Reinholz, Monica M.

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.

Abstract Src transduces activated HER tyrosine kinase and phosphorylates HER to further increase its activity. Src also is involved in osteoclast differentiation. We hypothesized that combining a HER tyrosine kinase inhibitor with a Src inhibitor would result in increased therapeutic activity. To address this hypothesis we performed a phase I trial of D and L to: 1)determine the maximally tolerated dose (MTD) of D combined with L; 2)describe the toxicities associated with this combination; 3)assess the pharmacokinetic (PK) interaction of L and D; 4) assess the effect of the L and D combination on circulating tumor cells (CTC) and on osteoclast precursor activation; 5)study the association of toxicity and/or tumor response with PK, and/or pharmacodynamic parameters; and 6)describe the responses of this combination. Eligibility criteria included ≥ 18 years of age, incurable solid tumors not amenable to other therapies, ANC ≥ 1500/ L, PLT ≥ 100,000/ L, total bilirubin ≤ ULN, AST ≤ 3 × ULN or AST ≤5 × ULN if liver involvement, creatinine ≤ 1.5 × ULN, Hgb ≥ 9.0 g/dL, K+ and Mg++ WNL, life expectancy ≥ 12 weeks, ejection fraction &gt; 50%, ECOG PS 0–2, and able to swallow pills whole. In addition to standard phase I exclusion criteria patients could not have received erlotinib, sunitinib, sorafenib, gefitinib, imatinib ≤ 4 weeks prior to registration. Each agent was administered once daily on a 28 day schedule. At the MTD patients were treated with D day 1, and day 9–28 and L day 2–28 to assess PK interaction. Dose levels (mg) were #1 D 70, L 1000, #2 D 100, L 1000, #3 D 140, L 1000. 12 patients were entered on the dose escalation portion of the trial and 15 were entered at the MTD. 5 patients entered at the MTD are too early to be fully evaluable. Characteristics of evaluable patients were: median age, 63; 46% female; PS 0 (64%) 1 (27%), 1 (9%); ≥ 3 prior chemotherapies (59%). DLT was seen in 2 patients at # 3 (1 patient, grade 3 hypermagnesemia, 1, grade 3 rash) and 1 patient at # 2 (grade 3 dyspnea). The MTD was determined to be # 2. No grade 4 or 5 events have been observed in this study. 6 patients had stable disease in the dose escalation cohort. 1 (RCC) was stable disease for 6 cycles, 2 (colorectal) were stable for 5 cycles, 1 (esophagus) was stable for 3 cycles, and 2 (GIST & leiomyosarcoma) were stable for 2 cycles. Analysis of drug levels, CTCs and circulating osteoclast precursors is ongoing. There was a consistent increase in the percentage of CD11b (monocyte/macrophage/osteoclast lineage marker) positive cells in circulation as treatment progressed. However, the late osteoclast lineage marker c-Fms was not similarly increased, suggesting that the elevated CD11b reflects an inflammatory response and not activation of osteoclastogenesis. D and L is a tolerable combination at nearly full doses of each drug. Funded by a grant from Glaxo Smith Kline and Bristol Myers Squibb. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B56.

Abstract Purpose: To investigate the associations between baseline and posttreatment circulating tumor cell (CTC) gene expression and outcome of patients enrolled in four North Central Cancer Treatment Group metastatic breast cancer (MBC) trials in which specimens were shipped (at 4°C) from community-based sites to a reference laboratory (Mayo Clinic, Rochester, MN). Experimental Design: Blood was collected at treating sites from MBC patients before (baseline), during, and at the end of treatment with erlotinib + gemcitabine (N0234), sorafenib (N0336), irinotecan + cetuximab (N0436), or paclitaxel-poliglumex + capecitabine (N0437). CTCs from 10 mL of EDTA blood were enriched with CD45 depletion, 24 to 30 hours postblood collection. Reverse transcription/quantitative PCR was used to determine cytokeratin-19 (CK19) and mammaglobin (MGB1) mRNA levels in CTCs from up to 13 (N0234), 16 (N0336), 18 (N0436), and 39 (N0437) patients. The gene expressions were normalized to β2-microglobulin and calibrated to healthy blood using the 2–ΔΔCq algorithm; positivity was defined as 2 or more. Results: CK19+mRNA cells were detected in 56% to 75% and MGB1+mRNA cells in 23% to 38% of 86 patients at baseline. CK19+mRNA cells were detected in 30% to 67% and MGB1+mRNA cells in 14% to 64% of 110 postbaseline serial samples. The presence of baseline CK19+mRNA cells (P = 0.01) but not MGB1+mRNA cells (P = 0.14) was significantly associated with shorter overall survival. A decrease in MGB1+mRNA levels (baseline-week 8) seemed to be associated with clinical response (P = 0.05). Conclusions: CTC gene expression analysis conducted by a reference laboratory is feasible when blood is collected from treating sites and processed 24 to 30 hours postcollection. The presence of baseline CK19+mRNA CTCs was associated with poor prognosis; a decrease in MGB1+mRNA CTCs may help predict response to therapy of MBC patients. Clin Cancer Res; 17(22); 7183–93. ©2011 AACR.

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.

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.

Purpose We examined associations between tumor characteristics (human epidermal growth factor receptor 2 [HER2] protein expression, HER2 gene and chromosome 17 copy number, hormone receptor status) and disease-free survival (DFS) of patients in the N9831 adjuvant trastuzumab trial. Patients and Methods All patients (N = 1,888) underwent chemotherapy with doxorubicin and cyclophosphamide, followed by weekly paclitaxel with or without concurrent trastuzumab. HER2 status was determined by immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) at a central laboratory, Mayo Clinic, Rochester, MN. Patients with conflicting local positive HER2 expression results but normal central laboratory testing were included in the analyses (n = 103). Results Patients with HER2-positive tumors (IHC 3+, FISH HER2/centromere 17 ratio ≥ 2.0, or both) benefited from trastuzumab, with hazard ratios (HRs) of 0.46, 0.49, and 0.45, respectively (all P < .0001). Patients with HER2-amplified tumors with polysomic (p17) or normal (n17) chromosome 17 copy number also benefited from trastuzumab, with HRs of 0.52 and 0.37, respectively (P < .006). Patients who received chemotherapy alone and had HER2-amplified and p17 tumors had a longer DFS than those who had n17 (78% v 68%; P = .04), irrespective of hormone receptor status or tumor grade. Patients with HER2-normal tumors by central testing (n = 103) seemed to benefit from trastuzumab, but the difference was not statistically significant (HR, 0.51; P = .14). Patients with hormone receptor–positive or –negative tumors benefited from the addition of trastuzumab, with HRs of 0.42 (P = .005) and 0.60 (P = .0001), respectively. Conclusion These results confirm that IHC or FISH HER2 testing is appropriate for patient selection for adjuvant trastuzumab therapy. Trastuzumab benefit seemed independent of HER2/centromere 17 ratio and chromosome 17 copy number.

Abstract Metaplastic breast carcinoma, a rare tumor composed of adenocarcinomatous and nonglandular growth patterns, is characterized by a propensity for distant metastases and resistance to standard anticancer therapies. We sought confirmation that this tumor is a basal-like breast cancer, expressing epidermal growth factor receptor (EGFR) and stem cell factor receptor (KIT). EGFR activating mutations and high copy number (associated with response to tyrosine kinase inhibitor gefitinib) and KIT activating mutations (associated with imatinib sensitivity) were then investigated. Seventy-seven metaplastic cases were identified (1976-2006); 38 with tumor blocks available underwent pathologic confirmation before EGFR and KIT immunohistochemical analyses. A tissue microarray of malignant glandular and metaplastic elements was constructed and analyzed immunohistochemically for cytokeratin 5/6, estrogen receptor, progesterone receptor, and p63, and by fluorescence in situ hybridization for EGFR and HER-2/neu. DNA isolated from individual elements was assessed for EGFR and KIT activating mutations. All assessable cases were negative for estrogen receptor, progesterone receptor, and (except one) HER2. The majority were positive for cytokeratin 5/6 (58%), p63 (59%), and EGFR overexpression (66%); 24% were KIT positive. No EGFR or KIT activating mutations were present; 26% of the primary metaplastic breast carcinomas were fluorescence in situ hybridization-positive, displaying high EGFR copy number secondary to aneusomy (22%) and amplification (4%). We report here that metaplastic breast carcinoma is a basal-like breast cancer lacking EGFR and KIT activating mutations but exhibiting high EGFR copy number (primarily via aneusomy), suggesting that EGFR tyrosine kinase inhibitors should be evaluated in this molecular subset of breast carcinomas. [Mol Cancer Ther 2008;7(4):944–51]