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Abstract Background: Trastuzumab (H) improves disease-free survival (DFS) in patients (pts) with HER2 positive breast cancer (BR Ca) when used with adjuvant chemotherapy. We herein compare the rates of local regional recurrence (LRR) in pts randomized to adjuvant chemotherapy with or without adjuvant H. Methods: The phase 3 randomized trial NCCTG N9831 enrolled 3505 pts with high risk HER2 positive Br Ca to evaluate the effect of adjuvant H on DFS. Pts were randomized to either doxorubicin (A) and cyclophosphamide (C) followed by paclitaxel (T); or AC→TH→H. RT was given concurrently with H after ACT chemotherapy. Pts analyzed underwent lumpectomy (L) + radiotherapy (RT), mastectomy (M) alone, or M+RT. All pts underwent sentinel lymph node biopsy alone and/or axillary dissection. 2816 pts were eligible for competing risk analysis of LRR as a first event (competing risks were distant recurrence, contralateral Br Ca, second primary cancer, or death). Median follow-up is 5.3 years. Results: Primary breast therapy included L+RT 1062 (38%), M 711 (25%), and M+RT 1043 (37%). Axillary dissection was performed in 90% of pts but less frequently with L+RT (83%) compared to M (88%) or M+RT (98%), chi-sq P<0.001. Stage at presentation was I 41%, II 51%, and III 8% with a significant greater proportion of higher stage pts undergoing M+RT, chi-sq P<0.001. Overall the 5-year LRR rate was 4.1% (95% CI 3.5-4.9%) and similar among the treatment groups: L+RT 4.7% (95% CI 3.6-6.1%), M 3.5% (95% CI 2.4-5.1%), and M+RT 2.3% (95% CI 1.6-3.4%). Among pts with a LRR, 66% were local recurrence only, 11% were local-regional, and 23% were regional only. In the L+RT patients with a LRR, the corresponding rates were 74%, 7%, and 19%, respectively. In the M patients with a LRR, the corresponding rates were 40%, 23%, and 37%, respectively. In the M+RT patients with LRR, the rates were 79%, 4%, and 17%, respectively. H is associated with a non-statistically significant reduction in the risk of LRR for pts who receive L+RT or M+RT (Table 1). No such trend was seen in the M alone group but the number of events was low. Table 1. LRR According to Local and Adjuvant Treatment Groups Conclusion: The LRR as the first reported site of failure was low with a median 5. 3 year follow-up. Adjuvant H was associated with a trend of lower LRR in the pts treated with L + RT or M+RT. This observation suggests an additive effect of RT and H on LRR that warrants further investigation. The small number of local-regional events in this one randomized trial supports further investigation of LRR across other adjuvant trastuzumab trials. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-13-02.

Abstract In the Breast International Group (BIG) and North Central Cancer Treatment Group (NCCTG) co-led phase III adjuvant breast cancer trial, ALTTO (Adjuvant Lapatinib and/or Trastuzumab Treatment Optimisation), two central laboratories, the European Institute of Oncology (IEO; Milan, Italy) and Mayo Clinic (Rochester, MN), are responsible for confirming the human epidermal growth factor receptor-2 (HER2), estrogen receptor α(ER), and progesterone receptor status of the primary breast tumors for the Rest of World (excluding China, which conducts a separate central review) and North American patients, respectively, prior to patient study entry. As of December 2009, discordance in HER2 and ER testing between local and central laboratories was observed by both central laboratories. For IEO, 14.5% of 8,037 HER2 cases locally positive [by either immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH)] were not able to be confirmed as centrally positive; whereas for Mayo, 5.8% of 412 HER2 cases locally positive were not able to be confirmed as centrally positive (Table 1). Central and local IHC ER discordance was 12.1% and 11.7% for 9,021 IEO screened cases and 419 Mayo screened cases, respectively (Table 2). In particular, IEO observed more false-positive (locally positive/centrally negative) HER2 findings than Mayo and Mayo observed more false-positive ER findings than IEO. Purpose: Motivated by the above findings, we launched a ring study to assess whether the central lab results of a subset of local/central discordant ALTTO cases could be confirmed in the other central lab. Methods: IEO and Mayo exchanged and retested a subset of FFPE breast tumors collected in ALTTO. IEO sent 20 HER2 false-positive, 5 ER false-positive, and 5 ER false-negative (locally negative/centrally positive) ALTTO breast tumors to Mayo. Mayo sent 5 HER2 false-positive, 20 ER false-positive, and 5 ER false-negative ALTTO breast tumors to IEO. IEO and Mayo performed IHC for ER according to their own methodology: DAKO cocktail of ER 1D5 and 2.123 monoclonal antibodies and monoclonal ER 1D5 antibody, respectively. The two laboratories performed IHC for HER2 according to the HercepTest® manufacturers’ instructions (Dako, Carpenteria, CA) and FISH for HER2 using the PathVysion HER2 DNA probe kit and the HER2/centromere 17 probe mixture (Abbott Molecular, Des Plaines, IL). Results: IEO and Mayo confirmed the central HER2-negative result in 100% of 25 cases. Analyses of ER are ongoing and ER results will be presented. Conclusions: In this subset of patients, enrollment ineligibility did not change when HER2 testing was performed by either IEO or Mayo Clinic central laboratories. Table 1. HER2 Local/Central Lab Results Table 2. ER Local/Central Lab Results Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-10-36.

Abstract Previous findings suggested that patients (pts) with copy number alterations of MYC (8q24) and centromere 8 (CEN 8) in the setting of HER2-positive breast cancer may be associated with improved outcome to adjuvant trastuzumab. Our tissue microarray (TMA) data suggested that alternate cutpoints for MYC copy number anomalies [of 802 pts, those with MYC:CEN8 ratio ≥1.3 or <1.3 & polysomy 8 (N=658; 82%) had a hazard ratio (HR) of 0.44 (P<0.001) and those with MYC copy number ≥2.5 (N=669; 84%) had a HR of 0.42 (P<0.001)] were potentially predictive of benefit from adjuvant trastuzumab when administered concurrently with chemotherapy in the NCCTG N9831 HER2+ phase III trial. Purpose: We were interested in determining whether MYC is the target protein responsible for the suggested benefit from trastuzumab. We evaluated the association between MYC protein expression and disease-free survival (DFS) of pts randomized to receive chemotherapy alone (Arm A) or chemotherapy with sequential (Arm B) or concurrent trastuzumab (Arm C) using N9831 TMAs. Patients/Methods: This analysis included 1220 pts from Arms A, B, and C. The percentage of strong (3+) nuclear staining was determined in TMA sections containing 3 cores per block using immunohistochemistry (IHC; clone 9E10, Sigma-Aldrich, St. Louis, MO). A patient's tumor was considered positive (MYC+) if the maximum nuclear 3+ staining percentage across cores was >30% a priori. Median follow-up was 5.8 years. Results: Of 1220 pts with completed IHC analyses, 557 (46%) were MYC+. MYC+ was associated with a higher rate of hormone receptor positivity (58% vs 48%, chi-sq P<0.001) and higher rate of nodal positivity (92% vs 84%, chi-sq P<0.001) but not associated with age, race, tumor histology, tumor grade, or tumor size. In comparing DFS between arms C and A, MYC+ and MYC-pts had HRs of 0.46 (p=0.002; A: 187 pts, 57 events, C: 161 pts, 25 events) and 0.70 (p=0.09; A: 213 pts, 59 events, C: 208 pts, 38 events), respectively (interaction p=0.20). In comparing DFS between arms B and A, MYC+ and MYC-pts had HRs of 0.74 (p=0.11; B: 209 pts, 52 events) and 0.81 (p=0.29; B: 242 pts, 53 events), respectively (interaction p=0.80) (Figure). In addition, pts with nuclear MYC staining of 3+ in 0-9%, 10-30%, 31-60%, and 61-100% of cells had HRs (C vs A) of 0.98 (95% CI 0.53-1.82), 0.54 (95% CI 0.30-0.97), 0.55 (95% CI 0.27-1.12), and 0.37 (95% CI 0.19-0.75). Conclusions: In our analysis of 1220 pts, we observed that MYC protein expression positively correlated with hormone receptor positivity and nodal status. We observed a trend towards greater trastuzumab benefit among MYC+ as compared to MYC-pts. A rising trend in trastuzumab benefit was observed with increasing MYC protein expression. Analyses including pts with whole sections and outcome by MYC protein and gene copy number will be presented. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-09-08.

Purpose This phase III study compared the efficacy and safety of bevacizumab (BV) when combined with several standard chemotherapy regimens versus those regimens alone for first-line treatment of patients with human epidermal growth factor receptor 2–negative metastatic breast cancer. Patients and Methods Patients were randomly assigned in 2:1 ratio to chemotherapy plus BV or chemotherapy plus placebo. Before random assignment, investigators chose capecitabine (Cape; 2,000 mg/m 2 for 14 days), taxane (Tax) -based (nab-paclitaxel 260 mg/m 2 , docetaxel 75 or 100 mg/m 2 ), or anthracycline (Anthra) -based (doxorubicin or epirubicin combinations [doxorubicin/cyclophosphamide, epirubicin/cyclophosphamide, fluorouracil/epirubicin/cyclophosphamide, or fluorouracil/doxorubicin/cyclophosphamide]) chemotherapy administered every 3 weeks. BV or placebo was administered at 15 mg/kg every 3 weeks. The primary end point was progression-free survival (PFS). Secondary end points included overall survival (OS), 1-year survival rate, objective response rate, duration of objective response, and safety. Two independently powered cohorts defined by the choice of chemotherapy (Cape patients or pooled Tax/Anthra patients) were analyzed in parallel. Results RIBBON-1 (Regimens in Bevacizumab for Breast Oncology) enrolled 1,237 patients (Cape cohort, n = 615; Tax/Anthra cohort, n = 622). Median PFS was longer for each BV combination (Cape cohort: increased from 5.7 months to 8.6 months; hazard ratio [HR], 0.69; 95% CI, 0.56 to 0.84; log-rank P < .001; and Tax/Anthra cohort: increased from 8.0 months to 9.2 months; HR, 0.64; 95% CI, 0.52 to 0.80; log-rank P < .001). No statistically significant differences in OS between the placebo- and BV-containing arms were observed. Safety was consistent with results of prior BV trials. Conclusion The combination of BV with Cape, Tax, or Anthra improves clinical benefit in terms of increased PFS in first-line treatment of metastatic breast cancer, with a safety profile comparable to prior phase III studies.

Abstract The number of non-coding transcripts (NCTs) exceeds the number of transcripts that code for protein. Their diverse and important functions are now being identified and utilized in the lab as well as in the clinic. Utilizing the Affymetrix Gene Chip Human Tiling 1.0R Array Set, we analyzed the transcription across the entire genome in both normal human bronchial epithelial cells (NHBE) alone and cells exposed to the tobacco carcinogen (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone), NNK. We focused our efforts on the identification and function of novel long non-coding transcripts that had increased expression after exposure to NNK. We report on the identification of 12 long NNK-induced non-coding transcripts (NiTs) that are 300 nucleotides and larger in length. We first validated that these transcripts were indeed stress responsive using quantitative real-time RT-PCR (qPCR) and by Northern blot analysis revealed that these transcripts were between two and four kilobases in length. We also show using qPCR that these transcripts are more abundantly expressed in normal rapidly growing tissues or tissues that are under cellular stress. We show that there is altered expression of NiTs in a panel of lung cancer cell lines and many of these transcripts had increased expression in many breast cancer cell lines. We further analyzed one of these transcripts, NiT5 and identified it to be a 2.5 kb antisense, polyadenylated transcript by RLM-RACE and by Northern Blot analysis. Isolating nuclear and cytoplasmic fractions in BT474 cells, we identified Nit5 to be localized in the nucleus and found it to be transcribed by RNA polymerase III. NiT5 also has increased expression in breast, ovarian and cervical cancer-derived cell lines relative to normals from these three sites, but not increased expression in lung cancer cell lines. In summary, our results suggest that these novel long non-coding transcripts play an important role in stress also may play a role in cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4083.

Abstract A personalized approach to breast cancer management is increasingly possible with state of the art diagnostics and treatments. However, many challenges still exist in choosing the right therapeutic strategy and the right dosing for a specific patient population. Biomarkers such as estrogen receptor, progesterone receptor, and HER-2 status are used to tailor therapy, but success rates with targeted therapy suggest that appropriate patient populations still require greater definition. The introduction of the genomic era has likewise advanced the ability to offer a tailored approach to breast cancer management, but much work is still needed in this area as well. Microarray-based gene expression profiles have demonstrated initial promise, but we need a better understanding of more complex tumor signaling pathways. Strategies include annotated tumor specimens, next-generation gene sequencing, proteomics, and metabolomics. An individualized approach to breast cancer management has the potential to offer clear clinical benefits and the ability to bring more tailored, cost-effective strategies to the market. The current issues facing the delivery of individualized breast cancer care will be the focus of this discussion.

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.

Purpose Patients with inflammatory breast cancer (IBC) or locally advanced breast cancer (LABC) were randomly assigned to 21-day doxorubicin and cyclophosphamide administered for five cycles (standard arm) versus weekly doxorubicin and daily oral cyclophosphamide administered with granulocyte colony-stimulating factor support for 15 weeks (continuous arm). All patients had subsequent weekly paclitaxel for 12 weeks before surgery. Patients and Methods Patients (n = 372) were randomly assigned to the standard arm (n = 186) or the continuous arm (n = 186) stratified by disease type (LABC, n = 256; IBC, n = 116). The primary outcome was microscopic pathologic complete response (pCR) at surgery. Secondary outcomes included disease-free survival, overall survival, and toxicity. Results More patients in the standard arm had grade 3 to 4 leukopenia and neutropenia, but there were more instances of stomatitis/pharyngitis and hand-foot skin reaction in the continuous arm. Assessed among 356 eligible patients, pCR was not different between the treatment groups stratified by disease type (P = .42). In subset analysis, higher pCR rates were observed in the continuous arm versus the standard arm only for stage IIIB disease (P = .0057) and in IBC (P = .06). Comparison of overall survival and disease-free survival showed no difference between treatment groups (P = .37 and P = .87, respectively). Conclusion No significant clinical benefit was seen for the investigational arm in this trial overall.

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 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.