Perez, EA

Background Tumor-infiltrating lymphocytes (TILs) are established determinants of clinical outcomes in breast cancer. 1–6 While stromal TILs (sTILs) are commonly evaluated due to easier scoring and higher interobserver reproducibility, intraepithelial TILs may provide additional insights into anti-tumor immunity. 7 We aimed to comprehensively characterize immunotype-associated cellular landscapes in triple-negative breast cancer (TNBC) and identify features associated with clinical outcomes. Methods 212 TNBC tumors were classified into three immunotypes based on immune infiltrate location and density: immune desert (ID), immune excluded (IE), and immune inflamed (IN). 8 Single-cell spatial transcriptomics was performed on 75 tumors to define the cellular composition and functional states of the tumor microenvironment across immunotypes. Findings were validated and integrated with bulk RNA sequencing data and clinical outcome datasets from FinXX, CALGB-40603, I-SPY 2, and Real-World Clinico-Genomic Data (RWCGD) cohorts. Results Patients with IN tumors had significantly improved outcomes compared with ID tumors. Despite high sTILs, IE tumors showed poor outcomes similar to ID tumors. Single-cell spatial analysis revealed that ID and IE tumors exhibited reduced major histocompatibility complex I/II expression and fewer tumor-resident plasmacytoid dendritic cells (pDCs) compared with IN tumors, which were enriched for interferon-alpha (IFNα) and interferon-gamma (IFNγ) responses. High IFN response scores were associated with favorable outcomes across multiple therapy types in independent datasets. Deconvolution of RWCGD bulk RNA-seq data confirmed that pDC abundance correlated with improved outcomes specifically in hormone receptor–negative subtypes. Conclusions Our study highlights pDCs and IFN signaling as hallmarks of effective anti-tumor immunity in TNBC. Immunotype-based profiling underscores the limited prognostic value of sTILs alone in immune-excluded tumors and supports pDCs and IFN pathways as potential biomarkers for prognosis and therapeutic development in TNBC.

564 Background: HER2+ ILC is rare (~5% of ILC), with limited data on long-term adjuvant trastuzumab outcomes. We investigated late recurrence and immune landscapes of HER2+ ILC vs invasive ductal carcinoma (IDC) in the phase III NCCTG N9831 trial (NCT00005970) and a real-world cohort. Methods: We evaluated 3304 early-stage HER2+ breast cancer patients (pts) enrolled in N9831 (122 ILC, 3182 IDC). Recurrence-free survival (RFS) and overall survival (OS) were estimated (Kaplan-Meier) and compared using multivariable Cox regression adjusting for age, tumor size, grade, hormone receptor (HR), and nodal status. Findings were validated in a real-world cohort of HER2+ pts (Caris Life Sciences CODEai platform). Immune microenvironments were profiled by WTS deconvolution (Quantiseq). Real-world overall survival was calculated from trastuzumab start to last contact. Results: In N9831, ILC pts were older (median 54 vs 49 y), more ER+ (74.6% vs 51.4%), and had more N3 disease (25.4% vs 12.7%) compared to IDC. ILC pts derived a comparable magnitude of benefit from adjuvant trastuzumab compared to the overall population (HR 0.58 vs 0.67). However, long-term outcomes diverged: while the 5-year RFS was similar (77.1% vs 82.7%), ILC pts experienced a steep decline between years 5-15 (77.1% to 57.7%; ~20% decline) vs IDC (82.7% to 72.7%; ~10% decline) after multivariable Cox analysis (Table). In the real-world early/advanced cohort (n=1328; 26 ILC vs 1302 non-ILC [subset IDC n=595]) treated with trastuzumab-based therapy, ILC pts had worse mOS (33.7 vs 68.6 m, HR 2.01, 95%CI 1.00-4.07, p=0.04) compared to non-ILC. However, there was no statistically significant difference when compared specifically to IDC (HR 1.77, 95% CI 0.87-3.61, p=0.11). ILC had: i) numerically lower tumor mutation burden (TMB)-high (4.2% vs 9.5%, p=0.38); ii) higher infiltration of B cells, Tregs, and M2 macrophages (p<0.05); iii) upregulated MHC class II expression (HLA-DOA, p<0.05); and iv) downregulated proliferative and DNA repair pathways (E2F, G2M, MYC; FDR <0.25) compared to non-ILC. Conclusions: HER2+ ILC carries late recurrence risk despite initial trastuzumab benefit. The unique immune landscape (Treg/M2 macrophage enrichment and lower proliferative signatures) distinguishes HER2+ ILC from IDC, highlighting the need for tailored strategies. Long-term RFS and OS in HER2-positive ILC vs IDC (N9831 trial). Population N 5-year% (95% CI) 10-year% (95% CI) 15-year% (95% CI) ILC 122 RFS 77.13% (69.9, 85.1) OS 84.04% (77.7, 90.9) RFS 70.57% (62.7, 79.4) OS 76.0% (68.7, 84.2) RFS 57.67% (44.2, 75.1) OS 65.22% (56.6, 75.2) IDC 3182 RFS 82.71% (81.4, 84.1) OS 90.18% (89.1, 91.2) RFS 77.45% (75.9, 78.9) OS 81.81% (80.4, 83.2) RFS 72.68% (70.77, 74.6) OS 76.68% (75, 78.4)

536 Background: Folate receptor alpha (FRα) is overexpressed in ~80% of triple-negative breast cancers (TNBC). We previously demonstrated that patients (pts) with breast cancer naturally mount immune responses to FRα. In prior Phase I and II studies, a GM-CSF-adjuvanted FRα peptide vaccine (FRPV) was safe and induced durable FRα-specific T-cell immunity in 83–90% of pts with breast and ovarian cancer. Methods: Eligible pts were women ≥18 years with TNBC (ER/PR ≤10%) with 1+ of the following: stage ≥T1c disease, node-positivity, or residual disease after neoadjuvant chemotherapy. Pts were randomized 2:1 to receive FRPV or placebo (GM-CSF alone) every 28 days for cycles 2-7 and then every six months for 7 booster doses, with cycle 1 consisting of cyclophosphamide 50mg orally twice daily for days 1-7 and 15-21 (in both arms). Randomization was stratified by chemotherapy setting (adjuvant only vs neoadjuvant) and stage (I/II vs III). FRα expression was evaluated by immunohistochemistry. The study was designed to detect (at 30 months after the last pt enrolled) a disease-free survival (DFS) difference of 14% assuming a 3.5-year DFS rate of 70% in the control group and with 78% power. Results: 280 pts were enrolled (median age 52). The majority had stage I or II tumors (n = 219, 78%). There were numerically more node-positive tumors in the vaccine arm, 78 (41%) vs 27 (32%), p = 0.15. Median follow-up was 3.5 years, and 33 (FRPV: 25, placebo: 8) DFS events were observed; 3.5-year DFS rate was 88% with FRPV and 90% with placebo (HR 1.42, 95% CI 0.64–3.15; p = 0.39). Overall survival (OS) also did not differ by arm (HR 2.48, 95% CI 0.55–11.20; p = 0.22). Among node-positive pts (n = 105), DFS did not differ between arms (HR 0.95, 95% CI 0.34–2.66; p = 0.92). However, a numerical DFS event rate improvement (14% for vaccine vs. 26% for placebo) was observed in 61 patients with stage III disease (HR 0.51, 95% CI 0.16–1.68; p = 0.26). DFS did not differ by chemotherapy setting or by receipt of anthracycline, capecitabine, and/or an immune checkpoint inhibitor. FRPV was well tolerated, with predominantly grade 1–2 adverse events (AEs) and no vaccine-related grade ≥4 AEs. FRα expression was available from tumor in 230 pts, with 72% expressing > 0. Overall, DFS was not significantly associated with FRα expression (0 vs > 0; HR 0.55, 95% CI 0.21–1.44; p = 0.21), and no significant differences in DFS by FRα expression were observed between treatment arms. Conclusions: FRPV following oral cyclophosphamide was safe, with no vaccine-related high-grade AEs. While no significant improvement in DFS or OS was observed overall, a numeric trend toward improved DFS was observed in pts with stage III TNBC. This finding is hypothesis-generating and supports further evaluation of FRα-targeted vaccination in higher-risk TNBC. Immune correlative analyses are ongoing to better define vaccine immunogenicity and its relationship to clinical outcomes. Clinical trial information: W81XWH-15-1-0293.

Abstract Background: Epidermal Growth Factor Receptor (EGFR, HER1) is known to heterodimerize with HER2 and may impact the effectiveness of trastuzumab. Historically, EGFR has been difficult to measure; thus there is no evidence regarding its effects on trastuzumab clinical therapy. Here we use a new, standardized, quantitative immunofluorescence assay and a novel EGFR antibody to evaluate the correlation between EGFR expression and clinical outcome in a cohort from the North Central Cancer Treatment Group (NCCTG, Alliance) N9831 trial. Methods: Of the 3505 women in the N9831 trial, tissue microarrays were constructed that allowed analysis of 1,444 patients randomly assigned to receive chemotherapy alone (Arm A), sequential trastuzumab after chemotherapy (Arm B) or concurrent trastuzumab with chemotherapy (Arm C). Fluorescence-based, standardized measurement of EGFR was done using the rabbit monoclonal EGFR antibody D38B1 on the AQUA platform (HistoRx). EGFR expression levels were analyzed as a continuous variable and dichotomized using recursive partitioning to define an optimal cut point. The cut point was validated on an independent retrospective cohort comprised of 130 patients with HER2-positive metastatic breast cancer who received trastuzumab. Results: EGFR assessed as a continuous variable shows an association with disease free survival (DFS) in Arm C (p = 0.002) but not in Arms A or B. Dichotomizing EGFR expression shows that high level expression is associated with worse outcome (HR = 2.2; 95% CI 1.33–3.59, p = 0.002) in arm C. Five year DFS within the low level EGFR expression group was 71%, 74% and 90% for Arms A, B and C, respectively (log-rank p-value=0.02) and within the high level EGFR expression (n = 213) group was 62%, 76% and 74% respectively (log-rank p-value 0.22). As validation, the same cut-point was used to define high EGFR expression in a retrospective metastatic breast cancer cohort. The median progression free survival difference between EGFR high group and EGFR low group was 6.1 months (p = 0.0063) and the hazard ratio of high EGFR was 1.92 (p = 0.0073). Conclusions: High expression of EGFR by the AQUA platform appears to be associated with decreased benefit from adjuvant concurrent (not sequential) trastuzumab and shorter PFS in the metastatic setting. The biological underpinning for these observations is being considered. Since there are other treatment options for HER2-driven tumors, this marker, once validated, could help select patients for alternative or additive therapy. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr S5-4.

Abstract Background: Intrinsic breast cancer subtypes determined by the PAM50 assay are reported to be prognostic independent of standard clinicopathological variables. The CALGB (Alliance) 9741 adjuvant trial randomized treatment in a 2×2 factorial design to (i) 2-wk (dose dense; DD) vs 3-wk therapy and (ii) sequential vs concurrent doxorubicin, cyclophosphamide, paclitaxel (A&gt;T&gt;C vs AC&gt;T). DD therapy improved disease-free survival and overall survival (OS) (Citron et al. JCO 2003.). A significant interaction between intrinsic breast cancer subtypes and the use of DD therapy was hypothesized. Methods: C9741 was conducted in collaboration with ECOG, NCCTG, and SWOG. Biospecimens were collected from 1652 of 2005 subjects. Multiplexed gene expression profiling (NanoString Technologies, Inc) generated PAM50 subtype calls (luminal A, luminal B, HER2-enriched, basal-like), risk of relapse (ROR) score, and proliferation score for 1321 cases (80%). Excluded samples were due to insufficient tumor (n = 181) or RNA (n = 150). The primary endpoint was relapse-free survival (RFS). The primary analysis to determine if the clinical benefit of DD therapy is dependent on intrinsic subtype was conducted as a test of interaction in a Cox proportional hazards model (3 degrees of freedom, df; alpha=0.05). Similar analyses were performed for ROR score and proliferation score as continuous measures of risk (1 df). PAM50 subtype, ROR score, and proliferation score were evaluated in separate multivariate models adjusting for number of positive nodes, menopausal status, dose density, and sequence of therapy. Results: Improved outcomes for DD therapy in the evaluable subset mirrored results from the complete dataset (HR = 1.20; 95%CI 0.99–1.44) with a median follow-up of 12.3 yrs. Subtype analysis identified 32% luminal A (n = 417), 26% luminal B (n = 341), 20% HER2-enriched (n = 267), and 22% basal-like (n = 296). Intrinsic subtypes were prognostic of RFS (p &lt; 0.0001) irrespective of treatment assignment with HRs relative to the luminal A subgroup of 1.50 (luminal B), 1.70 (HER2-enriched) and 1.66 (basal-like). No subtype specific treatment effect on RFS was identified (p = 0.44). ROR scores (range 0–100, median 60) were also prognostic of RFS (HR = 1.12 for a 10-unit change; 95% CI 1.07–1.18; p &lt; 0.0001), but no association with DD therapy benefit was seen (p = 0.58). Similar results were obtained for the proliferation score, for OS as a secondary endpoint, and from multivariate models with clinical covariates. Conclusion: The prognostic value of PAM50 subtype, ROR score, and proliferation score remains highly significant in patients treated with contemporary adjuvant anthracycline and taxane based chemotherapy. Intrinsic subtype did not predict for improved survival with the 2-wk DD vs 3-wk treatment schedule; we hypothesize that this is because the prognostic differences by subtype outweighed the modest but significant clinical benefit of DD chemotherapy administration for the overall population. Planned clinical validation studies will assess the ability of PAM50 and other gene signatures to stratify patients and individualize systemic therapy regimens based on the expected risk of distant recurrence. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-10-01.

Abstract Background A Phase III trial (Study 301; NCT00337103) in MBC comparing eribulin (E) with capecitabine (C) showed a trend for improved overall survival (OS) (hazard ratio [HR] 0.88; 95% CI 0.77, 1.00; P = 0.056) but not progression-free survival (PFS) (HR 1.08; 95% CI 0.93, 1.25; P = 0.30) with E. In order to investigate this apparent discordance, post-hoc analyses assessed the effect of PPT and events defining disease progression on OS. Methods Pts with locally advanced or MBC who had received an anthracycline and a taxane (≤2 prior chemotherapy regimens for advanced disease), were randomized to E (554) or C (548). Co-primary endpoints were OS and PFS. The impact of PPT on OS was assessed by analysis of exploratory PPT subgroups. Analyses of OS sensitivity censoring at time of PPT, and OS adjusted by PPT as a time-dependent covariate in a Cox regression model, were performed. The relationship between OS and progression events (appearance of a “new” lesion or metastasis [NM]; increase in size of pre-existing lesions [IPEL]; or other [death, clinical progression, or censored]) was investigated using Cox regression. Results PPT was received by 70% and 62% of E and C pts, respectively. For E pts, there were no significant OS differences by PPT (Table 1). Excluding 56 pts (22 E, 34 C) who received anti-HER2 therapy as PPT from the intent-to-treat OS analysis, OS was longer with E (HR 0.86; 95% CI 0.76, 0.99; nominal P = 0.03). OS sensitivity analysis censoring at time of PPT was consistent with the overall study results (HR 0.82; 95% CI 0.65, 1.03). Assessment of the overall effect of PPT, adjusting by PPT as a time-dependent covariate in a Cox regression model, showed that OS was longer with E (HR 0.85; 95% CI 0.75, 0.97; nominal P = 0.02). Progression due to NM or IPEL occurred in 271 and 147 of E pts, and 285 and 129 of C pts, respectively. Median OS was similar between arms in pts with IPEL and longer with E vs C in pts with NM (Table 2). Pts who progressed due to NM were at higher risk of death (HR 2.12; 95% CI 1.84, 2.43; nominal P&lt;0.01). Conclusions Treatment with C or any other PPT after progression on E did not account for the trend in OS benefit with E observed in the primary analysis. Pts who progressed with NM had a worse prognosis than those with IPEL. The appearance of NM was highly correlated with OS, and the apparent discordance between PFS and OS seems to be related to these different progression events. Tab 1: OS by PPT after EPPTAll ptsC as 1stOther than C as 1stC at any timeOther than C at any timeNoneN554221169275115164Median OS, months (95% CI)15.9 (15.2, 17.6)18.3 (15.8, 20.9)19.9 (17.6, 24.0)19.6 (17.6, 21.5)18.0 (15.4, 22.7)7.4 (6.2, 9.1) Tab 2: OS by progression event Progression event NMIPELOther ECECECN271285147129136134Median OS, months (95% CI)15.5 (14.2, 17.5)12.9 (11.3, 14.5)17.4 (14.4, 19.7)17.4 (15.3, 20.9)16.7 (14.8, 24.2)15.5 (11.7, 18.3)HR (95% CI)0.81 (0.68, 0.97)1.13 (0.87, 1.46)0.78 (0.59, 1.03)Nominal P -value0.020.350.08Time to NM or death, months (95% CI)5.8 (5.2, 6.5)5.2 (4.3, 5.9)NANAHR (95% CI)0.90 (0.77, 1.05)NANANominal P-value0.17NANA Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-13-03.

Abstract Background: Hsp90 is a molecular chaperone protein required for the stabilization and activation of many proteins, referred to as Hsp90 ‘clients’, such as HER2, HIF1-a, EGFR, ER, PI3K, AKT, P53 and VEGFR. The drug candidate, ganetespib is a novel triazolone inhibitor of Hsp90, with over 700 patients treated to date. Ganetespib has shown activity in preclinical models of HER2+, ER+/PR+ and TNBC. Early clinical trials documented ganetespib single agent activity in heavily pretreated HER2+ and TNBC patients. Ganetespib has been well tolerated in clinical trials with a favorable safety profile. This efficacy-screening study is designed to provide further evidence of ganetespib activity and identify potentially predictive biomarkers in metastatic breast cancer (BC). Methods: The ENCHANT-1 Trial is an international, first-line 2-cohort Phase 2 study in BC patients: Cohort A, HER2 amplified (n = 35), and Cohort B, TNBC (n = 35). Patients who present with previously untreated metastatic disease are eligible for treatment with ganetespib at 150 mg/m2 twice weekly on 3 out of 4 wks, for a total of up to 12 wks. Primary endpoint: ORR assessed using RECIST1.1 criteria. Key secondary endpoints include metabolic response as assessed by PET/CT at wk 3 utilizing modified EORTC criteria. Disease progression (PD) at wk 3 by PET imaging indicates discontinuation of study therapy, and is performed to quickly offer patients with metabolic PD a standard of care treatment. The study is designed as Simon 2-stage requiring at least one OR in 15 patients for the respective cohort to expand to 35 patients. A Steering Committee is established to oversee the overall study and review the interim results. Results: The study was initiated in 23 centers globally. At the time of submission, a total of 17 patients had been enrolled; TNBC (n = 15) and HER2 (n = 2). Here we report the interim analysis in the TNBC cohort. The median age was 54 years (range 30 -77) with ECOG PS 0 (n = 7/15). Most patients (n = 9) presented with de novo metastatic disease. 5 patients were not evaluable for PET assessment (3 had not yet reached wk 3 and 2 withdrawn before wk 3 for clinical progression), and 9 patients were not evaluable for objective response at wk 6 (3 withdrawn before or at wk 3 for clinical progression and 6 had not yet reached wk 6 evaluation). In the 10 patients with evaluable PET imaging, 9 patients achieved metabolic (m) response (2 mPR, 4 mSD with dominant tumor shrinkage and 3 SD) and one patient with mPD. In the 6 patients evaluable for OR at wk 6, one patient achieved PR, 2 SD and 3 PD. Treatment with ganetespib was well tolerated; the most common AEs were mild or moderate diarrhea (8/15, 53%), fatigue (5/15, 33%), decreased appetite (4/15, 27%), insomnia (4/15, 27%), and nausea (4/15, 27%). Conclusion: Ganetespib single agent was generally well tolerated and showed anti-tumor activity TNBC patients as early as 3 weeks following treatment. PET seems to be a good tool to screen antitumor activity of new agents in early settings rather that in heavily pretreated patients. The TNBC cohort has met the protocol criteria for proceeding to stage 2. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-16-23.

Abstract Background: We have previously reported the results of surgical excision without irradiation for selected patients with DCIS in ECOG E5194, where the 5-year rates of local recurrence varied with age, grade, and lesion size (Hughes et al. J Clin Oncol 27:5319, 2009). New methods are needed to provide more accurate and reproducible assessment of recurrence risk. Methods: ECOG E5194 included 670 eligible patients with DCIS treated with surgical excision (≥ 3 mm negative margins) without irradiation, 228 of whom received tamoxifen. Patients had low or intermediate grade DCIS ≤ 2.5 cm, or high grade DCIS ≤ 1 cm. The Oncotype DX® assay was performed by quantitative RT-PCR using formalin fixed paraffin embedded tumor specimens from 327 patients (49% of the parent study). Recurrence Score® (RS) was calculated using the published algorithm. A new, prespecified DCIS Score™ was designed to predict recurrence using an optimized gene expression algorithm. The primary objective was to determine whether there was a significant association between the risk of an ipsilateral breast event (IBE) and the continuous DCIS Score in Cox models. 46 patients had an IBE (defined as ipsilateral local recurrence of DCIS [n=20] or invasive cancer [n=26]). Median follow-up was 8.8 years. Results: The 10-year IBE rates were 15.4% for low/intermediate grade DCIS and 15.1% for high-grade DCIS (as determined by central pathology review), and for invasive IBE, 5.6% and 9.8%, respectively. Comparison between local and expert grading showed substantial disagreement. Continuous DCIS Score was significantly associated with IBE (HR 2.34 per 50 units; 95% CI 1.15, 4.59; p=0.02) when adjusted for tamoxifen use (prespecified primary analysis) and with invasive IBE (HR 3.73; CI 1.34, 9.82; p=0.01). DCIS Score was significantly associated with outcome when evaluated by the prespecified risk groups (see Table). Similar results were observed with and without adjustment for tamoxifen use or for negative margin width. Features associated with IBE in multivariate models included menopausal status (HR 0.49; 95% CI 0.27, 0.90; p=0.02), tumor size (HR 1.52 per 5 mm; 95% CI 1.11, 2.01; p=0.01), and continuous DCIS Score (HR 2.41; 95% CI 1.15, 4.89; p=0.02). The standard RS, which is calculated using thresholding of many genes unlike the DCIS Score, was not associated with IBE or invasive IBE (p &gt; 0.6). Conclusions: We have prospectively validated a multigene assay that quantifies recurrence risk and complements traditional clinical and pathologic factors in selected patients with DCIS treated with surgical excision without irradiation. The DCIS Score provides a new clinical tool for individualized selection of treatment for patients with DCIS. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr S4-6.

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&lt;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 Abstract #56 Background: Recent findings from NSABP B-31 suggested that breast cancer (BC) patients (pts) with c-MYC (MYC)/HER2 co-amplification in their primary tumors may benefit more in terms of disease-free survival (DFS) from adjuvant trastuzumab (n=471; p&lt;0.0001, HR 0.24; 13 vs. 51 events) than those pts with only HER2 amplification in their primary tumors (n=1078, p=0.007, HR 0.63; 55 vs. 82 events).&#x2028; Purpose: The overall objective of this analysis is to determine the association between MYC/HER2 co-amplification and outcome of pts randomized to receive chemotherapy alone (Arm A) or chemotherapy with concurrent trastuzumab (Arm C) on N9831.&#x2028; Patients/Methods: This analysis included 868 pts from the HER2+ NCCTG N9831 Intergroup adjuvant trastuzumab phase III clinical trial on Arms A or C. Fluorescent in situ hybridization (FISH) was performed at a central laboratory, Mayo Clinic Clinical Cytogenetics Laboratory, using dual probe mixtures for HER2 (17q11.2-12/centromere 17) and for MYC (8q24/centromere 8). The HER2 amplification status in whole tissue sections was evaluated to determine eligibility of pts for N9831. Tissue microarrays containing three cores from each patient's tumor were analyzed for MYC amplification status. The gene and centromere copy number as well as the distribution of the signals for each marker were determined in sixty non-overlapping interphase nuclei.&#x2028; Results: Of 273 pts with completed FISH analyses, 34 (12%) pts had MYC amplification. The rate of MYC amplification was similar across arms (18/135 [13%] vs. 16/138 [12%]; ChiS p=0.66). Co-amplification of MYC/HER2 was observed in 32 (12%) pts with similar co-amplification rates across arms (17/135 [13%] vs. 15/138 [11%]; ChiS p=0.66). Based on preliminary analyses, MYC amplification and MYC/HER2 co-amplification do not appear to be associated with race, menopausal status, hormone receptor status, nodal status, tumor histology, tumor grade, or tumor size (all ChiS p&gt;0.05); however, MYC amplified pts were significantly younger than MYC non-amplified pts (mean 46.4 vs. 51.0, t-test p=0.02) and MYC/HER2 co-amplified pts were significantly younger than MYC/HER2 non-co-amplified pts (mean 45.9 vs. 51.0, t-test p=0.01).&#x2028; Conclusions: In our initial analyses of 239 pts, we observed a lower frequency of MYC/HER2 co-amplification than the previously reported 30% for NSABP B-31. Complete FISH analyses of 868 pts in N9831 and association with DFS will be presented. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 56.