all node-positive and estrogen receptor-positive. The HER2 status of the tumors was determined using the Dako Hercept Test following the 2007 ASCO/CAP guidelines for HER2 positivity. The non-background corrected probe-expression data were exported from Illumina BeadStudio software and normalized using the software R and the function fastlo. Log2 transformed expression values were used to obtain the relative gene expression per probe. Spearman correlations were calculated between both types of replicates. Unequal-variance t-tests were conducted to test for differences in expression levels between HER2+ and HER2- tumors. An overall ERBB2 gene expression level was calculated by taking the average of the three ERBB2 probes for each tumor across all replicates. To simulate the possible outcome if only one replicate of each tumor was measured for gene expression, differential expression was tested for all possible combinations of single replicate probe expression values. Results: Spearman correlations for technical and biological replicates within tumor samples ranged from 0.9804 – 0.9946, and 0.9822 – 0.9945, respectively. The overall ERBB2 gene expression was significantly lower in HER2- tumors than in HER2+ tumors (p = 0.0000018). The top five most differentially expressed genes (p &lt; 0.0005) included ERBB2, GRB7, C17ORF37, PERLD1, and ASF1B. For each of the three ERBB2 probes, there were 65,536 possible ways to combine the individual probe expression values. The p-values for testing differential expression in each of these 65,536 combinations of HER2+ tumors versus HER2- tumors ranged from 0.00029 – 0.0091 for ILMN_1717902, 0.0000078 – 0.00026 for ILMN_1728761, and 0.000000002 – 0.0000032 for ILMN-2352131. Conclusions: Illumina's Whole Genome Platform provided highly reproducible gene expression values among biological and technical replicates of breast tumors. This platform also demonstrated significant differential ERBB2 gene expression between HER2+ and HER- breast tumors, which correctly classified the breast tumors as HER2+ and HER2-. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6127.

Abstract Background: Despite the impressive results of the trastuzumab adjuvant therapy trials, 15% of patients (pts) with HER2 overexpressing or amplified breast cancer developed tumor relapse at 4 years. Lapatinib is a reversible tyrosine kinase inhibitor against ErbB1 and ErbB2. The current study was developed to assess the cardiac safety and feasibility of adding lapatinib to paclitaxel and trastuzumab following doxorubicin and cyclophosphamide as part of adjuvant therapy.Methods: A single-arm phase II study of doxorubicin (A, 60 mg/m2 day 1) and cyclophosphamide (C, 600 mg/m2 day 1) [q2w or q3w for 4 cycles]; followed by PTL: paclitaxel (P, 80 mg/m2 weekly), trastuzumab (T, 4 mg/kg loading dose then 2 mg/kg weekly), and lapatinib (L, 1000 mg PO daily which was later amended to 750 mg) [total of 12 weeks of treatment]; followed by T (6 mg/kg day 1, every 3 weeks) and L (1000 mg PO daily) [total of 40 weeks of treatment] was conducted. The primary endpoint was severe symptomatic congestive heart failure (CHF: New York Heart Association Class III or IV with a drop in left ventricular ejection fraction [LVEF] of at least 10 percentage points to below 50% confirmed by cardiologist) with secondary endpoints of cardiac death and grade 3+ cardiac adverse events (AEs).Results: From April 2007 to October 2008, 109 evaluable pts were enrolled and initiated study treatment. Median age was 51 (range 28-72). Median follow-up is 10.2 months. No pts experienced CHF or cardiac death while on active treatment. One pt had CHF and 1 pt died due to myocardial infarction (MI) after they went off study treatment. The pt with CHF refused further treatment after completing 2 cycles of PTL and had CHF 1.8 months post PTL discontinuation with LVEF of 45% and dyspnea on exertion. The pt with MI went off study treatment due to non-cardiac AEs after completing 2 cycles of PTL and died 2.9 months post PTL discontinuation. Mean LVEF at baseline was 63.6% (N=109, SD=5.7); end of AC was 63.3% (N=104, SD=6.4); end of PTL was 59.9% (N=89, SD=8.3); and cycle 12 was 59.7% (N=66, SD=7.1). Grade 3+ non-hematologic AEs with incidence &gt;5% include diarrhea (N=42, 39% & included both 1000mg & later amended 750mg dosing), fatigue (N=15, 14%), nausea (N=7, 6%), vomiting (N=7, 6%), acne (N=6, 6%), and hypokalemia (N=6, 6%). Pre-treatment cardiac marker data (troponin-T, troponin-I, brain natriuretic peptide, creatine kinase MB isoenzyme) will also be presented.Conclusions: Preliminary data suggest that the inclusion of L does not add to the cardiac profile of T. Data from ALTTO and long-term follow data are needed to confirm the cardiac safety of this regimen. Cardiac marker data are being analyzed in the context of changes in LVEF. We gratefully acknowledge support from the Breast Cancer Research Foundation and GlaxoSmthKline for this study. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3086.

Abstract Long-term outcomes and hence the role of adjuvant therapy in patients with small (≤1 cm), node-negative breast cancer remain unclear. This study's objective was to evaluate whether human epidermal growth factor receptor (HER)-2 status is an independent, poor prognostic marker in patients with these tumors and to identify a subgroup of patients with these small tumors who might benefit from adjuvant systemic therapy. All patients with a diagnosis of a node-negative breast tumor measuring ≤1 cm and available HER-2 test results between January 1, 2001, and December 31, 2005, at the three Mayo Clinic sites were identified. Clinicopathologic data were compared in three groups: HER-2−, HER-2+, and triple-negative (TN) tumors. Of the 421 tumors identified, 364 (86.5%) were HER-2−, 28 (6.7%) were HER-2+, and 29 (6.9%) were TN. The median follow-up time was 1,015 days (range, 1–2,549 days). Groups were balanced in terms of patient age and tumor histology. Eleven patients with HER-2− tumors (3.0%), seven with HER-2+ tumors (25.0%), and eight with TN tumors (27.6%) received adjuvant chemotherapy. Follow-up data were available for 357, 28, and 28 patients in the three groups, respectively. Death rates in the three groups were 6.4% (23 of 357) (one recurrence-related death), 0% (0 of 28), and 7.1% (2 of 28) (one recurrence-related death), respectively. During follow-up, the tumor recurred in nine patients: four were HER-2− tumors (1.1%), two were HER-2+ tumors (7.1%), and three were TN tumors (10.7%). Patients with small, node-negative breast tumors have an excellent prognosis, but HER-2+ and TN tumors appear to have a higher recurrence rate, warranting consideration for broad use and optimization of systemic adjuvant treatments.

ABSTRACT Coccidioides spp. are dimorphic fungal pathogens endemic to the semiarid regions of North, Central, and South America. Currently, direct smear and culture are the most common means of identifying Coccidioides spp. While these methods offer relatively sensitive and specific means of detecting Coccidioides spp., growth in culture may take up to 3 weeks, potentially delaying the diagnosis and initiation of appropriate antifungal therapy. In addition, growth of the organism represents a significant safety risk to laboratory personnel. The need for a rapid and safe means of diagnosing coccidioidomycosis prompted us to develop a real-time PCR assay to detect Coccidioides spp. directly from clinical specimens. Primers and fluorescent resonance energy transfer (FRET) probes were designed to target the internal transcribed spacer 2 region of Coccidioides . The assay's limit of detection is below 50 targets per reaction. An analysis of 40 Coccidioides sp. clinical isolates grown in culture demonstrated 100% sensitivity of the assay. A cross-reactivity panel containing fungi, bacteria, mycobacteria, and viruses was tested and demonstrated 100% specificity for Coccidioides spp. An analysis of 266 respiratory specimens by LightCycler PCR demonstrated 100% sensitivity and 98.4% specificity for Coccidioides spp. compared with culture. Analysis of 66 fresh tissue specimens yielded 92.9% sensitivity and 98.1% specificity versus those of the culture method. The sensitivity of the assay testing 148 paraffin-embedded tissue samples is 73.4%. A rapid method for the detection of Coccidioides spp. directly from clinical material will greatly assist in the timely diagnosis and treatment of patients, while at the same time decreasing the risk of accidental exposure to laboratory personnel.