Ingle, James N.

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: N9831 is the only randomized phase III trial comparing safety and efficacy of the addition of trastuzumab (H) to doxorubicin and cyclophosphamide then paclitaxel (Arm A: AC→T) either following (Arm B: AC→T→H) or starting concurrently with paclitaxel (Arm C: AC→T+H→H) for women with resected Stage I-III invasive HER2+ breast cancer. The 3 yr cumulative incidence of NYHA class III or IV congestive heart failure or sudden cardiac death was previously reported: 3.3% in Arm C, 2.8% in Arm B (Perez EA, et al. JCO 2008). The comparison of AC→T to AC→T+H→H was reported in a joint analysis of N9831 and NSABP B-31 in 2005 and updated in 2007, demonstrating a 52% reduction in risk of a disease event (Romond E et al., NEJM 2005; Perez EA, et al. ASCO 2007).Materials and Methods: Primary endpoint is disease-free survival (DFS). At the second planned interim analysis of Arm A vs. Arm B, the O’Brien-Fleming boundary (OFB) was crossed. NCCTG Independent Data Safety Monitoring Committee approved the release of these data as well as the data pertaining to Arm B vs. Arm C due to slow pace of events [expected 647 events in 4 yr follow-up period (f/u) vs. actual 334 events in 4.5 yr f/u]. Shortly thereafter, there were sufficient events to perform the first planned interim analysis of B vs. C. We present the results of each of these pairwise comparisons taking into account the potential for crossover to Arm C after the release of the joint analysis findings in 2005.Results: From 5/2000 to 4/2005, 2448 eligible women were enrolled for the Arm A (n=1087) vs. Arm B (n=1097) comparison. Median f/u is 5.5 yrs. with 386 events. The addition of trastuzumab sequentially to AC→T significantly improved DFS, univariately [HR(Arm B/Arm A)=0.70, 95% CI: 57-86%, logrank p=0.0005] and after adjusting for age, tumor size, number of positive nodes, and ER [PPH: HRadj=0.67 (95% CI: 0.55-0.82)]. 5 yr DFS was increased from 72% with AC→T to 80% with AC→T →H.From 5/2000 to 4/2005, 1903 eligible women were enrolled for the Arm B (n=954) vs. Arm C (n=949) comparison. Median f/u is 5.3 yrs. with 312 events. The log-rank p-value testing whether DFS differs with respect to starting time of trastuzumab was 0.019. [Not crossing pre-specified OFB for statistical significance]. After adjusting for tumor size, number of positive nodes, and ER, HRadj(Arm C/Arm B)=0.75 (95% CI: 0.60-0.94)]. 5 yr DFS was increased from 80% with AC→T →H to 84% for AC→T+H →H.Conclusions: DFS is significantly improved with the addition of 52 weeks of H (sequentially or concurrently) to AC→ T. There is a statistically significant 33% reduction in the risk of an event with the sequential addition of H following AC→T. There is a strong trend for a 25% reduction in the risk of an event with starting H concurrently with T relative to sequentially after T. Therefore, based on a positive risk/benefit ratio, we recommend that trastuzumab be incorporated in a concurrent fashion with T chemotherapy.Acknowledgements: NIH CA25224, Breast Cancer Research Foundation, Genentech. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 80. NOTE: This abstract was accepted for presentation at the Symposium after the Abstract Book went to press.

Abstract It has long been known that immune suppression is induced by the presence of a tumor, resulting in compromised T cell and dendritic cell (DC) functionality. What is not well understood is how soon the immune system recovers normal functionality following surgical removal of tumors and adjuvant therapy. This timing is of critical importance, as the effectiveness of therapeutic immune strategies relies on optimal presentation of antigens and activation of T cells. Blood from breast cancer patients was collected prior to surgery and at 3 and 6 months post adjuvant therapy. Patients were treated with surgery (breast conservation or mastectomy) and chemotherapy (CT) (n=22) or surgery without chemotherapy (n=38). Most patients had radiation therapy (RT). Blood from 22 healthy woman of similar age served as controls. T cell functionality (TCF) was determined following stimulation with plate-bound anti-CD3 (1 µg/ml) and anti-CD28 (0.5 µg/ml) and proliferation was measured by 3H-thymidine uptake. DC functionality (DCF) was determined by ability to present allo-antigens in a mixed lymphocyte reaction. Values for the normal samples determined the normal ranges (NR). Responses were grouped into 4 categories based on their status at 6 months: Group 1 - remained in NR; Group 2 - climbed into NR; Group 3 - remained below NR; Group 4 - fell below NR. Analysis of immunosuppression will be based on lymphocyte subsets, cytokine, COX-2, and PGE2 metabolite levels.T Cell Proliferation AllWith CTNo CT N=60N=22N=38Group 120%14%24%Group 223%23%24%Group 338%41%37%Group 418%23%16% DC Function AllWith CTNo CT N=58N=20N=38Group 143%35%47%Group 219%25%16%Group 317%15%18%Group 421%25%18% Forty-three percent of patients had normal levels (Groups 1 and 2) for TCF and 62% for DCF by 6 months post adjuvant therapy, suggesting that breast cancer patients are potentially amenable to vaccine therapy. 28% of patients had both TCF and DCF in NR; 50% had either TCF or DCF in NR; and 22% had neither in NR. The percentage of patients who had TCF in NR by adjuvant therapy was: 35% CT+RT (6/17); 33% CT (1/3); 52% (17/33) RT; and 20% (1/5) neither. The percentage of patients who had DCF in NR by adjuvant therapy was: 67% (10/15) CT+RT; 33% (1/3) CT; 67% (22/33) RT; and 40% (2/5) neither. Proper understanding of the effects of tumor, chemotherapy and radiation therapy on immune function, especially the effects on T cells and DCs, may enable us to identify the appropriate patients in whom to study immunotherapy approaches in women with early breast cancer and to examine strategies to counteract cancer-related defects in immune function. (supported by P50CA116201) Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4131.

Abstract Background: The third-generation aromatase inhibitors (AIs) are established adjuvant therapy for postmenopausal women with early stage breast cancer. Musculoskeletal adverse events (MS AE) have become increasingly recognized as an issue for many women receiving AIs resulting in decreased quality of life and, in some instances, discontinuation of the AI. MA.27 is an ongoing randomized clinical trial comparing anastrozole with exemestane that accrued 7576 patients (pts), including 6827 from North America, about 79% of whom provided DNA and consent for genetic testing. A review of withdrawals related to adverse events revealed that the most common cause was MS AE. The hypothesis for our study was that a genome-wide association (GWA) case control study would identify single nucleotide polymorphisms (SNPs) associated with MS AE.Methods: Cases were defined as pts with grade 3 or 4 MS AE (NCI CTCAE v3.0) or who went off treatment for any grade MS AE within the first 2 years and were matched to 2 controls based on treatment arm, length of follow-up, prior chemotherapy, age and prior celecoxib (at least 3 months prior to MS AE). The study was restricted to the 94% of pts self-identified as Caucasian. Genotyping was performed with the Illumina Human610-Quad. Genotype quality control prior to analysis required call rates ≥98%. Eigenstrat analyses were performed to control for population stratification.Results: The GWA study included 293 cases and 586 controls with 6 pts (0.7%) (4 cases, 2 controls) excluded for call rates &lt;98%. 551,358 SNPs were used in the analyses after removing 29,439 for a minor allele frequency &lt;1% and 82 with Hardy Weinberg Equilibrium p-values &lt;10-6. Eigenvectors did not impact the results when used as covariates. Four SNPs with the lowest p-values (3.3-8.2 x 10-6, Armitage test), 3 in high LD (r2&gt;.8) on chromosome (ch) 14 (rs7158782, rs7159713, rs2369049) and 1 on the X ch (rs6637820) were identified. The gene closest (4000-7000 bp) to the 3 SNPs on ch 14 was T-Cell Leukemia 1A (TCL1A), encoding a protein known to augment AKT kinase activity. Expression array data from lymphoblastoid cell lines from 300 subjects of 3 ethnicities revealed that all 3 SNPs were associated with decreased TCL1A expression after adjusting for ethnicity using 2 probe sets (p=0.006 to 0.03). Electromobility shift assay (EMSA) showed that 2 of these SNPs (rs7158782, rs7159713) displayed a shift and the former SNP showed substantially less binding for variant than wild type. The gene closest to the X ch SNP was Immunoglobulin Superfamily 1 (IGSF1), but EMSA did not show a shift for this SNP.Conclusions: MS AEs represent a major impediment to optimal use of AIs in women with breast cancer and this GWA study identified SNPs on ch 14 that provide a focus for further research to identify pts at risk for, and means to ameliorate, this adverse event.(Supported in part by NIH grants U01GM61388, U01GM63173, P50CA116201 and U10CA77202) Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 15.

Abstract Background: Analyses of adjuvant endocrine therapy suggest that the appearance of new vasomotor or joint symptoms within 3 months of initiating either tamoxifen or aromatase inhibitor therapy predict for lower subsequent recurrence. We hypothesized that new vasomotor or joint symptoms may predict outcomes with anastrozole or exemestane in participants in the ongoing NCIC CTG Trial MA.27. We performed a pooled analysis across treatment arms of the MA.27 trial, to examine the effects of early onset new or worsening symptoms on relapse-free survival (RFS).Methods: 7576 postmenopausal women with early breast cancer were accrued to MA.27, a randomized adjuvant trial of exemestane and anastrozole. The second interim analysis data with a 3 year median follow-up was used for this analysis. The trial's primary endpoint is event-free survival, but for this investigational analysis is RFS, which excludes non-breast cancer deaths. All 7118 participants known to be hormone receptor-positive who received trial therapy, and had no relapse in the first 3 months, were included. The association of vasomotor and joint symptoms with patient characteristics was examined with chi-square tests. Stratified log-rank and stratified univariate and multivariate Cox modeling were used to examine the association of early vasomotor and joint symptoms on disease outcomes.Results: Of 4749 women who were asymptomatic at baseline, 230 (5%) experienced vasomotor or joint symptoms at 3 months. The RFS stratified univariate Cox hazard ratio (HR) for vasomotor or joint-related symptoms was 1.184 (95% CI, 0.675-2.079). When all 7118 women were included, 361 (5%) developed new or worsening symptoms at 3 months. RFS was not statistically significant between the women with or without new symptoms (HR 1.129, 95% CI, 0.708-1.800). Higher T stage was the only factor included in the multivariate Cox RFS model that was associated with new or worsening symptoms (p&lt;0.001).Conclusions: In contrast to prior findings, we did not observe a correlation between early emergence of symptoms and improved RFS. Longer follow-up and analysis by treatment arm and at different time points are required. Additional symptoms and time points will be added to the analysis prior to the meeting. Until further data are available, clinicians should not use treatment-emergent symptoms to counsel their patient whether to continue or change their prescribed medication. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 14.

Abstract Background: MA17 showed that adjuvant letrozole after 5 years of tamoxifen markedly reduced the risk of recurrence in women with ER+ early stage breast cancer and improved overall survival in women presenting with node +ve disease. Most trials of early adjuvant aromatase inhibitor therapy required women to be postmenopausal at diagnosis to be eligible. We report here on a subset of women in the MA17 trial who were premenopausal at initial diagnosis and in whom subsequent menopause, prior to randomization, may have influenced their outcome on extended adjuvant letrozole.Methods: Women randomized to MA17 were divided into 2 groups: 1) pre-menopausal (&lt; 50 years of age but underwent bilateral oophorectomy at time when tamoxifen treatment started or &lt; 50 years of age at the start of tamoxifen treatment but became amenorrheic during adjuvant chemotherapy or tamoxifen treatment) and 2) post-menopausal. Disease-free survival (DFS) from time of randomization for women in these two groups were summarized by Kaplan-Meier curves and compared based on log-rank test and Cox model.Results: 889 women were identified as premenopausal and 4,277 as postmenopausal. Treatment, age, race, duration of tamoxifen, prior adjuvant chemotherapy, and mastectomy were independently associated with menopausal status on multivariate analysis. The interaction between treatment and menopausal status was statistically significant for DFS (p=0.02), indicating that women diagnosed with pre-menopausal breast cancer had significantly greater benefit (Hazard ratio=0.25, 95% CI: 0.12-0.51) with letrozole treatment in terms of DFS than those with post-menopausal status (hazard ratio=0.69 with 95% CI from 0.52 to 0.91). In comparison with women treated with placebo, pre-menopausal women on letrozole had a significantly higher incidence of arthralgia (24 vs. 16%; p=0.004) but a lower incidence of vaginal bleeding (10 vs. 16%; p=0.01). Compared with placebo post-menopausal women receiving letrozole had a higher incidence of hot flashes/flashes (55 vs. 50%; p=0.001), arthralgia (25 vs. 21%; p=0.002), myalgia (15 vs. 12%; p=0.007) and alopecia (5 vs. 3%; p=0.003).Conclusions: Letrozole after tamoxifen was more effective in improving DFS in women who were premenopausal at primary diagnosis than those who were postmenopausal. Letrozole was well tolerated in premenopausal women. These data indicate that women who are premenopausal at the time of diagnosis but become postmenopausal anytime before, or during, adjuvant tamoxifen should be considered for extended adjuvant therapy with letrozole. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 13.

Abstract Abstract #57 Background: ABCSG 8 is the only prospective trial examining the value of switching patients (pts) to an aromatase inhibitor after tamoxifen (Tam) in which randomization occurred prior to therapy. This trial demonstrated that 2 years (yrs) of Tam followed by 3 yrs of anastrozole (TAM to A) was superior to 5 yrs of Tam (Jakesz SABC 2005). Tam is metabolically activated by CYP2D6 to endoxifen and pts with impaired CYP2D6 metabolism have a higher risk of recurrence (Goetz JCO 2005). The HOXB13/IL17BR (H/I) index (Ma Cancer Cell 2004) is associated with Tam resistance while a 5-gene molecular grade index (MGI) distinguishes outcome in grade II tumors. H/I and MGI are complementary prognostic factors that define low (low MGI), intermediate (low H/I, high MGI) and high risk groups (high H/I and MGI) (Ma Clin Cancer Res 2008). We sought to validate these biomarkers in ABCSG Trial 8, and hypothesized that they may predict the benefit of sequencing TAM to A versus Tam alone.&#x2028; Methods: From 1996-2004, ABCSG 8 randomized 3,901 pts with ER positive breast cancer. The primary endpoint was event-free survival. Assuming 40% of pts had reduced CYP2D6 metabolism, we designed a nested case control study in which 110 Tam treated cases matched to 2 controls would yield 80% power to detect a relative risk of 2.3 for pts with reduced CYP2D6 metabolism. Additionally, we sought to perform an exploratory analysis of the benefit of switching from Tam to A in pts with impaired vs non-impaired CYP2D6 metabolism. DNA and RNA derived from paraffin embedded sections were used to genotype for CYP2D6 and to obtain H/I and MGI RT-PCR profiles.&#x2028; Findings: Thus far, CYP2D6 genotyping is complete in 345 pts corresponding to 53 cases/106 controls (Tam) and 62 cases/124 controls (Tam to A). DNA and RNA yields were excellent with a CYP2D6 genotype call rate of &gt;95%. CYP2D6 allele frequencies were as expected: *3 (.01), *4, (.23), *6 (.01), *10 (.02), *17 (.00), and *41 (.10). 44% of pts had reduced CYP2D6 metabolism (at least one CYP2D6 null allele or intermediate metabolizer) and 8% were CYP2D6 poor metabolizers. For H/I and MGI, 340 pts were profiled with 64%, 20% and 16% in the low, intermediate, and high risk groups, respectively. Tissue sectioning/genotyping for additional cases/controls is ongoing.&#x2028; Discussion: This is the first retrospective analysis of a large Tam versus Tam to A trial to evaluate the association between 1) CYP2D6, HOXB13/IL17BR, and MGI with TAM treatment outcome and 2) the predictive benefit of these biomarkers in pts randomized to switch from Tam to AI versus Tam alone. Final results will be presented at the meeting. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 57.

Purpose We performed a case-control genome-wide association study (GWAS) to identify single nucleotide polymorphisms (SNPs) associated with musculoskeletal adverse events (MS-AEs) in women treated with aromatase inhibitors (AIs) for early breast cancer. Patients and Methods A nested case-control design was used to select patients enrolled onto the MA.27 phase III trial comparing anastrozole with exemestane. Cases were matched to two controls and were defined as patients with grade 3 or 4 MS-AEs (according to the National Cancer Institute's Common Terminology Criteria for Adverse Events v3.0) or those who discontinued treatment for any grade of MS-AE within the first 2 years. Genotyping was performed with the Illumina Human610-Quad BeadChip. Results The GWAS included 293 cases and 585 controls. A total of 551,358 SNPs were analyzed, followed by imputation and fine mapping of a region of interest on chromosome 14. Four SNPs on chromosome 14 had the lowest P values (2.23E-06 to 6.67E-07). T-cell leukemia 1A (TCL1A) was the gene closest (926-7000 bp) to the four SNPs. Functional genomic studies revealed that one of these SNPs (rs11849538) created an estrogen response element and that TCL1A expression was estrogen dependent, was associated with the variant SNP genotypes in estradiol-treated lymphoblastoid cells transfected with estrogen receptor alpha and was directly related to interleukin 17 receptor A (IL17RA) expression. Conclusion This GWAS identified SNPs associated with MS-AEs in women treated with AIs and with a gene (TCL1A) which, in turn, was related to a cytokine (IL17). These findings provide a focus for further research to identify patients at risk for MS-AEs and to explore the mechanisms for these adverse events.

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.

Evidence has emerged that the clinical benefit of tamoxifen is related to the functional status of the hepatic metabolizing enzyme cytochrome P450 2D6 (CYP2D6). CYP2D6 is the key enzyme responsible for the generation of the potent tamoxifen metabolite, endoxifen. Multiple studies have examined the relationship of CYP2D6 status to breast cancer outcomes in tamoxifen-treated women; the majority of studies demonstrated that women with impaired CYP2D6 metabolism have lower endoxifen concentrations and a greater risk of breast cancer recurrence. As a result, practitioners must be aware that some of the most commonly prescribed medications coadministered with tamoxifen interfere with CYP2D6 function, thereby reducing endoxifen concentrations and potentially increasing the risk of breast cancer recurrence. After reviewing the published data regarding tamoxifen metabolism and the evidence relating CYP2D6 status to breast cancer outcomes in tamoxifen-treated patients, we are providing a guide for the use of medications that inhibit CYP2D6 in patients administered tamoxifen.