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BACKGROUND: TMEM (Tumor Microenvironment of Metastasis) are the portal for tumor cell intravasation into the circulation and subsequent metastasis (Harney et al Cancer Discov 2015). The potent Tie2 kinase inhibitor rebastinib inhibits intravasation at TMEM sites, reduces circulating tumor cell (CTC) burden, increases angiopoietin (Ang) 1/2 levels, prevents distant metastases, and improves survival in breast cancer animal models when added to either P or E (Harney et al MCT 2017), and circumvent chemotherapy-induced pro-metastatic changes in the tumor microenvironment mediated by TMEM (Karagiannis et al STM 2017). We sought to determine the safety of rebastinib combined with antitubulin therapy (P or E) in patients with HER2- MBC. We also hypothesized that addition of rebastinib would reduce CTC burden and increase Ang levels by blocking Ang-mediated stimulation of VEGF release from TMEM-associated macrophages. METHODS: We aimed to determine the safety and recommended phase 2 dose (RP2D) of rebastinib (2 dose levels: 50 mg or 100 mg PO BID) in combination with P (80 mg/m2 x 12 weeks) or E (1.4 mg/m2 on day 1 & 8 q 21 days) using a standard 3+3 design (1 cycle = 21 days). Secondary objectives included evaluating the effect of the P/E + rebastinib combination on CTCs (TelomeScan) and Ang levels. Dose limiting toxicity (DLT) was defined as grade 3-4 febrile neutropenia, thrombocytopenia, and non-hematologic toxicity during the first 6 weeks of therapy. Eligibility included HER2- MBC, ECOG PS 0-1, CDK4/6 inhibitor progression if ER+. Patients with ≤ 2 prior non-taxane chemotherapy regimens received P+ rebastinib, whereas those with ≥ 2 chemo regimens (including a taxane) received E+ rebastinib. RESULTS: Of 11 treated patients, 6 received rebastinib + P and 5 received rebastinib + E (2 non-evaluable due to rapid disease progression and non-compliance). Among 11 patients who received 60 treatment cycles, only 1 patient (treated with eribulin) had grade 3 events (anemia and neuropathy after week 6) potentially related to treatment. When combined with P, the RP2D of rebastinib was 100 mg PO BID, with DLT occurring in 0/6 patients. When combined with E, 0/3 evaluable patients had a DLT at 50 mg BID of rebastinib (accrual ongoing for 100mg BID). Best response included partial response/stable disease in 4(2PR/2SD) of 6 treated with P+ rebastinib, and 1(1PR) of 5 treated with E+ rebastinib. CTCs decreased during therapy (median decrease 99.7 %) and 4/8 patients converted from CTC+ to CTC-. Ang1 levels increased during therapy in 8 patients (0.2-7.0 fold), while Ang2 levels were also increased in 8 patients (0.2-1.4 fold). CONCLUSIONS: When combined with P x 12 weeks, the RP2D of rebastinib is 100 mg PO BID. When combined with E, the RP2D of rebastinib is at least 50mg PO BID; however, the 100 mg PO BID dose level is still accruing patients. The P/E + rebastinib combinations are associated with antitumor activity and exhibit pharmacodynamic evidence indicating blockade of Tie2 (increased Ang) and TMEM function (reduced CTCs) We plan to further evaluate the P+ rebastinib combination as neoadjuvant therapy in the I-SPY program, and continue further evaluation of P/E + rebastinib combinations in MBC. Citation Format: Anampa JD, Xue X, Oh S-y, Kornblum N, Sadan S, Oktay MH, Condeelis J, Sparano JA. Phase Ib study of rebastinib plus antitubulin therapy with paclitaxel (P) or eribulin (E) in patients with HER2-negative metastatic breast cancer (MBC) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-18-22.

Background: Tumor microenvironment of metastasis (TMEM) is a microanatomical structure composed by 3 cells in direct contact, including a tumor cell expressing the actin-regulatory protein Mammalian-enabled (Mena), a perivascular Tie2hi/Vegfhi-expressing macrophage, and an endothelial cell. TMEM are intravasation sites that function as doorways for hematogenous tumor cell dissemination and metastases (Harney et al. Cancer Discovery 2015). TMEM may be identified and enumerated by triple immunohistochemistry in mouse and human mammary carcinomas. High TMEM score is associated with increased risk of distant metastasis in early stage breast cancer, and provides complementary prognostic information to IHC4 (Rohan et al. JNCI 2014) and Oncotype DX Recurrence Score in ER+, HER2-negative breast cancer (Sparano et al. NPJ Breast Cancer, 2017). Neoadjuvant chemotherapy (NAC) increases TMEM score in breast carcinoma in animal models and humans, indicating a previously unrecognized mechanism of resistance to cytotoxic therapy (Karagiannis et al. Science Trans Med 2017). Intravasation at TMEM sites may be inhibited using agents that block release of VEGF from TMEM-associated TIE2-hi, VEGF-hi macrophages (Harney et al. Mol Cancer Ther, 2017). Here we investigated whether TMEM score in post-NAC treated breast carcinoma is prognostic of distant recurrence in localized breast cancer after NAC, and thus provides a foundation for testing agents that block TMEM function in combination with NAC. Methods: We determined TMEM score in 80 evaluable patients' post-NAC specimens with residual invasive ductal carcinomas of at least 0.5 cm. Approximately 60% of patients had ER+/HER2-negative, 28% had triple negative and 12% had HER2+ disease. Most of the patients received doxorubicin/cyclophosphamide + taxane and an anti-HER2 therapy if applicable. Tissue sections from residual tumors were stained for TMEM using triple immunohistochemistry for Mena-expressing cancer cells, CD31-expressing endothelial cells and CD68-expressing macrophages. The stained slides were scanned, and the images were analyzed by three pathologists, blinded to outcome, who independently determined the tissue areas appropriate for TMEM scoring. TMEM was scored within these areas using an automated algorithm. Results: TMEM score was significantly higher in patients with distant recurrence (average TMEM=106), compared to patients without distant recurrence (average TMEM=71) (p50 yrs. vs. Conclusion: TMEM score may provide independent prognostic information for distant recurrence in patients with residual invasive carcinoma after NAC. These results support the use of agents that block TMEM function in combination with NAC, as planned in the I-SPY2 trial. Citation Format: Oktay MH, D'Alfonso T, Ginter P, Lanjewar S, Entenberg D, Pastoriza JM, Wang Y, Lin Y, Karagiannnis GS, Lin J, Ye X, Anampa J, Xue X, Rohan TE, Sparano JA, Condeelis JS. Tumor microenvironment of metastasis (TMEM) score in residual breast carcinoma post-neoadjuvant chemotherapy as an independent prognosticator of distant recurrence [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-08-18.

Background: Metastasis is the primary cause of death in breast cancer, yet no specific therapies are available that inhibit the metastatic process. TMEM (Tumor Microenvironment of Metastasis) are microanatomic structures formed by a Mena-expressing tumor cell, Tie2-expressing macrophage, and endothelial cell in direct content, which serve as the primary portal for tumor cell intravasation into the circulation and subsequent metastasis. High TMEM score in the primary tumor is associated with higher risk of recurrence in ER+, HER2- early breast cancer. Paclitaxel induces the formation of TMEM in the primary tumors of patients treated with neoadjuvant chemotherapy, and in the primary tumor and distant metastases in the PyMT/PDX models. Tumor cell intravasation is mediated by release of VEGF that promotes focal vascular leakiness specifically at TMEM sites, and is derived from TMEM-associated Tie2HI/VEGFHI macrophages that release VEGF upon binding of the Tie2 receptor to angiopoietin2 (ANG2), which is elaborated by TMEM-associated endothelial cells. Moreover, ANG2-stimulated release of IL-10 by tumor-associated macrophages suppresses T cell proliferation, increases the ratio of CD4+T cells to CD8+ T cells, and promotes the expansion of CD4+CD25highFOXP3+ cells. The Tie2 inhibitor rebastinib inhibits intravasation at TMEM sites, reduces circulating tumor cell (CTC) burden, prevents distant metastases, and improves survival in breast cancer animal models when added to either paclitaxel or eribulin. We therefore hypothesize that the addition of a potent Tie2 inhibitor (rebastinib) to antitubulin therapy in patients with HER2 negative metastatic breast cancer (MBC) will prevent hematogenous dissemination and distant metastasis by inhibition of TMEM function, reduction in CTC burden, and inhibition of immune-system suppression resulting in improvement in breast clinical outcomes Methods: Primary objective of this phase Ib study (NCT02824575) is to evaluate safety and tolerability of rebastinib in two dose levels (DL) (50mg or 100mg po BID) combined with paclitaxel IV 80mg/m2 (day 1, 8 and 15) or eribulin IV 1.4mg/m2 (day1 and 8) for four 21-day cycles. Key eligibility includes histologically confirmed HER2 negative MBC. ≤ 2 non-taxane chemotherapy regimens are allowed for rebastinib plus paclitaxel arm, while ≥ 2 chemotherapy regimens (including a taxane) are required for eribulin plus rebastinib arm. ≥ 2 endocrine regimens, including an approved CDK4/6 inhibitor, is required for ER+ disease. Patients require ECOG PS 0 or 1 and normal organ and marrow function. Exclusion criteria include significant ocular disease, significant history of cardiac disease or concomitant use drugs that prolong QTc interval. Pharmacodynamic biomarkers to be measured during cycle 1-3 include CTCs, ANG 1/2 levels and Tie-2 expressing monocytes. Tissue biopsy after two treatment cycles in 6 patients who have accessible tumors will be performed to evaluate TMEM score and function. With two DL of rebastinib, and 3-6 patients at each DL, it is anticipated that 6-12 patients will be required. This trial has enrolled three patients assigned paclitaxel arm (DL1) and one patient in eribulin arm(DL1). Citation Format: Anampa JD, Xue X, Oktay M, Condeelis J, Sparano JA. Phase Ib study of rebastinib plus antitubulin therapy with paclitaxel or eribulin in patients with metastatic breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr OT2-06-04.

Background: EarlyR is a prognostic gene signature score in ER+ breast cancer (BC) computed from the expression values of ESPL1, SPAG5, MKI67, PLK1 and PGR using a nonlinear mathematical formula. EarlyR has been validated in multiple cohorts profiled on Affymetrix and Illumina microarrays and by RNA-seq. This study sought to assess the prognostic features of EarlyR in a cohort of E2197. Patients and Methods: Illumina DASL assay was used to measure gene expression in FFPE tissue of primary BC from a case-cohort sampling subset of women in E2197 treated with doxorubicin plus docetaxel (AT) or doxorubicin plus cyclophosphamide (AC). ER+ patients received hormone therapy at physician's discretion. After 79.5 months median follow-up, disease-free survival was 85% in both treatment arms. Among patients centrally reviewed with sufficient RNA material for the DASL assay, 319 with ER+ status and assessed for EarlyR are included in the analytic cohort. EarlyR scores and pre-specified risk strata (≤25=low, 26-75=intermediate, >75=high) were computed, while blinded to clinical data. The analysis endpoint was disease-free survival (DFS), defined as the time from randomization to date of invasive BC recurrence or death from any cause within 8 years. Weighted Cox proportional hazards models were used to associate EarlyR score or risk strata with DFS. Variances of the estimated coefficients were adjusted to account for the case-cohort design. Results: The distribution of the EarlyR risk groups was 59% low, 11% intermediate and 30% high risk in this ER+ cohort. The continuous EarlyR score was significantly prognostic of DFS up to 8 years after randomization (p = 0.02). Patients with low EarlyR score (≤ 25) had significantly lower risk of BC recurrence within 8 years (p = 0.031, univariate HR=0.562, 95%CI: 0.334-0.948) compared to those with high EarlyR score (> 75). Analysis within the AC arm showed that patients with low EarlyR score had significantly lower risk of 8-year BC recurrence (p = 0.023, univariate HR=0.392, 95%CI: 0.175-0.878) compared to those with high EarlyR score. Within the AT arm there was no significant difference in 8-year DFS prognosis between any of the EarlyR risk groups. Conclusions: This study confirmed the prognostic significance of EarlyR using FFPE tissue in a cohort of patients treated with AC chemotherapy from E2197. Patients with high EarlyR score who were treated with AC had significantly higher risk of recurrence than low EarlyR score patients treated with AC. On the other hand, prognosis of high EarlyR score AT-treated patients was not significantly lower than the prognosis of low EarlyR score AT-treated patients. Further study in a larger cohort is needed to assess the relative benefits of AC versus AT within the EarlyR high risk group and the EarlyR low risk group. Citation Format: Badve S, Wang V, Willis S, Leyland-Jones B, Gokmen-Polar Y, Shulman L, Martino S, Sparano J, Davidson N, Goldstein L, Buechler S. Independent validation of EarlyR gene signature in E2197: A randomized clinical trial comparing doxorubicin plus docetaxel to doxorubicin plus cyclophosphamide as adjuvant chemotherapy in breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-06-08.

Purpose: The immune system influences breast cancer prognosis. Analysis of the international BIG 02-98 trial suggests that TILs are associated with disease-free survival (DFS) in patients with operable triple negative breast cancer (TNBC, Loi et al, JCO 2013). Here we seek to validate the association of increased lymphocytic infiltration of primary breast tumors with an improved prognosis in TNBCs in two adjuvant Phase III trials conducted by the Eastern Cooperative Group. Methods: “Whole” H&E stained sections of tumors from E2197 and E1199 were evaluated by two independent pathologists blinded to outcome data for density of TILs in intratumoral (iTIL) and stromal compartments (sTIL). Cases from E2197 and E1199 were randomly selected based on TNBC status (E2197 central review, E1199 local determination) and availability of tumor sections. The association of DFS with TIL scores was determined by fitting proportional hazards models stratified on study. Nonrecurrences from E2197 were given a relative weight of 1.43, to reflect that they had been undersampled, and robust variance estimators were used. Results: 500 cases were obtained for which 481 were evaluable (190 for E2197, 291 for E1199). The median age of patients was 49 with 54% being premenopausal, 59% nodal involvement, 66% of tumors being > 2cm and all patients received anthracycline-based chemotherapy. 80% of tumors had at least 10% lymphocytic infiltration in the stroma (range 10-80%) but only 15% of cancers had at least 10% TILs intratumorally (range 10-50%). The lymphocyte-predominant breast cancer (LPBC) phenotype, defined as ≥50% stromal or intratumoral lymphocytic infiltration was seen in 4.4% of TNBC (21/481). At a median follow-up of 10.6 years, 190 DFS events were reported. Higher TIL scores were associated with a better prognosis: for every 10% increment in sTIL, a 14% reduction of risk for recurrence or death was observed (p = 0.02) and for every 10% increment in iTIL, a 28% risk reduction was seen (p = 0.06). Multivariate analysis confirmed sTIL to be an independent prognostic marker of DFS. Hazard Ratio (95% CI)p-valuesTIL (10% increase)0.84 (0.74,0.95)0.005Tumor Size < = 2.0 vs. 2.1-5.0cm0.59 (0.42,0.82)0.002Tumor Size < = 2.0 vs. >5.0cm0.46 (0.27,0.78)0.004Node-neg vs. 1-3 N+0.53 (0.37,0.78)0.001Node-neg vs. >3 N+0.23 (0.14,0.38)60 vs. 24-402.12 (1.29,3.49)0.003Table: Estimated DFS Hazard Ratios, 95% CI and Individual Term P-values for the Multivariate Model for the Linear Effect of the Stromal TIL Score The presence versus the absence of stromal TILs as binary marker was also a strong prognostic marker with a 31% reduction of risk of recurrence or death for the group with sTILs (95% CI 2 to 51, p = 0.04). Conclusion: We confirm in two independent data sets from national randomized clinical trials using contemporary adjuvant chemotherapy that stromal lymphocytic infiltration constitutes a robust prognostic factor in TNBCs. The data provide strong evidence for the incorporation of this feature into routine histopathological assessment as a prognostic factor for patients with TNBCs. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr S1-07.

Background: Although AIs are an effective treatment for HR-positive MBC, whether used alone or in combination with CDK4/6 inhibitors, resistance to therapy and disease progression invariably develops. Therapeutic options for AI-resistant disease include the m-TOR inhibitor everolimus in combination with the steroidal AI exemestane, or the selective estrogen receptor downregulator (SERD) fulvestrant alone. We hypothesized that the combination of fulvestrant and everolimus would be more effective than fulvestrant alone in AI-resistant MBC. Methods: Major eligibility criteria included post-menopausal women with HR-positive, HER2-negative MBC, recurrence or progression while receiving prior non-steroidal or steroidal AI therapy, ECOG PS 0-1, and ≤1 prior chemotherapy regimen for metastases. All patients received fulvestrant (500mg IM q2 weeks for 3 doses, then q4 weeks) plus oral everolimus (10mg) or placebo (1:1 randomization). Prophylactic corticosteroid mouthwash was not used. Tumor assessment was performed at baseline and every 12 weeks. Treatment continued until progressive disease (PD) by RECIST 1.1 criteria. Patients who discontinued everolimus/placebo due to toxicity continued fulvestrant until PD. The primary endpoint was progression-free survival (PFS), defined as time from start of treatment until progression or death. With accrual of 130 patients (120 eligible), the trial had 90% power to detect an improvement in median PFS from 5.4 months to 9.2 months (1-sided stratified log-rank test, type I error rate10%), with analysis planned after 98 PFS events. Results: Of 130 patients treated (64 everolimus, 66 placebo), median age was 61 years (range 35-92), and treatment arms were balanced for stratification factors used in randomization, including ECOG PS 0 vs. 1 (59%/41%), measurable disease (66%), and prior chemotherapy for metastasis (17%). Grade 3/4 AEs were more common in the everolimus arm (53%/3% vs. 23%3%), including hyperglycemia (16%/0% vs. 0%), stomatitis (11%/0% vs. 0%), hypertriglyceridemia (9%/2% vs. 0%), lymphopenia (9%/0% vs. 0%), and pneumonitis (6%/2% vs. 0%). There were 3 grade 5 events (2 everolimus, 1 placebo arm), none of which were attributed to therapy. Selected grade 2 events of interest included fatigue (17% vs. 6%), hyperglycemia (6% vs. 0%), and stomatitis (6% vs. 0%). Everolimus/placebo was discontinued for adverse events, patient withdrawal, or physician discretion in 39% in the everolimus arm and 21% in the placebo arm. After 98 PFS events, median PFS was 10.4 months in the everolimus arm versus 5.1 months in the placebo arm (hazard ratio: 0.61, 95% C.I. 0.40 – 0.92; stratified logrank p= 0.02). Conclusions: The addition of everolimus to fulvestrant significantly improved PFS in post-menopausal women with AI-resistant MBC. Everolimus used without prophylactic corticosteroid mouthwash exhibited a similar rate of oral mucositis and overall AE profile when combined with fulvestrant as when combined with exemestane. Keywords: advanced breast cancer, PrECOG 0102, endocrine resistance, everolimus, exemestane, hormone receptor-positive, mTOR inhibitor, postmenopausal. Citation Format: Kornblum NS, Manola J, Klein P, Ramaswamy B, Brufsky A, Stella PJ, Burnette B, Telli M, Makower DF, Leach J, Truica CI, Wolff AC, Soori GS, Haley B, Nagarajan A, Wassenaar TR, Goldstein L, Miller KD, Sparano JA. PrECOG 0102: A randomized, double-blind, phase II trial of fulvestrant plus everolimus or placebo in post-menopausal women with hormone receptor (HR)-positive, HER2-negative metastatic breast cancer (MBC) resistant to aromatase inhibitor (AI) therapy [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr S1-02.

Population-based studies have suggested improved survival for patients diagnosed with MBC in recent years, presumably due to the availability of new and more effective therapies (Chia et al. Cancer 2007; Dawood et al. JCO, 2008). The objective of this analysis was to determine if survival improved for patients who participated in Eastern Cooperative Oncology Group (ECOG) adjuvant trials and later developed MBC. Methods: Adjuvant trials coordinated by the ECOG that accrued patients between 1978 and 2002 were reviewed (n=12), which included followup until 2010. Cytotoxic and biologic agents approved for MBC during this time included paclitaxel (1994), capecitabine and trastuzumab (1998), docetaxel and gemcitabine (2004), lapatinib and ixabepilone (2007), and bevacizumab (2008). Survival following distant recurrence was estimated for 4 time periods ranging from 6–10 years, and adjusted for baseline covariates in a Cox proportional hazards model. Because distant relapse free interval (DRFI) was the covariate most strongly associated with survival after recurrence, and the potential for “gap time” bias this could introduce, logrank tests for other covariates and estimates of effects were computed stratified on DRFI (0-3, >3-6, > 6 years). HER2 status was not routinely available and thus not included. Results: The 12 trials included 14,752 patients (93% received adjuvant chemotherapy); 3711 (25.2%) developed distant recurrence. Median survival after distant recurrence was 20 months; the estimated 5 and 10-year survival rates were 16.3% and 6.1%, respectively. Median survival by time period is shown in the table, stratified by DRFI. Median survival did not significantly change over time by DRFI (≤3 years, p=0.15; >3 yr, p=0.57). In a Cox proportional hazards model, factors associated with inferior survival after adjusting for other covariates included shorter DRFI ( 6 vs. < 3 years — HR 2.23, p 9 vs. 0 nodes — HR 1.51, p Conclusions: In contrast to reports from population-based studies, we do not observe any improvement in survival over time for patients who develop distant recurrence after adjuvant chemotherapy. There remains a critical unmet need for new therapies for MBC, especially for those who recur after adjuvant chemotherapy. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-08-01.

Background We have previously reported that TOP2A RNA expression was prognostic in patients with operable estrogen receptor (ER) positive breast cancer treated with anthracycline containing adjuvant chemotherapy plus endocrine therapy in trial E2197, and provided complementary prognostic information in those who had a mid-range Recurrence Score (RS) (Sparano et al. Clin Cancer Res 2009; 15; 7693). The purpose of this analysis was to provide additional evidence regarding this observation. Methods: We evaluated TOP2A RNA expression in 4 independent data sets individually and in a pooled analysis including all data. We also used an algorithm to simulate the Oncotype DX Recurrence Score (SimRS). The analysis included 752 patients with early stage breast cancer that was ER-positive, HER2−negative defined by gene expression. Results are expressed as the hazard ratio (HR) for estimates of the effect of a one standard deviation increase in the value of the log gene expression (x + 1SD vs. x) as a continuous function. Five-year relapse free survival (RFS) rates were also evaluated for patients with a mid-range SimRS for high vs. low TOP2A expression (above vs. below the median, which was prognostic in E2197). The distribution of SimRS was categorized to be concordant with the distribution of actual RS in E2197 (47%, 34%, 19%, respectively for RS 31 - high). Results: The HR for models including TOP2A alone and joint models including TOP2A and SimRS are shown in the table. TOP2A expression was significantly associated with recurrence in all 4 datasets when evaluated individually (HR 1.56, p Conclusions: This analysis provides additional evidence that TOP2A expression provides prognostic information in patients with ER-positive, HER2−negative disease, a population known to have low incidence of TOP2A gene alterations. These findings also suggest that TOP2A expression provides information that is complementary to RS, and may be useful for identifying high risk subjects who have a mid-range RS. These findings require prospective validation in other prospective or prospective-retrospective trials using the actual rather than simulated RS. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-12-13.

Background: Metastasis is the primary cause of death in ESBC. We have shown in mouse models that a subpopulation of tumor cells expressing invasive Mena isoforms stream, form microanatomic structures (“TMEM”) with endothelial cells and macrophages, intravasate into the circulation at TMEM sites, and metastasize (Harney et al. Cancer Discovery, 2015). Further, TMEM sites (“MetaSites”) are identifiable in human ESBC, and “MetaSite score” [MS] is positively associated with distant recurrence in HR+/HER2- ESBC independent of clinicopathologic features, including IHC4 (Rohan et al. JNCI 2014). Here we determined the association between MS and recurrence in an independent ESBC cohort (E2197; NCT00003519). Methods: We evaluated primary tumors from 600 patients (median followup 14.8 years) with ESBC (weighted % = 50% T1, 54% N0, 46% N1) treated with surgery and 4 cycles of adjuvant chemotherapy (AC or AT) and endocrine therapy. Grade, ER, PR, and HER2, and Oncotype DX Recurrence Score (RS) were evaluated in central labs (Badve et al. JCO 2008), and MS was determined in a CLIA-certified lab using an analytically validated, fully automated digital pathology/image analysis method that identifies Mena expressing tumor cells in direct contact with CD68+ macrophages and CD31+ endothelial cells (ie, “TMEMs”, or “MetaSites”). The objectives were to determine the association between MS and distant relapse free interval (DRFI) and relapse free interval (RFI). Kaplan-Meier survival curves were used to estimate time-to-event distributions. Cox proportional hazards models were used to assess hazard ratio associated with MS while controlling for covariates, and allowing time-varying association with MS. Both Kaplan-Meier and Cox regression methods addressed stratified sampling by incorporating proper weights. All analyses were performed in R 3.2.3. Results: MS ranged from 0-199; the weighted mean MS was lower in HR+/HER2- than TN (16.1 vs. 23.8, p=0.001) and HER2+ disease (26.2, p=0.003). MS was not associated with T or N status, and correlated poorly with RS (r=0.29). Proportional hazards models revealed a significant positive association between continuous MS and DRFI (p=0.001) and RFI (p=0.00006) in HR+/HER2- disease in years 0-5 (and by MS tertiles for DRFI [p=0.04] and RFI [p=0.01]), but not after year 5 or in TN or HER2+ disease. Proportional hazards models including clinical covariates (N0 vs. N1; T1 vs. T2; high vs. int. vs. low grade) also revealed significant positive associations for continuous MS with RFI (p=0.04), and borderline association with DRFI (p=0.08). Similar findings for MS (RFI p=0.05;DRFI p=0.10) were noted in a joint model including categorical RS (30). Conclusions: MS, a novel metastasis biomarker reflecting interaction between streaming and metastasizing tumor cells and microenvironment, provides prognostic information complementary to classical clinicopathologic features and RS in HR+/HER2- ESBC. Further evaluation is warranted in order to identify patients at highest risk of recurrence within 5 years most likely to benefit from adjuvant chemotherapy or novel therapies. (Supported by BCRF and NCI CA21115, CA180794, CA23318, CA66636, CA180820). Citation Format: Sparano JA, Gray R, Oktay MH, Entenberg D, Rohan T, Xue X, Donovan M, Peterson M, Shuber A, Hamilton D, D'Alfonso T, Goldstein LJ, Gerlter F, Davidson N, Condeelis J, Jones J. Tumor microenvironment of metastasis (TMEM) score is associated with early distant recurrence in hormone receptor (HR) positive, HER2-negative early stage breast cancer (ESBC) [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr S4-04.

Background: Treatment for mTNBC is limited, and significant challenges persist in treating this disease, as outcomes remain largely dependent on chemotherapy without any effective targeted treatment. Pembrolizumab (MK-3475) is a highly selective, humanized monoclonal antibody against PD-1, blocking the negative immune regulatory signaling of the PD-1 receptor that is usually expressed by T-cells. Recent data showed that some patients with mTNBC may benefit from immune-based therapies (PD-1 or PD-L1 antibodies). Cumulative evidence suggest that stromal tumor infiltrating lymphocytes (sTILs) have a prognostic and predictive role in response to treatment in subsets of TNBC, particularly in response to carboplatin use. Preclinical data revealed that blocking PD-1/PD-L1 pathway in combination with platinum containing cytotoxic therapy improved response rates and survival. High levels of sTILs and an increased PD-L1 expression make mTNBC a candidate for PD-1–targeted therapy. As studies showed that the subset of TNBC with better response rates to carboplatin are heavily infiltrated with sTILs, pembrolizumab, becomes a very attractive drug to be tested in combination with carboplatin, with the goal of improving outcomes in mTNBC. A Phase II multicenter, randomized, trial has been initiated to evaluate the efficacy and safety of combining pembrolizumab with carboplatin and gemcitabine in patients with mTNBC. Methods: A safety run-in will assess the safety and tolerability of combining pembrolizumab with carboplatin and gemcitabine in patients with mTNBC. Following the completion of the safety run-in, patients will be randomized 2:1 to receive pembrolizumab (200 mg IV) on day 1 along with carboplatin (AUC 2, day 1 and day 8, IV) plus gemcitabine (800 mg/m2, day 1 and day 8, IV) of a 21-day cycle, or carboplatin plus gemcitabine (same aforementioned dose) alone. Patients will have histologically documented unresectable mTNBC. Prior systemic therapy for mTNBC, for up to 2 lines is allowed, and patients will have ECOG PS 0–2 and measurable disease (RECIST v1.1). Prior carboplatin/gemcitabine or cisplatin therapy is allowed in the adjuvant or neoadjuvant setting, as long as it occurred more than 12 months from the beginning of their enrollment. Subjects whose tumors progressed while on treatment with carboplatin or cisplatin are excluded. Known CNS disease (except asymptomatic treated metastases), autoimmune disease or prior immune checkpoint blockade therapy is an exclusion to enrollment on this trial. Primary endpoint is assessing the objective response rate according to RECIST v1.1 . Other endpoints include clinical benefit rate (CBR), progression-free survival (PFS), overall survival (OS), duration of response (DOR), and safety. Tumor biopsies will be obtained at baseline and just prior to initiation of cycle 3 to assess biomarkers of response and immune escape. PD-L1 expression will be evaluated in exploratory analysis with a planned assessment of response based on PD-L1 status. This trial will enroll 6-12 patients in the safety run-in portion, and 75 patients in the randomized part, at 7 sites in the United States. Clinical trial information: NCT02755272 www.clinicaltrials.gov. Citation Format: Obeid E, Miller KD, Sparano JA, Blackwell K, Goldstein LJ. A Phase II randomized trial of pembrolizumab with carboplatin and gemcitabine for treatment of patients with metastatic triple-negative breast cancer (mTNBC) [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr OT2-01-17.

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 > 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.

PURPOSE: Several groups have reported that the combination of doxorubicin plus paclitaxel given as a 3-hour intravenous (IV) infusion for up to eight cycles produces a high response rate (> 80%) and complete response rate (> 20%) in metastatic breast cancer, but is also complicated by a 20% incidence of congestive heart failure (CHF). The purpose of this phase II trial was to evaluate the antineoplastic activity of the regimen in a multi-institutional setting and to reduce the incidence of cardiotoxicity by limiting treatment to a maximum of six cycles. PATIENTS AND METHODS: Fifty-two patients with advanced breast cancer received doxorubicin (60 mg/m2 by IV injection) followed 15 minutes later by paclitaxel (200 mg/m2 by IV infusion over 3 hours) every 3 weeks for four to six cycles. RESULTS: Objective responses occurred in 25 of 48 assessable patients (52%; 95% confidence interval [CI], 38% to 66%), including four complete responses (8%; 95% CI, 0% to 16%). The median cumulative doxorubicin dose given was 240 mg/m2 (range, 132 to 360 mg/m2). Eleven patients (21%) were documented as having a decrease in the LVEF below normal, including three patients (6%; 95% CI, 0% to 12%) who developed CHF. CONCLUSION: The doxorubicin/paclitaxel regimen that we used is unlikely to produce an objective response rate of more than 70% and a complete response rate of more than 20% in patients with metastatic breast cancer, and proved to be excessively cardiotoxic for use in the adjuvant setting.

270 Background: Neuropathy is a common and potentially enduring and disabling complication of adjuvant taxane therapy. Recent studies have identified candidate host single nucleotide polymorphisms (SNPs) associated with taxane-induced neuropathy (Schneider et al. ASCO 2011, abstr. 1000). We therefore sought to determine whether neuropathy was associated with breast cancer recurrence. Methods: This study included 4,950 eligible women with axillary lymph node positive or high-risk node-negative breast cancer who received up to 4 cycles of AC (doxorubicin 60 mg/m2 and cyclophosphamide 600 mg/m2) every 3 weeks, followed by either: (1) paclitaxel 175 mg/m2 every 3 weeks x 4 (P3), (2) paclitaxel 80 mg/m2 weekly x 12 (P1), (3) docetaxel 100 mg/m2 every 3 weeks x 4 (D3), or (4) docetaxel 35 mg/m2 weekly x 12 (D1). Chemotherapy doses were based on actual body weight. Cox proportional hazards model were used to determine the relationship between neuropathy and disease free survival (DFS) and overall survival (OS) treating neuropathy status as a time dependent covariate and using a landmark analysis. Results: Of 4,702 patients who received at least 1 taxane dose, grade 2-4 neuropathy developed in 20%, 27%, 16%, and 16% in the P3, P1, D3, and D1 arms, respectively. In a model including age, tumor size, nodal status, treatment arm, neuropathy, and the neuropathy- treatment interaction, there was no relationship between neuropathy and DFS and OS in the entire population, for any of the individual treatment arms, or for any breast cancer subtypes, whether analyzed as a time-dependent covariate or using a landmark analysis. Baseline covariates associated with an increase rate of neuropathy included black race (25% vs. 19% grade 2-4, p=0.02) and obesity (21% vs. 19%, p=0.04), but not age. Conclusions: There was no association between taxane-induced neuropathy and DFS or OS in patients treated with contemporary AC-taxane therapy, including weekly paclitaxel. These findings show that taxane-induced neuropathy is not associated with outcome, thus suggesting that validation of SNPs predictive of neuropathy may be useful in identifying patients at higher risk for neuropathy but not taxane benefit and thereby improve therapeutic individualization.

1000 Background: Neuropathy, one of the most common toxicities associated with taxane therapy, may be severe, function-limiting, and sometimes irreversible. Established predictors for increased risk include advanced age, diabetes, and type/dose/schedule of taxane. No established biomarkers have been identified to predict patients at greatest risk. Methods: E5103 is a randomized phase III trial comparing standard adjuvant chemotherapy for patients with early stage breast cancer with the same chemotherapy plus concurrent bevacizumab or concurrent and sequential bevacizumab. All 3 arms include weekly paclitaxel for 12 weeks. A GWAS was performed using the Infinium Human Omni1 array on 2204 patients. The primary hypothesis was to identify SNPs associated with time to report of first grade 2−4 neuropathy. SNP data underwent rigorous review to assess both SNP and sample quality. Analyses were performed using Cox regression model including established clinical trial covariates. Bonferroni corrections for multipl...

Background: Obesity has been associated with worse outcomes in operable breast cancer. We evaluated the effect of obesity on outcomes in several adjuvant trials coordinated by the ECOG which included doxorubicin/cyclophosphamide plus other agents, including E1199 (NEJM 2008; 358: 1663), E5188 (JCO 2005; 23: 5973) and E3189 (JCO 10998; 16: 2382). Methods: The relationship between body mass index (BMI in kg/m2) and disease free survival (DFS) and overall survival (OS) was evaluated in multivariate models adjusted for covariates including age, tumor size, nodal status, race (black vs. other), surgery type, prior RT, menopausal status, treatment arm, and treatment adherence. Results are expressed as hazard ratios (HR) from Cox's proportional hazards models (HR > 1 indicates a worse outcome). All p-values are two sided. Results: The study characteristics and results are shown in the table below. We first evaluated results for the E1199 trial (for the 75% of those with BMI data available) and found a non-significant trend toward inferior DFS (HR 1.10, 95% CI 0.96, 1.26, p=0.18) and OS (HR 1.13, 95% CI 0.96, 1.33, p=0.15) for obese subjects compared with others. When evaluated by breast cancer subtype, however, obese subjects with ER and/or PR-positive/HER2-negative disease had significantly inferior DFS (HR 1.24, p=0.026) and OS (HR 1.42, p=0.003), an effect that was not seen for HER2-positive and triple negative disease. In a model including obesity, ER and/or PR-pos/HER2-neg disease, and their interaction, the interaction term was significant for OS (p=0.017) but not DFS (p=0.21). There was no evidence for differing chemotherapy delivery or endocrine therapy adherence, or differences in toxicity for obese subjects in the E1199 trial. We then evaluated the relationship between obesity and outcomes in two prior ECOG studies which included only premenopausal women with ER positive, node positive breast cancer (E5188) and women of any age with ER/PR negative, node positive disease (E3189), and confirmed a relationship between obesity and poor outcomes only in women with hormone receptor positive disease. HR-hazard ratio; ER/PR-estrogen/progesterone receptor Conclusions: Obesity was associated with significantly inferior DFS and OS in hormone receptor positive operable breast cancer treated with adjuvant chemotherapy. These findings suggest that hyperinsulinemia or other factors associated with obesity may predispose to recurrence specifically in hormone receptor positive disease, and requires further evaluation in prospective studies in order to identify modifiable factors contributing to this effect. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr S2-1.

Marrow red cells (MRBCs) from 56 autologous bone marrow harvests were rescued after processing and transfused as the sole transfusion support after surgery. There was no correlation between volume of harvest and total mononuclear cell (MNC) count. The marrow collection induced a significant decrease in hematocrit values (mean, 6.1; range, -0.3-12.3; p less than 0.001) unrelated to the patient's diagnosis, age, or gender. Processing of the marrows resulted in a mean transfused MRBC mass of 258 mL (range, 101-494 mL), representing 78 percent (range, 43-100%) of the MRBC mass collected. The amount of MRBCs transfused correlated with total MNC count (p less than 0.01). All autologous MRBC transfusions were well tolerated. It can be concluded that autologous MRBC transfusions are safe and may eliminate the need for homologous blood transfusions after marrow harvest.

500 Background: Triple negative breast cancer (TNBC) is associated with a higher risk of recurrence and earlier recurrences than other breast cancer phenotypes. We evaluated the genotypic features of TNBC compared with hormone receptor (HR)-positive disease, and also evaluated genotypic features associated with recurrence. Methods: RNA extracted from tumor samples obtained from 764 patients with stage I-III breast cancer was analyzed by RT-PCR for 371 genes. All patients received adjuvant chemotherapy (plus hormonal therapy in HR-positive disease) in trial E2197; HR and HER2 expression were evaluated by immunohistochemistry (IHC) in a central lab (J Clin Oncol 26:2473–2481). An unsupervised clustering analysis was performed in all samples (N=764). Cox proportional hazard models were used to identify differences in gene expression in TNBC versus HR-positive disease, and with recurrence in phenotypically defined (by IHC) TNBC (N=246) and HR-positive (N=465) disease. Results: Unsupervised analysis revealed two major clusters that differed with regard to HR expression by IHC. Supervised analysis comparing the TNBC vs. HR-positive phenotypes revealed 269 genes (73%) with significantly different expression (p [Table: see text]

557 Background: Although E2197 showed no difference in recurrence or overall survival, an exploratory analysis suggested differential benefit for AC in PR-pos disease and AT in PR-neg disease (Proc...

22009 Background: Laboratory assessment of HER2 is of marked clinical importance. Accurate quantification remains problematic within and between laboratories. Here we report central laboratory HER2 results comparing IHC and quantitative RT-PCR using Oncotype DX in patients enrolled in a large adjuvant breast cancer trial. Methods: 755 patients with 0 to 3 positive lymph nodes from Intergroup study E2197 were studied. Central IHC for HER2 was performed using duplicate 1.0 mm microarrays (HercepTest; Dako). Percent positive cells and staining intensity (0–3+) was assessed, where IHC positive cases exhibited 3+ staining in >30% cells, IHC equivocal cases exhibited 3+ staining in 10.7

1036 Background: Approximately 30% of human cancers have mutated Ras genes that produce proteins that remain in an active state causing uncontrolled proliferative signals. Post-translational modification of Ras include farneyslation catalyzed by FT. Tipifarnib (R115777) is an oral FTI active against human tumor cell lines and exhibiting modest single agent activity in pts with previously treated MBC. A previous phase I trial reported that CT inhibited farneyslation in peripheral blood mononuclear cells without affecting the pharmacokinetics of either agent. Objective: To evaluate objective response rate (ORR) of CT in taxane refractory MBC and to secondarily evaluate associated toxicity and progression-free survival (PFS). Methods: Pt with measurable MBC, previously treated (rx) with an anthracycline and relapse on a taxane or within 30 days (d). Study rx: T- 300 mg, po BID × 14 d plus C- 1,000 mg/m2, po BID × 14 d, followed by 7 d rest. Tumor reassessment was repeated q 3 cycles. The study was designed to detect improvement in ORR from 25% with C alone to 40% for the CT combination (90.5% power; type I error rate of 9.9%; 21 responses in 64 eligible pt needed to be promising. Results: 66/71 pt are available for primary analysis. Median age 50 yrs. Performance status: 0–1, 100%. ORR: PR-4.8% (3/62) [95% CI 0.01, 0.13], SD - 21% (13/62) [ 95% CI 0.12, 0.33]. Median survival - 10.6 months. Toxicity (%): anemia - 8(G3/4), neutropenia - 30 (G3/4), thrombocytopenia - 8 (G3/4), HFS-8 (G3), nausea/vomiting - 11(G3), diarrhea - 8 (G3), sensory neuropathy - 5 (G3). Conclusion: CT in taxane -refractory MBC has low antitumor activity without excessive toxicity. More mature data, including PFS, will be presented. No significant financial relationships to disclose.

516 Background: Evidence suggests that docetaxel is more effective than paclitaxel, and paclitaxel is more effective when given weekly than every 3 weeks in metastatic breast cancer (BC). Methods: Eligibility included axillary lymph node positive or high-risk (tumor at least 2 cm) node-negative BC. All patients received 4 cycles of AC (doxorubicin 60 mg/m2 and cyclophosphamide 600 mg/m2) every 3 weeks, followed by either: (1) paclitaxel 175 mg/m2 every 3 weeks × 4 (P3), (2) paclitaxel 80 mg/m2 weekly × 12 (P1), (3) docetaxel 100 mg/m2 every 3 weeks × 4 (D3), or (4) docetaxel 35 mg/m2 weekly × 12 (D1). The primary comparisons included taxane (P vs. D) and schedule (every 3 weeks vs. weekly), and secondary comparisons included P3 vs. other arms. The trial had 86% power to detect a 17.5% decrease in disease-free survival (DFS) for either primary comparison, and 80% power to detect a 22% decrease for the secondary comparisons (2-sided nomimal 5% level tests corrected for multiple comparisons). Results: A total of 4,950 eligible patients were accrued. There was no difference in the primary comparisons afer 856 DFS events and 483 deaths after a median follow-up of 46.5 months at the 4th interim analysis ( www.sabcs.org , abstract 48). This is the final pre-specified analysis for the primary comparisons after 1,042 DFS events and 650 deaths (with 1,020 DFS events at this time, to be updated at the meeting). After a median followup of 60.2 months, there remains no significant difference in the hazard ratio (HR) for the taxane (1.02; p=0.73) or schedule (1.07; p=0.30) (as in the first analysis). In secondary comparisons of the standard arm (P3) with the other arms (HR > 1 favoring the experimental arms), the HRs were 1.30 (p = 0.003) for arm P1, 1.24 (p=0.02) for arm D3, and 1.09 (p=0.33) for arm D1. Analysis of interaction by hormone-receptor status will be presented. The incidence of worst grade toxicity (grade 3/4) was 24%/6% for arm P3, 24%/3% for arm P1, 21%/50% for arm D3, and 38%/6% for arm D1. Conclusions: There were no differences in DFS when comparing taxane or schedule overall. DFS was significantly improved in the weekly paclitaxel and every 3-week docetaxel arms compared with the every 3-week paclitaxel arm. [Table: see text]

21022 Background: Central and local laboratory concordance for hormone and HER2 receptor measurement is of national interest. This study compares ER/PR/HER2 by local laboratories using immunohistochemistry (IHC) and central laboratories (IHC & quantitative RT-PCR). Methods: Of 2952 patients in E2197, a case-cohort sample of 776 patients who either did (N=179) or did not recur was studied. Central IHC for ER/PR/HER2 was performed using single 0.6 mm microarrays; Allred score (AS) was used for ER/PR (AS>2 = positive). Positive HER2 was 3+ staining in >10% cells for Central IHC and 2+ or 3+ for Local IHC. RT-PCR analysis by Oncotype DX™ for ER/PR/HER2 was performed using pre-defined cutoffs of 6.5, 5.5 and 11.5 units, respectively. Hormone receptor (HR) pos was defined as ER &/or PR pos. Results: Results from Local IHC (ER/PR in 776 & HER2 in 517 pts) were compared with Central IHC (760 pts) and RT-PCR results (776 pts). The discordance between HR positivity by Local IHC and RT-PCR was very low. However, 12% of HR neg pts by Local IHC (38/321) & Central IHC (39/326) were HR pos by RT-PCR. The relationship between ER and recurrence as a function of AS was examined. Patients with AS of 3–4 were found to be closer to the AS=2 group than to the AS>4 group Patients with AS of 3–4 were found to be closer to the AS ÿ 2 group than to the AS > 4 group (Est.HR for ER 0.97 for AS 3–4 vs. 0–2 and 0.46 for AS 5–8 vs. 0–2, and for PR were 0.84 for AS 3–4 vs. 0–2 and 0.41 for AS 5–8 vs. 0–2). Conclusions: There is a high degree of overall concordance among Local IHC, Central IHC, and Central RT-PCR for ER and PR. The degree of concordance is even greater for HR compared to ER or PR alone. Although the concordance with local labs for HER2 testing was poor, the concordance between Central IHC and RT-PCR was very high. The relatively high incidence (12%) of IHC HR neg pts who are HR pos by RT- PCR is notable. [Table: see text] [Table: see text]

1033 Background: Activation of EGF receptor has been associated with resistance to trastuzumab in breast cancer cell lines. EGFR tyrosine kinase inhibitors inhibit HER2 phosphorylation and synergize with trastuzumab in HER2+ cell lines that co-express EGFR. Methods: Pts with MBC and HER2 overexpression by immunohistochemistry (3+) and/or HER2 gene-amplification by FISH, 0–2 prior chemotherapy regimens for met disease, LVEF 50%, and no prior trastuzumab were treated with trastuzumab 2 mg/kg/wk and gefitinib 250- 500 mg/day until disease progression, unacceptable toxicity or withdrawal of consent. The phase I portion of the trial used a 3+3 design to determine MTD. In the phase II portion of the trial, patients were stratified based upon prior chemotherapy exposure (Group 1= no prior exposure to chemotherapy, Group 2= prior exposure to 1–2 chemotherapy regimens). Response measured using RECIST criteria. The primary endpoint was to increase proportion progression free from 50 to 65% at 6 months in Group 1 and from 50 to 70% at 3 months in Group 2. Results: Phase I: DLT (Grade 3 diarrhea) occurred in 2/3 patients treated at the 500 mg/day dose level of gefitinib in combination with weekly trastuzumab. 0/3 patients treated at the 250 mg/day dose level experienced DLT. This was considered MTD and was the dose selected for the Phase II portion of the trial. Phase II: 36 eligible pts were enrolled. Most patients were ECOG PS of 0 and had visceral organ involvement. Of the patients enrolled in Group 1, one pt achieved a CR, one PR and 7 had SD (≥ 24 weeks). Median time to progression (TTP) was 2.9 months (95% CI, 2.5–4). In Group 2 no responses were observed with a median TTP of 2.5 months (95% CI, 1.5- 2.7). Most common severe toxicities were rash (grade 3, 14%) and diarrhea (grade 3, 30%). No grade 3 cardiac toxicity was encountered. Conclusions: Trastuzumab in combination with gefitinib at doses of 250 mg/day demonstrated an acceptable toxicity profile; however, during planned interim analysis, the TTP did not meet predetermined statistical endpoints required for study continuation. These results do not support the further use of this combination and have implications for other trials using trastuzumab and EGFR TK inhibitors simultaneously. [Table: see text]