Osborne CK Research Articles

Abstract P6-17-12: Neratinib in combination with trastuzumab is superior to each alone and to pertuzumab plus trastuzumab in HER2-positive in vivo breast cancer models

Abstract Background: Lapatinib (L) plus trastuzumab (T) alone or with endocrine therapy for HER2+/ER+ tumors but without chemotherapy, yielded complete tumor eradication in xenograft models. In neoadjuvant trials (NCT00548184, 00999804, 01973660), a substantial number of patients achieved pathologic complete response with this same strategy. The irreversible pan-HER inhibitor neratinib (N) has been recently approved by the FDA for early stage HER2+ breast cancer and has shown greater potency compared to L in the preclinical setting. However, the therapeutic efficacy of N in combination with T (N+T) and how it compares to pertuzumab (P) +T (without chemotherapy) has not been well studied. We hypothesize that dual HER2 inhibition using N+T will be highly efficacious and more effective than P+T due to more complete blockade of the HER pathway. Here, we evaluate the therapeutic efficacy of N, P, and T, either alone or in combination, with a primary focus on comparing N+T vs. P+T in established cell line- and patient-derived xenograft (PDX) models. Methods: Athymic nude and SCID/Beige mice bearing BT474-AZ cell line (ER+/HER2+), and BCM-3963 PDX tumors (ER-/HER2+, wild-type PIK3CA), respectively were randomized to vehicle, N (20mg/kg, 5 days/week), T (10mg/kg, twice a week), P (6mg/kg, once a week), N+T, or P+T, with simultaneous estrogen (E2) deprivation (ED) in BT474-AZ model. Treatment response was assessed by biweekly tumor measurements. Study endpoints included time to tumor regression (TTR) and progression (TTP) (tumor halving/doubling over baseline, respectively), and the rate and time of complete response (CR and TCR, respectively). Results were analyzed using survival analysis (Kaplan-Meier estimates) and generalized Wilcoxon tests. Results: In the BT474-AZ model, mice treated with E2+vehicle and ED+vehicle showed steady tumor growth, with a median TTP of 8 and 25 days, respectively. While tumor regression was observed in 100% of mice treated with N, P, T, N+T, and P+T, tumors treated with N+T regressed faster compared to P (p<0.001), T (p=0.004), and P+T (p=0.044). Further, N+T was superior to N (p=0.018) and T (p=0.007) alone in achieving accelerated CR. In the BCM-3963 model, tumors treated with vehicle, T, P, and P+T continued to grow with a median TTP of 11, 16, 19, and 17 days, respectively. In contrast, CR was achieved in 100% of N and N+T treated mice. Importantly, combining N with T accelerated the attainment of CR compared to N alone (p=0.026). Molecular and pathologic analysis of short-term treated tumors in both models to evaluate alterations in HER signaling, cell proliferation, and apoptosis is ongoing. Model/TreatmentN of miceMedian TTP (Days)Median TTR (Days)Median TCR (Days)CR (%)BT474-AZ E2+Vehicle98--0ED+Vehicle1025--0ED+N13-214100ED+T12-519100ED+P12-185492ED+N+T13-210100ED+P+T14-414100BCM-3963 Vehicle1511--0N15-417100T1416--0P1319--0N+T19-614100P+T1617--0 Conclusions: Our findings establish the preclinical efficacy of combining N with T for HER2+ breast cancer and warrant further clinical testing to investigate the efficacy of N+T without chemotherapy in the neoadjuvant setting for patients with HER2+ breast cancer. Citation Format: Veeraraghavan J, Sethunath V, Qin L, Shea MJ, Mitchell T, De Angelis C, Nanda S, Diala I, Lalani AS, Hilsenbeck SG, Rimawi MF, Osborne CK, Schiff R. Neratinib in combination with trastuzumab is superior to each alone and to pertuzumab plus trastuzumab in HER2-positive in vivo breast cancer models [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 P6-17-12.

Abstract P4-15-05: Biomarkers of response to neoadjuvant endocrine therapy with anastrozole (Ana) alone or in combination with fulvestrant (Ful) in ER-positive (ER+) HER2-negative (HER2-) breast cancer (PACT01 trial)

Abstract Background: In recent years, several clinical trials showed that fulvestrant (Ful), alone or in combination with an aromatase inhibitor (AI), is more effective than an AI alone. PACT01 is a randomized neoadjuvant trial of Anastrazole (Ana) alone or in combination with Ful in ER+/HER2- breast cancer. Methods: Patients with newly diagnosed ER+/HER2- breast cancers, 2 cm or larger in size, were randomized to 16 weeks of Ana (1 mg orally every day) alone or in combination with Ful (500mg IM days 1, 15, 29, and every 28 days thereafter) for 16 weeks. Patients then proceeded to surgery. Tumor tissue was collected at baseline, day 28 (D28), and at the time of surgery. Primary endpoint was the reduction of Ki67 in tumor tissue between baseline and D28. Baseline and D28 samples were stained for ER, PR, HER2, and Ki67. ER and PR were scored for intensity and percentage (H-score), HER2 was scored for intensity of membrane staining; and Ki67 was scored as percentage. Data were summarized descriptively. Changes in biomarkers from baseline to D28 were calculated and compared by Wilcoxon signed rank test. Results: PACT01 trial enrolled 72 patients. Three of them did not start treatment. Baseline samples were collected from the remaining 69 patients, and D28 samples from 60 patients (5 refused, 2 withdrew, 1 lost to follow up, 1 unknown). Samples from 18 patients had no tumor (5 at baseline, 9 at D28, 4 at both). Of the 42 patients with paired samples, 20 received Ana and 22 received Ana+Ful. All cases except one were centrally confirmed to be ER+, and all were HER2-. Table 1 summarizes median expression of Ki67, ER, and PR. Both treatment regimens led to a significant reduction in Ki67 between baseline and D28. However, Ana+Ful did not reduce Ki67 more effectively than Ana alone. Ki67 was reduced to <10% in 60% of the Ana arm and 68% of the Ana+Ful, which was not statistically significant.PR was similarly reduced in both treatment arms. ER was significantly reduced at D28 in the Ana+Ful arm (p=0.0004) but not in the Ana alone arm. Safety profile of both treatment arms was consistent with package insert and published studies. Median expression of Ki67, ER and PR in Anastrazole and Anastrazole + Fulvestrant Arms at Baseline and Day 28ARMTimepointNKi67 (%)ER H-scorePR H-scoreAnaBaseline2024.8182.5100.3 Day 28205.6*170.025.0Ana + FluBaseline2225.6198.120.5 Day 28225.1*117.50.0* p=0.0004. Other comparisons were not stastistically significant Conclusions:In this small neoadjuvant trial, the addition of Ful to Ana did not increase Ki67 suppression at D28. This may be due to untreated primary tumors being exquisitely sensitive to Ana and that fulvestrant may not add to it. It is also possible that the effect of Ful may be noted later in the course of treatment. Further biomarker data on tissue collected at the end of treatment will be presented at the meeting. Citation Format: Dhamne S, Nagi C, Wang T, Pavlick AC, Reusser B, Schiff R, Julie N, Niravath P, Silberfein EJ, Sedgwick EL, Sepulveda KA, Gutierrez C, Hilsenbeck SG, Chang JC, Osborne CK, Rimawi MF. Biomarkers of response to neoadjuvant endocrine therapy with anastrozole (Ana) alone or in combination with fulvestrant (Ful) in ER-positive (ER+) HER2-negative (HER2-) breast cancer (PACT01 trial) [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 P4-15-05.

Abstract P6-09-02: Effects of palbociclib on thymidine kinase-1 (TK1) in hormone receptor positive (HR+) breast cancer cell lines

Abstract Introduction: TK1 plays a crucial role in DNA synthesis and is a well-established marker of cell proliferation. We and others have previously described the potential role of TK1 activity (TKa) as predictive biomarker of response to endocrine therapy in HR+/HER2 negative metastatic breast cancer patients. TK1 synthesis is regulated by the E2F pathway, the target pathway of CDK4/6 inhibitors, and TKa has recently been reported as a potential circulating pharmacodynamic marker of CDK4/6 inhibition in breast cancer. However, modulations of TK1 levels and activity during palbociclib treatment and in the development of treatment resistance are unknown. Here, we report how TK1 expression and TKa are modulated in response to palbociclib in a panel of HR+ breast cancer cell lines: both palbociclib-sensitive (PDS) and with acquired resistance to (PDR). Material and methods: We used a panel of 7 PDR HR+ breast cancer models previously developed in our lab via chronic exposure of parental cells (MCF7, T47D, ZR75-1, BT474, MDAMB361 and two MCF7 endocrine resistant derivatives) to escalating doses of palbociclib, from a Starting Treatment Concentration (STC) of 50 nM or 350 nM according to the cell line, up to 1 μM. We analyzed gene expression profiles of PDS cells treated with drug vehicle (DMSO) as a control or palbociclib at STC for 3 days, and PDR cells grown with palbociclib 1 μM. Cell proliferation was assessed by methylene blue assay in MCF7 and BT474 PDS and PDR treated for 3, 6 and 9 days with DMSO, palbociclib STC and 1 μM. In parallel, TKa was measured in cell lysates at 3 days of treatment using the DiviTumTM assay (Biovica, Sweden). Results: Among E2F target genes, gene expression data demonstrated that TK1 was one of the most differentially expressed genes between PDR and PDS treated cells. In PDS cells compared to control, treatment with palbociclib resulted in reduced TK1 expression, with the HER2 positive models (BT474 and MDAMB361) showing the highest reduction. In PDR cells, TK1 expression was higher, but remained slightly inhibited compared to untreated PDS cells. TKa was significantly reduced in PDS cells treated with palbociclib for 3 days compared to vehicle (p<0.05). TKa response to palbociclib was more dramatic in BT474 cells as compared to MCF7. As expected, palbociclib inhibited cell proliferation in PDS models, with a significant reduction observed only after 6 days of treatment, suggesting that TKa may be an early marker of growth inhibition in response to palbociclib. No significant alterations in TKa were observed in PDR cells, at any dose of palbociclib. Similarly, proliferation rate was not affected by palbociclib in PDR cells. Conclusions: TK1 expression and activity are regulated by palbociclib in HR+ breast cancer cell lines, particularly in HER2 positive models. Ongoing studies of TKa in patients treated with palbociclib will assess the role of TKa as a circulating biomarker for predicting and monitoring response to CDK4/6 inhibitors. Citation Format: Bonechi M, Migliaccio I, Benelli M, Romagnoli D, Bergqvist M, Mattsson K, Boccalini G, Capaccioli G, De Luca F, Galardi F, Biagioni C, Risi E, McCartney A, Rossi L, Osborne CK, Schiff R, De Angelis C, Guarducci C, Di Leo A, Malorni L. Effects of palbociclib on thymidine kinase-1 (TK1) in hormone receptor positive (HR+) breast cancer cell lines [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 P6-09-02.

Abstract PD6-02: A randomized, controlled trial of high dose vs. standard dose vitamin D for aromatase inhibitor-induced arthralgia in breast cancer survivors

Abstract Background: Approximately half of women on aromatase inihbitor (AI) therapy develop AI-induced arthralgia (AIA), and many discontinue the medication because of this common side effect. While Vitamin D has been studied as a treatment for AIA, trial results have been conflicting thus far. Patients and Methods: All subjects were post menopausal women who were beginning adjuvant AI therapy for stage I-III hormone receptor positive breast cancer. Patients were randomized 1:1 to receive standard dose vitamin D3 (800 IU daily for 52 weeks) or high dose vitamin D3 (50,000 IU weekly for 12 weeks, followed by 2000 IU daily for 40 weeks). All patients also took oral calcium 600 mg daily. The primary endpoint was development of AIA, as defined by pre-specified changes in the Health Assessment Questionnaire II (HAQ-II). Secondary endpoints include compliance with AI therapy, and correlation between grip strength and development of AIA. Exploratory endpoint was measurement of inflammatory cytokine reduction in each arm. The trial was designed to enroll 184 patients, but this futility analysis was performed after 93 patients were enrolled. The futility boundary for stopping the trial early was calculated as p = 0.47. Results: All 93 patients (46 in the high dose arm, and 47 in the standard dose arm) enrolled in the study at the time of the interim analysis were evaluable. The HAQ-II was completed at 12 weeks in 76% on the high dose arm, and 68% in the standard dose arm. Subjects who did not complete the questionnaire were deemed as study failures (i.e. development of AIA was assumed). In the high dose arm, 25 patients (54%) developed AIA, compared to 27 patients (57%) in the standard dose arm. The one-tailed p value is 0.3818, and the Z-score is 0.3, yielding only a 38% conditional power that that study would find a significant difference between the two arms. Thus, the study was terminated early for futility. There was no significant difference between the two arms in adherence to AI therapy. The grip strength and inflammatory cytokine data are pending at this time. They will be ready by the time of the conference. Conclusions: There was no significant signal for benefit of high dose vitamin D supplementation, as compared to standard dose vitamin D, for AIA prevention in post menopausal women taking adjuvant AI therapy. These results further characterize the role of Vitamin D in AIA, and they inform future clinical trials in this arena. Further research is necessary, as this remains an important cause of non-adherence to this highly effective therapy. Citation Format: Niravath P, Wang T, Hilsenbeck SG, Lipscomb K, Pavlick A, Jiralerspong S, Nangia J, Ellis M, Ademuyiwa F, Cherian M, Frith A, Ma C, Park H, Rigden C, Suresh R, Osborne CK, Rimawi MF. A randomized, controlled trial of high dose vs. standard dose vitamin D for aromatase inhibitor-induced arthralgia in breast cancer survivors [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 PD6-02.

Abstract P2-02-14: Metabolic regulation and drug resistance in c-Src activated triple negative breast cancer

Abstract c-Src (Src) is a proto-oncogene involved in signaling that culminates in the control of multiple biological functions. Src is also one of the most frequently upregulated pathways in triple negative breast cancer (TNBC). Dysregulation of Src has been detected in TNBC and is strongly associated with tumor metastasis and poor prognosis. However, even after promising preclinical studies, Src inhibitors did not show major clinical advantage in unselected TNBC populations. We have previously published that metastatic TNBC has high energy-dependency to mitochondrial fatty acid beta-oxidation (FAO) and FAO activates Src by inducing autophosphorylation at Y419. However, our recent analysis suggests that as observed with the Src inhibitors, TNBC tumors treated with FAO inhibitors also develop drug-resistance and continue tumor growth. Evaluation of their drug resistance mechanism revealed that while short-term inhibition of FAO or Src induces autophagic and apoptotic cell deaths, long-term inhibition results in autophagy-mediated drug resistance and survival. Further analyses suggest that FAO and Src inhibitors activate mitogen-activated protein (MAP) kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway via the induction of cellular reactive oxygen species (ROS) in TNBC. Activated MEK/ERK then induces survival pathways for drug resistance and tumor survival. Validation of in vitro findings using in vivo TNBC models confirmed that combination of FAO/Src inhibitors with MEK/ERK inhibitors can provide significant benefit to overcome the therapeutic resistance of TNBC. These findings open-up new therapeutic opportunities to manage TNBC patients with currently non-targetable metastatic tumors. Citation Format: Jung KH, Park JH, Sirupangi T, Jia D, Gandhi N, Pudakalakatti S, Elswood J, Porter W, Putluri N, Zhang XH-F, Chen X, Bhattacharya PK, Creighton CJ, Lewis MT, Rosen JM, Wong L-JC, Das GM, Osborne CK, Rimawi MF, Kaipparettu BA. Metabolic regulation and drug resistance in c-Src activated triple negative breast cancer [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 P2-02-14.

Abstract P2-09-12: Independent validation of the HER2-enriched subtype as a predictor of pathological complete response following trastuzumab and lapatinib without chemotherapy in early-stage HER2-positive breast cancer

Abstract Background: HER2-positive breast cancer consists of 4 intrinsic molecular subtypes (Luminal A, Luminal B, HER2-enriched [HER2-E], and Basal-like) and a Normal-like subtype, with the HER2-E subtype having the highest activation of the EGFR-HER2 pathway. Concordant with this, the HER2-E subtype was significantly associated with pathological complete response (pCR) following lapatinib and trastuzumab without chemotherapy on the PAMELA phase II neoadjuvant trial (Lancet Oncol 2017). Here, we aimed to further validate this observation in a different cohort using tumor samples from the TBCRC023 trial. Methods: TBCRC023 (NCT00999804) was a randomized phase II trial combining a Simon Phase 2 design in the experimental arm with a pick-the-winner design, not powered for direct comparison. Ninety-seven women with HER2+ breast cancer measuring 2 cm or larger (median = 5 cm) were randomized in a 1:2 ratio to 12 vs. 24 weeks of lapatinib and trastuzumab. Letrozole (along with ovarian suppression if premenopausal) was added in patients whose tumors were also estrogen receptor (ER)-positive. All evaluable patients were assessed for pCR, defined as no residual invasive carcinoma in the breast. Intrinsic molecular subtypes from tumor biopsy formalin-fixed, paraffin-embedded samples taken at baseline (day 0) were determined with the nCounter-based PAM50 predictor. Gene expression PAM50 data was performed at Hospital Clínic in Barcelona blinded from clinical data. The primary outcome was the association of the HER2-E subtype (vs. non-HER2-E) with pCR. A logistic regression model adjusted for tumor size, ER status, nodal status and treatment arm was performed. Results: A total of 85 of the 97 (87.6%) baseline tumor samples were available. Most patients had the HER2-E subtype (51 [60.0%]), followed by Normal-like (12 [14.1%]), Basal-like (11 [13.0%]), Luminal B (7 [8.2%]) and Luminal A (4 [4.7%]). The proportion of patients with HER2-E tumors within ER+ and ER-negative disease was 54.9% and 67.7%, respectively. At the time of surgery, 17 of 85 patients (20.0%; 95% confidence interval [CI] 0.13-0.30) had a pCR in the breast. Fourteen of 51 patients with the HER2-E subtype (27.5%; 95% CI 0.17 to 0.41) and 3 of 34 patients with non-HER2-E subtypes (8.8%; 95% CI 0.03 to 0.23) achieved a pCR at the time of surgery (adjusted odds ratio 4.33; 95% CI 1.08-17.41; P=0.039). No other clinical-pathological variable was found significantly associated with pCR in the multivariable model. Conclusions: In an independent validation study performed while blinded to clinical outcomes, HER2-E subtype confirms its ability to identify patients with HER2-positive breast cancer who are likely to benefit from dual HER2 blockade therapies without chemotherapy. Further studies should be performed to prospectively validate this biomarker, alone or in combination with other biomarkers. Citation Format: Prat A, De Angelis C, Pascual T, Gutierrez C, Wang T, Paré L, Rexer B, Cortes J, Forero A, Llombart A, Wolff AC, Krop I, Galván P, Pavlick AC, Villagrasa P, Hilsenbeck SG, Schiff R, Osborne CK, Rimawi MF. Independent validation of the HER2-enriched subtype as a predictor of pathological complete response following trastuzumab and lapatinib without chemotherapy in early-stage HER2-positive 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 P2-09-12.

Abstract P4-04-03: Hyperactive FOXA1 activates super-enhancer-engaged HIF2α/EPAS1 to promote endocrine-resistant metastatic ER-positive breast cancer

Abstract Background: We have recently reported that acquired endocrine resistance (Endo-R) in multiple ER+ breast cancer (BC) Endo-R cell models is driven by high levels of FOXA1 (High-FOXA1), via gene amplification and/or overexpression (OE), leading to coordinated reprogramming of the FOXA1 genomic binding (cistrome) and transcriptome. Forced FOXA1 OE in parental (P) cells induced similar transcriptional reprogramming leading to Endo-R and metastasis. Recent clinical data showing enrichment of FOXA1 amplification in ER+ metastases further support the clinical importance of our findings. However, the molecular components and the mechanism of High-FOXA1-induced transcriptional reprogramming in Endo-R and metastasis are unknown. Methods: High-FOXA1-containing MCF7 tamoxifen-resistant (TamR) and P/FOXA1-OE cells were used in this study. An integrative multi-OMICS approach was employed to analyze transcriptome (RNA-seq), FOXA1 cistrome, and histone H3K27 acetylation (ac) (ChIP-seq). Intersection of High-FOXA1-induced transcriptome and distinct FOXA1 cistrome-predicted genes defined a High-FOXA1 core gene signature (CGS). Gene Set Enrichment Analysis (GSEA) and Gene Ontology (GO) were used for functional annotation. Cell growth and migration/invasion were measured by a bright-field automated cell counter and Transwell insert system. Altered gene expression was measured by RT-qPCR. High-FOXA1 signaling inhibition included gene knockdown (siRNA) or pharmacologic blockade (the EPAS1 inhibitor PT2385). The predictive role of EPAS1 and the associated gene signature were analyzed using publicly available BC datasets. Results: FOXA1 OE reprogrammed the FOXA1 cistrome in P cells to resemble that of the TamR cells. The FOXA1 cistrome was significantly correlated with the deposition of H3K27ac in TamR vs. P cells (P<2.2e-16). Similarly, the differentially expressed genes in TamR vs. P cells were enriched for FOXA1 binding at enhancers demarcated by H3K27ac (P=8e-125). The FOXA1-CGS was linked to multiple metastasis-related GO terms including “hypoxia response”, enriched for the cancer secretome gene set (P=4.1e-16), and highly represented in the Endo-R transcriptome across our multiple cell models (MCF7, 600MPE, and CAMA1) (P<0.01). Integrative analysis of H3K27ac-defined super-enhancers (SEs) and altered cistrome/transcriptome upon High-FOXA1 nominated EPAS1, a hypoxia-inducible transcription factor (TF), as a top candidate of SE-activated TFs amplifying High-FOXA1 signaling. EPAS1 blockade markedly repressed the secretome genes (e.g., IL8 and S100P) and cell migration and invasion in TamR cells. Primary ER+ tumors (TCGA) with high EPAS1 are enriched for a cancer secretome gene set (P=3e-4). High EPAS1 predicts poor distant metastasis-free survival in ER+ BC treated with endocrine therapy (P=.034). Conclusions: High-FOXA1 induces transcriptional reprogramming by coordinating histone enhancer marks to activate EPAS1 via an SE mechanism, which in turn mediates transcriptional reprogramming, partly via inducing a pro-metastatic secretome, to promote Endo-R and metastasis. Targeting the High-FOXA1/EPAS1 axis to block transcriptional reprogramming may offer a new therapeutic strategy to prevent and treat Endo-R metastatic ER+ BC. Citation Format: Fu X, Pereira R, De Angelis C, Veeraraghavan J, Shea MJ, Nanda S, Feng Q, Jeselsohn R, O'Malley BW, Brown M, Osborne CK, Schiff R. Hyperactive FOXA1 activates super-enhancer-engaged HIF2α/EPAS1 to promote endocrine-resistant metastatic ER-positive 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 P4-04-03.

Abstract PD2-04: FOXA1 induces a pro-metastatic secretome through ER-dependent and independent transcriptional reprogramming in endocrine-resistant breast cancer

Abstract Background: Metastasis in ER-positive (+) breast cancer (BC) occurring years to decades after initial diagnosis presents a daunting challenge for clinical care and preclinical research due to limited known key players and experimental models. FOXA1 is a pioneer factor for ER-chromatin binding and function, and is highly expressed in ER+ BC metastases, yet the underlying mechanism is unclear. Tumor-secreted proteins play a crucial role in the reciprocal interplay between cancer cells and host microenvironmental factors at both primary and secondary sites. We hypothesized that high FOXA1 provokes an ER-dependent transcriptional program that includes a unique pro-tumorigenic secretome essential for promoting ER+ BC metastasis. Methods: A lentiviral doxycycline (Dox)-inducible FOXA1 overexpression vector and a dual luciferase/GFP (LG) tracking vector were integrated to construct a stable MCF7-LG/FOXA1 cell model. Ovariectomized nude mice bearing MCF7-LG/FOXA1 xenografts in the presence of exogenous estrogen (E2) were randomized to ± Dox, each with continued E2, E2 deprivation (ED), or tamoxifen (Tam). Survival surgery removing the therapy-naïve (E2 arm) and relapsed (ED/Tam arms) tumors was performed when tumors reached ∼1000 mm3. All mice then received ED/Tam 'adjuvant' therapy, with longitudinal luminescence imaging to monitor local/distant recurrences. Mice were or will be euthanized at the ethical end-point. Integrative bioinformatics was performed using RNA-seq and FOXA1/ER ChIP-seq data from our preclinical models to identify secretome targets for functional intervention. Times to tumor regression (TTR) and progression (TTP) were defined by when the tumor reached half or twice the volume at randomization. Results: Median (m) TTR was achieved in ED (31/34 days, -/+Dox, P = 0.184) but not in Tam groups — Tam delayed tumor growth but failed to prevent progression in all mice with mTTP of 94/93 days (-/+Dox, P = 0.517). Despite no difference in mTTP at Tam-/+Dox, a quarter of +Dox tumors (3/12) had volume doubled by day 11. No metastases were observed by imaging in any of the mice before surgery ('neoadjuvant' setting). Local relapse and lymph-node/lung metastases were detected after surgery ('adjuvant' setting). At day 90 in the adjuvant Tam group with previously relapsed tumors, +Dox mice succumbed to metastasis more often than -Dox mice (7/8 vs. 3/10, P = 0.023). Compared to the adjuvant Tam+Dox mice with previous therapy-naïve tumors, the Tam+Dox with previously relapsed tumors showed higher distant metastasis rate (7/8 vs. 5/14, P = 0.026). Analysis of the ED setting is pending due to late recurrence. Data integration and functional study revealed a set of cytokines, growth factors, and extracellular matrix components (including IL-8, CTGF, and LOX), regulated by FOXA1 often in conjunction with ER, that are highly involved in FOXA1-induced metastasis. Global secretome profiling by mass spectrometry and target validation are ongoing. Conclusions: FOXA1 overexpression increases metastatic potential in ER+ BC. We established a pertinent metastatic xenograft mouse model to characterize a pro-metastatic secretome with diagnostic and therapeutic potential for treating metastatic ER+ BC. Citation Format: Fu X, Pereira R, Zhao D, Jung SY, Jeselsohn R, Creighton CJ, Shea M, Nardone A, Angelis CD, Tsimelzon A, Wang T, Gutierrez C, Huang S, Edwards DP, Rimawi MF, Hilsenbeck SG, Brown M, Chen K, Osborne CK, Schiff R. FOXA1 induces a pro-metastatic secretome through ER-dependent and independent transcriptional reprogramming in endocrine-resistant breast cancer [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 PD2-04.

Abstract S4-01: A novel BRD4 inhibitor enhances endocrine therapy efficacy and circumvents endocrine-resistance in estrogen receptor-positive breast cancer models

Abstract Background: The selective estrogen receptor (ER) down-regulator (SERD) fulvestrant (Ful) antagonizes ER activity and degrades ER protein in a dose-dependent manner in the preclinical and clinical settings, though its efficacy is limited by an incomplete abolition of ER protein levels. Therefore additional strategies are needed to achieve a more complete suppression of ER level and activity. The bromodomain-containing protein 4 (BRD4), a member of the BET family that is required both for ESR1 gene expression and for ER-mediated gene transcription, represents an attractive therapeutic target for ER+ breast cancer (BC). Here, we investigated the efficacy of the novel BRD4 inhibitor GS-626510 (GS-6510) in a panel of ER+ BC parental and endocrine-resistant (EndoR) cell lines and in a patient-derived xenograft (PDX) model. Materials and Methods: The effects of GS-6510 (25nM – 290nM) alone or in combination with endocrine therapies were tested in ER+ MCF7, T47D, and ZR75-1 cell lines, as well as in their derivatives made resistant to estrogen (E2) deprivation (EDR), tamoxifen (TamR), or Ful (FulR). Cell growth (by methylene blue) after 6 days of GS-6510 and protein levels (by Western blot) after 2 days of GS-6510 were assessed. The in vivo efficacy of GS-6510, Ful, and the combination was tested in the ER+ HCBx34 PDX model. The mRNA levels of genes associated with cell cycle, ER signaling, and endocrine-resistance were assessed using the NanoString platform. Results: A dose-dependent inhibitory effect of GS-6510 was observed in all of the experimental settings. At a clinically relevant dose, GS-6510 reduced E2-stimulated cell growth and enhanced the efficacy of endocrine therapies in all parental cell lines. In the endocrine-sensitive HCBx34 PDX model, GS-6510 reduced tumor growth and, in combination with Ful, induced tumor regression and inhibited the expression of ER-dependent and cell cycle related genes including CCND1, MYC, and BCL2. Notably, the addition of GS-6510 to EndoR cell models that continue to rely on and express ER (MCF7 EDR and TamR) led to a substantial inhibition in cell growth (85%; 98%, respectively). Though the combination of GS-6510 and Ful was not associated with a significantly greater cell growth inhibition compared to GS-6510 alone in these models, a better suppression of ER levels was observed. Interestingly, GS-6510 also remained effective in EndoR models that exhibited an ER-independent growth, including all FulR lines, though its efficacy varied among the different cell lines and resistant derivatives. Conclusion: Our findings suggest that the epigenetic regulator BRD4 is a suitable target for therapeutic intervention in ER+ BC. The anti-tumor efficacy of GS-6510 in endocrine sensitive and especially in ER-dependent EndoR models is worthy of further clinical investigation. The growth inhibitory effects observed in some of the ER-independent EndoR models suggests that additional genes/pathways involved in endocrine resistance could be affected by GS-6510. Identifying these pathways and determining their predictive role are needed to guide patient selection for future clinical trials. Citation Format: De Angelis C, Nardone A, Cataldo ML, Fu X, Trivedi M, Yi S, Breckenridge D, Chamnsess GC, Vitorino P, Osborne CK, Schiff R. A novel BRD4 inhibitor enhances endocrine therapy efficacy and circumvents endocrine-resistance in estrogen receptor-positive breast cancer models [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 S4-01.

Abstract S5-02: Scalp cooling alopecia prevention trial (SCALP) for patients with early stage breast cancer

Abstract Background Adjuvant chemotherapy decreases the risk of breast cancer recurrence. However, it is associated with distressing side effects, including alopecia. Women consider chemotherapy-induced alopecia as one of the most severe side effects. In many countries, scalp cooling is used to prevent chemotherapy-induced alopecia. It causes cutaneous vasoconstriction, which reduces blood flow to hair follicles during peak plasma concentrations of the drug and may reduce cellular uptake of these agents. It also results in reduced biochemical activity, which makes hair follicles less susceptible to the damage of chemotherapy. There are no randomized trials assessing this approach with modern scalp cooling and success rates in non-randomized trials have varied. Methods We conducted a multi-center randomized non-blinded controlled prospective trial to evaluate the safety and efficacy of the Orbis Paxman Hair Loss Prevention System (OPHLPS) in reducing chemotherapy-induced alopecia. Women with stage I-II breast cancer planned to receive neoadjuvant or adjuvant anthracycline- or taxane- based chemotherapy for at least four cycles were eligible. Participants were randomized in a 2:1 ratio to scalp-cooling or no cooling. Scalp-cooling was done using the OPHLPS 30 minutes prior to, during and 90 minutes after each chemotherapy. The primary efficacy endpoints were hair preservation, defined as CTCAE 4 alopecia <2 (assessed by an independent and blinded evaluator), and device safety. We planned to enroll 235 patients to provide 85% power to detect a 20% difference in hair preservation (i.e. 15% with no cooling and 35% with scalp-cooling). Secondary endpoints included wig/scarf use and quality of life assessed by the EORTC QLQ-30, HADS and BIS. Participants will be followed for 5 years post-study for time to first recurrence, overall survival, site of first recurrence, and incidence of isolated scalp metastasis. One interim analysis was planned to allow the study to stop early for efficacy (superiority) after 95 and 47 patients were enrolled to cooling or no cooling, respectively, and have been evaluated for the primary endpoint. To maintain the overall type 1 error rate, an O'Brien-Fleming spending function has been used to set the superiority boundaries (interim boundary was calculated as p=0.0061 (or Z=2.509)). Results This is the first prospective randomized trial with modern scalp cooling in the world. At the time of the interim analysis, 95 patients in the cooling group and 47 patients in the no cooling group were evaluable and had completed 4 cycles of chemotherapy. Among them, 48 (50.5%) out of 95 in the cooling group and 0 (0%) out of 47 in the no cooling group had hair preservation. The one-tailed p-value from the Fisher's exact test is <0.0001, which crosses the superiority boundary (p=0.0061). Thus, on 9/26/2016, the DSMB decided to stop the study early and release the results. Our trial shows that scalp cooling using OPHLP is highly effective in hair preservation and should become available for patients who receive chemotherapy for early breast cancer. These data and secondary endpoint analysis data will be presented. We are seeking FDA approval for the OPHLPS. Citation Format: Nangia JR, Wang T, Niravath PA, Otte K, Osborne CR, Papish S, Holmes FA, Abraham J, Goldfarb S, Courtright JG, Paxman RJ, Rude MB, Hilsenbeck SG, Osborne CK, Rimawi MF. Scalp cooling alopecia prevention trial (SCALP) for patients with early stage breast cancer [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 S5-02.

Abstract P6-02-07: Metabolomic analysis by nuclear magnetic resonance spectroscopy discriminates hormone receptor positive/HER2 negative breast cancer cell lines resistant to palbociclib

Abstract Introduction: Clinical trials of palbociclib in combination with endocrine therapy have recently shown unprecedented activity for the treatment of hormone receptor positive/HER2 negative (HR+/HER2neg) advanced breast cancer. However, de novo and acquired resistance to palbociclib limit its clinical utility. Moreover, combining palbociclib with endocrine therapy increases toxicity and costs of the treatment. Identifying patients more likely to benefit from this compound and understanding the mechanisms of resistance to palbociclib is critical. In this study we investigated whether metabolomic profiles of breast cancer cell lines with acquired resistance to palbociclib (PDR) differ from their sensitive counterpart (PDS). In addition we sought to identify metabolic biomarkers of sensitivity to palbociclib by analyzing a breast cancer cell line unable to acquire resistance to palbociclib. Material and methods: We have established in our lab three PDR HR+/HER2neg breast cancer models (MCF7L, T47D and ZR75-1) by chronically exposing cells to escalating doses of palbociclib. PDR derivatives show IC50 values 6 to 30 times higher than their PDS counterparts. One additional model, CAMA-1, was unable to develop resistance. Whole-cell lysates and conditioned cell culture media from five replicates of each of the PDS and PDR models and from CAMA-1 were analyzed by nuclear magnetic resonance (NMR). Principal component analysis (PCA) was used as first exploratory analysis and as dimension reduction technique. Canonical Analysis (CA) was used to discriminate different groups. Differentially expressed metabolites between PDR and PDS models and between CAMA-1 and PDS cells were analyzed. Results: Unsupervised PCA analyses of H NMR spectra, in which no information about PDS and PDR was inserted in the statistical model, correctly identified individual cell lines on both whole-cell lysates and conditioned media. However this analysis did not discriminate PDS from PDR within each model. Using a supervised approach, in which the statistical model was trained to discriminate between PDS and PDR, these groups were categorized with accuracy of 80% using whole-cell lysates and of 65% using conditioned media, using a cross-validation analysis by repeatedly testing the model on blind samples. CAMA-1 was correctly identified as a PDS model; however it showed a distinct metabolic profile compared to other PDS models. Over 30 metabolites were identified as differentially expressed between PDS and PDR models in lysates and conditioned media, but only glycerophosphocholine levels in conditioned media remained significantly higher in PDR compared to PDS models after correction for multiple testing. Conclusions: In this study we show that analysis of metabolic profile of cells lysates discriminates PDR from PDS cell lines with a high accuracy. Analysis of metabolic pathways implicated in resistance/sensitivity to palbociclib is ongoing and might help identifying new targets to overcome resistance. Additionally, metabolites associated with palbociclib resistance may be potentially tested in clinical samples as biomarkers for patients stratification. Further studies are warranted. Citation Format: Bonechi M, Guarducci C, Meoni G, Tenori L, Biagioni C, Schiff R, Osborne CK, Luchinat C, Di Leo A, Malorni L, Migliaccio I. Metabolomic analysis by nuclear magnetic resonance spectroscopy discriminates hormone receptor positive/HER2 negative breast cancer cell lines resistant to palbociclib [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 P6-02-07.

Abstract P3-04-07: The new oral SERD AZD9496 is efficacious in antagonizing ER and circumventing resistance to endocrine therapy

Abstract Background: The selective estrogen receptor (ER) degrader (SERD) fulvestrant (Ful) is a potent ER antagonist that upon binding to ER induces its degradation. Ful has shown clinical efficacy in metastatic disease upon progression on previous endocrine therapies and superior activitycompared to an aromatase inhibitor as first line therapy when given at a high dose, 500mg. However, major clinical limitations of Ful are its low bioavailability and its route of administration. Here, we assess the efficacy and the mechanism of action of the new oral SERD AZD9496 compared to Ful in our panel of endocrine-sensitive and -resistant (EndoR) in vitro and in vivo models. Methods: The effects of AZD9496 and Ful were studied in vitro in various ER+ MCF7, ZR75-1, T47D, 600MPE, and MDAMB415 parental lines and in MCF7 and T47D derivatives made resistant (R) to estrogen deprivation (ED), tamoxifen (Tam), or Ful. Cell growth, Western blot, Q-RT-PCR, and ERE-reporter assays were conducted to assess treatment efficacy as well as ER levels and activity. Xenografts of parental MCF7 cells were established in ovariectomized nude mice with exogenous estrogen (E2). Mice were then randomized to continued E2 or ED, with and without AZD9496 or Ful. Mice bearing transplantable MCF7 EDR and TamR xenografts were randomized to continue original treatment or to switch to Ful or AZD9496, and tumor size was followed. Expression of classic and nonclassic/indirect ER-regulated genes was evaluated in RNA extracts of short-term-treated xenografts using the BioMark FLUIDIGM platform. Results: AZD9496 inhibited cell growth (50-100%) of all ER+ parental cells and greatly, though not fully, degraded ER protein levels. AZD9496 also potently reduced ER-dependent exogenous and endogenous gene/protein expression in presence and absence of E2. In parental MCF7 xenograft-bearing mice, 10 days of AZD9496 resulted in a greater inhibition of tumor growth and in a greater reduction of levels of ER-dependent targets in comparison to Ful in the presence of E2. The effects of the 2 SERDs were similar in the absence of E2. In EndoR models that retain ER, AZD9496 inhibited cell growth in vitro by degrading ER, similar to Ful. Both SERDs also delayed tumor growth of EDR and TamR xenografts and effectively reduced levels of ER and ER-induced proteins, though no tumor regression was observed in the TamR model. Notably, AZD9496 failed to inhibit growth of FulR cells and xenografts. Expression analysis showed that the 2 SERDs potently inhibited classic ER activity, while simultaneously increasing expression of some genes known to be regulated by the nonclassic/indirect ER activity, including genes involved in escape pathways of endocrine resistance. Conclusions: The oral SERD AZD9496 is a potent antiestrogen that antagonizes and degrades ER. AZD9496, like Ful, inhibits ER-dependent transcription and tumor growth in both naïve and resistant EDR and TamR models, but shows cross-resistance in FulR models. Both AZD9496 and Ful failed to completely reduce ER protein expression and to induce TamR tumor regression, suggesting that additional strategies to reduce ER levels and to enhance the inhibition of ER signaling and/or of co-operating survival mechanisms may be needed to improve treatment outcome. Citation Format: Nardone A, Weir H, De Angelis C, Cataldo ML, Fu X, Shea MJ, Mitchell T, Trivedi M, Chamness GC, Osborne CK, Schiff R. The new oral SERD AZD9496 is efficacious in antagonizing ER and circumventing resistance to endocrine 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 P3-04-07.

Abstract PD7-07: Discovery of molecular predictors of late breast cancer specific events (BCSE) in ER+, node+ breast cancer – new transcriptome expression whole gene analysis of the phase III adjuvant trial SWOG S8814

Abstract BACKGROUND: Unique genes and pathways were identified for prognosis on tamoxifen (T, 5 yrs) and prediction on CAF-T vs T in S8814 using whole transcriptome RNA-Seq from archival FFPE tissue. (Albain, et al; Cherbavaz, et al; SABCS 2015) Discovery was robust for early DFS events but sparse for late. The aims of this new analysis were to 1) utilize a new endpoint BCSE for gene discovery of late events, prognosis and prediction and 2) add intronic counts to the previous exonic results to define whole genes impacting on late BCSE. METHODS: Charts of patients (pts) on CAF-T (212) vs T (142) were reviewed to define the BCSE endpoint (local/regional, contralateral, distant). Deaths without BC were treated as competing risks. BCSE models (including metagenes) of late prognosis and prediction used cumulative incidence functions. Consolidated intronic regions counts within genes were added to exonic regions counts. Using these “whole gene” (WG) counts, association of gene expression with time to BCSE was assessed by Cox regression. A multiple WG score (MWGS) for BCSE prognosis beyond 5 yrs (to 12.5 yrs) was constructed and evaluated for 1-3 and 4+ node (N) groups. False discovery rate was controlled at 10%. RESULTS: More exons and WG were discovered for prognosis on T alone over 12.5 yrs with the BCSE endpoint than DFS. For prognosis of late BCSE after 5 yrs, more genes were discovered using WG (n=111) than by exons (n=9). There were significantly fewer genes for late BCSE on CAF-T (8, WG; 0, exons). The functions of WG prognostic for late BCSE were: cell cycle/proliferation-26 genes, chromosome segregation/mitotic spindle-22, DNA repair/maintenance-10, transcriptional/translational control-5, cell adhesion/migration-4, immune-3, diverse/unknown-32 and growth factor/hormone receptor signaling-9 (this group was only found by WGs, not exons). Of these 111 WG, a MWGS prognostic for late BCSE on T used 57 previously discovered genes pre-specified for this analysis. Probability of BCSE beyond 5 yrs for low vs high MWGS was 8% vs 21% in N1-3+ and 17% vs 42% in N4+. Late prognosis on T differed by low vs high risk defined in a metagene model: cumulative BCSE at year 10 was 0% vs 47% (low vs high risk, p=0.001). Prediction of 10-yr incidence of BCSE varied by risk level by treatment in a metagene model: low risk- CAF-T=47%, T=0% (p=0.045); high risk- CAF-T=35%, T=45% (p=0.027). CONCLUSIONS: Gene discovery for prognosis of late BCSE is enhanced with a novel WG transcriptome expression approach. Use of chemotherapy (CT) before T significantly attenuated gene discovery, so that molecular tools for decisions on extending endocrine therapy (ET) may not be reliable in a setting of prior CT. Some pts on ET for 5 yrs may not require either longer ET or CT, given a N+ cohort was defined with no BCSE observed over 12.5 yrs. For prediction of CT benefit, CAF-T appeared to be inferior to T in a low risk metagene model for BCSE. In sum, these results add more evidence that ET alone may be sufficient (perhaps better) in select N+ settings. Validation in SWOG S1007 (RxPONDER) is planned. SUPPORT: NCI CA180888, 180819, 180821, 180820, 180863; in part, Genomic Health, Inc. Citation Format: Albain KS, Crager MR, Barlow WE, Baehner FL, Bergamaschi A, Rae JM, Ravdin PM, Tripathy D, Gralow JR, Livingston RB, Osborne CK, Ingle JN, Pritchard KI, Davidson NE, Carey LA, Cherbavaz DB, Sing AP, Shak S, Hortobagyi GN, Hayes DF. Discovery of molecular predictors of late breast cancer specific events (BCSE) in ER+, node+ breast cancer – new transcriptome expression whole gene analysis of the phase III adjuvant trial SWOG S8814 [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 PD7-07.

Abstract S3-06: A phase III trial evaluating pCR in patients with HR+, HER2-positive breast cancer treated with neoadjuvant docetaxel, carboplatin, trastuzumab, and pertuzumab (TCHP) +/- estrogen deprivation: NRG Oncology/NSABP B-52

Abstract Background: Preclinical evidence has shown that in xenograft models with estrogen receptor (ER)+/HER2+ breast cancer, signaling through the ER pathway can be enhanced in the presence of anti-HER2 treatment and lead to treatment resistance. Concurrent targeting of ER and HER2 has led to enhanced treatment efficacy and complete tumor disappearance. We hypothesized that targeting ER with endocrine therapy concurrently with chemotherapy plus dual HER2 inhibition will not be antagonistic and can overcome ER-mediated resistance and result in higher pCR as neoadjuvant treatment of ER+/HER2+ breast cancer. NRG Oncology/NSABP B-52 is a phase III, multicenter, randomized neoadjuvant therapy trial designed to determine whether the addition of estrogen deprivation to neoadjuvant therapy consisting of docetaxel, carboplatin, trastuzumab, and pertuzumab (TCHP+Est-Dep) yields a greater rate of pCR (breast and nodes) than TCHP alone. Methods: A total of 315 patients (pts) were randomly assigned between January 15, 2014 and March 17, 2016 to receive neoadjuvant therapy consisting of TCHP with or without estrogen deprivation therapy. Pts with locally advanced, hormone receptor-positive, HER2+ invasive breast cancer with no evidence of metastatic disease were eligible. Premenopausal women randomized to estrogen deprivation therapy received ovarian function suppression with goserelin (LHRH agonist) or equivalent plus an aromatase inhibitor (AI). Postmenopausal women received an AI. The determination of pCR (breast and nodes) was defined as the absence of any invasive component in the resected breast specimen and all resected lymph nodes following completion of neoadjuvant therapy. Following the intent-to-treat principle, the difference between the rates of pCR (breast and nodes) was tested using the binomial test for the difference between two proportions. The study was designed to have a statistical power of 80% to detect an increase in the pCR rate from 45% in the TCHP alone group to 60% in the TCHP+Est-Dep group. Results: The groups were balanced, with 57% clinically node positive and 50% premenopausal. Assessments for pCR were available from 308 of 315 randomized patients. The pCR (breast and nodes) for TCHP alone and TCHP+Est-Dep were 40.9% and 46.1%, respectively (p=0.36). The pCR (breast) were 44.2% and 47.4%, respectively (p=0.57). Grade 3/4 toxicities included diarrhea (23%, <1% vs 21%,0%) vomiting (8%, <1% vs 5%, 0%), and febrile neutropenia (5%, <1% vs 7%,1%) for TCHP vs TCHP plus estrogen deprivation. Conclusion: The addition of estrogen deprivation to neoadjuvant chemotherapy is not antagonistic. It improved pCR rates numerically, but the improvement was not statistically significant. The combination did not increase toxicity and may be a reasonable approach since all patients will receive endocrine therapy after neoadjuvant therapy. Correlative science studies including evaluation of residual cancer burden (RCB) and long-term outcomes will help define the role of estrogen deprivation in the treatment of HER2+ early breast cancer. Support: U10CA180868, -180822; UG1CA189867, Genentech. Citation Format: Rimawi MF, Cecchini RS, Rastogi P, Geyer, Jr CE, Fehrenbacher L, Stella PJ, Dayao Z, Rabinovitch R, Dyar SH, Flynn PJ, Baez-Diaz L, Paik S, Swain SM, Mamounas EP, Osborne CK, Wolmark N. A phase III trial evaluating pCR in patients with HR+, HER2-positive breast cancer treated with neoadjuvant docetaxel, carboplatin, trastuzumab, and pertuzumab (TCHP) +/- estrogen deprivation: NRG Oncology/NSABP B-52 [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 S3-06.

Abstract P1-01-01: Analytical validation of a standardized scoring protocol for Ki67: Phase-3 of an international multicenter collaboration

Abstract Aims: (i) To determine if between-pathologist agreement for Ki67 is adequate for clinical application, following a standardised scoring protocol. (ii) To compare between-pathologist agreement of scoring hot-spots vs a global method averaging Ki67 across each section. Background: The nuclear proliferation biomarker Ki67 has multiple potential roles in breast cancer, including aiding decisions based on prognosis, but has unacceptable between-laboratory variability. The International Ki67 Working Group has undertaken a systematic program to determine whether Ki67 measurement can be analytically validated and standardized across labs. In phase 1 variability in visual interpretation was the most important source of variability. Phase 2 showed that significant improvements in agreement could be achieved when scoring the same tumors on tissue microarrays by following clearly defined scoring methods. We now assess whether acceptable performance can be achieved on core-cut biopsies using a standardised method. Methods: Three adjacent sections from each of 30 primary ER+ breast cancers were centrally stained for Ki67 to assemble three sets of 30 stained tumor sections, circulated around 22 laboratories in 11 countries. Ki67 was scored by 2 methods by all labs: (a) global: 4 fields of 100 cells each were selected to represent any heterogeneity (b) hotspot: the field with highest Ki67 staining percentage was selected and 500 cells scored. Ki67 scores were log2-transformed for statistical analyses and back-transformed for presentation. The primary objective was to assess if either method could achieve an intraclass correlation coefficient (ICC) significantly greater than 0.8, considered substantial to almost-perfect agreement. Secondary objectives were to assess which method had highest observed ICC and to assess whether pathologists identified the same "hotspots". Results: The ICC for the global method was 0.88 (95%CI: 0.81-0.93) and therefore met the prespecified success criterion. The ICC for the hotspot method was 0.84 (95%CI: 0.77-0.92) and therefore had a CI which extended below the success criterion. Across the 22 labs, geometric mean value of the 30 scores ranged from 14.4 to 27.9 for the global method and from 17.4 to 40.2 for the hotspot method. The overall mean (95% CI) of these values was 19.8 (18.5-21.3) and 26.4 (24.6-28.3), respectively. Visually, there was moderately strong agreement in location of selected hotspot in the core-cuts across laboratories. The impact of variability of the Ki67 scores for estimating prognosis using the integrated IHC4 + clinical treatment score will be assessed. After selection of the areas to score, the median times for cell counting were 3 and 4 minutes for the global and hotspot methods, respectively. Conclusions: The global method met the prespecified criterion of success; it should now be evaluated for clinical validity in appropriate cohorts of samples. The hotspot method showed slightly less agreement between labs. The time taken for scoring is practical using counting software we are making publicly available. Establishment of external quality assessment schemes is likely to improve the agreement between labs further. (Supported by a grant from the Breast Cancer Research Foundation). Citation Format: Dowsett M, Leung SCY, Zabaglo L, Arun I, Badve SS, Bane AL, Bartlett JMS, Borgquist S, Chang MC, Dodson A, Enos RA, Fineberg S, Focke CM, Gao D, Gown AM, Grabau D, Gutierrez C, Hugh JC, Kos Z, Lænkholm A-V, Lin M-G, Mastropasqua MG, Moriya T, Nofech-Mozes S, Osborne CK, Penault-Llorca FM, Piper T, Sakatani T, Salgado R, Starczynski J, Viale G, Hayes DF, McShane LM, Nielsen TO. Analytical validation of a standardized scoring protocol for Ki67: Phase-3 of an international multicenter collaboration. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-01-01.

Abstract P5-07-01: Successful whole transcriptome analysis of 25-year-old breast tumor samples from the phase III trial SWOG-8814 by next generation sequencing (NGS): Standardized analytical methods for exploratory and validation studies

Abstract BACKGROUND: We previously reported that low 21 gene Recurrence Scores (RS) identify patients with ER-positive, lymph node-positive breast cancer who may not benefit from anthracycline-based adjuvant chemotherapy added to tamoxifen (SWOG-8814A NCI correlative science study; Albain et al. Lancet Oncol 2010). New exploratory and comprehensive quantitative analyses now permit whole transcriptome NGS on residual RNA extracted from FFPE blocks 12-18 years post-fixation. Herein, we report methodology details and feasibility results (see companion abstract, Albain et al., for clinical outcomes correlations). METHODS and RESULTS: Sequencing was carried out in Illumina HiSeq 2000 instruments, yielding 4.2 trillion data points. Messenger RNA expression was quantified using 3rd quartile normalization. Both Library (RNA) and Sequencing Standards showed high quality coverage as measured by median uniquely mapped reads over a 13 month window (168M and 182M, respectively, including duplicate reads). The median absolute deviation (MAD) of the relative log expression (RLE) of mapped reads for the Library and Sequencing Standards was 0.22 and 0.05, respectively. The Library Standard variation was greater than the Sequencing Standard, as library preparation was manual. Of 360 patient samples with sufficient RNA (≥ 100 ng total RNA), 354 (98.3%) were successfully sequenced and included in the final analysis data set. Average library yield was 39 ng/μL. Only 5 libraries failed yield requirements and one library failed expression quality metrics. The median insert length was 120 bp with the first and third quartiles 93 and 152 bp, respectively. After removal of duplicate reads, 82% of reads were uniquely mapped, and the median library size was 8.95M (number of unique mapped reads). Sequences with counts <10 for all 354 patients were excluded. The medians of the 1st, 2nd and 3rd quartiles for exons mapped to the RefSeq database were 20, 40 and 78 counts, and for introns, 72, 134 and 244 counts, respectively. The majority of exonic (86.7%) and intronic (95%) sequences were mapped. There were 20,101 RefSeq mapped exons with counts ≥10. Of these exons, 988 passed additional filtering criteria and were subjected to hierarchical clustering, with Gene Ontology and pathway analysis performed on selected gene expression patterns (for results, see companion abstract of Albain et al.). CONCLUSIONS: High quality whole transcriptome NGS is feasible from decades-old clinical trial FFPE specimens that have not been stored in any special fashion. Controlled laboratory, bioinformatics and biostatistics methods, with inclusion of appropriate process controls, ensure robustness and reliability of the NGS process. This in turn results in the discovery and validation of biologically and clinically relevant variations from prior landmark clinical trials. SUPPORT: NCI CA 180888, CA180819, CA180821, CA180820, CA180863; in part, Genomic Health, Inc. Citation Format: Cherbavaz DB, Hayes DF, Qu K, Crager MR, Barlow WR, Goddard AD, Beasley EM, Jeong J, Collin F, Liu M-L, Rae JM, Ravdin PM, Tripathy D, Gralow JR, Livingston RB, Osborne CK, Ingle JN, Pritchard KI, Davidson NE, Carey LA, Sing AP, Baehner FL, Hortobagyi GN, Shak S, Albain KS. Successful whole transcriptome analysis of 25-year-old breast tumor samples from the phase III trial SWOG-8814 by next generation sequencing (NGS): Standardized analytical methods for exploratory and validation studies. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-07-01.

Abstract OT3-02-08: Scalp cooling alopecia prevention trial (SCALP) for patients with early stage breast cancer

Abstract Background Adjuvant chemotherapy treats micro-metastatic disease and decreases the risk of breast cancer recurrence. However, it may be associated with distressing side effects, including alopecia. Women with breast cancer rate chemotherapy-induced alopecia as one of the most severe, troublesome, and distressing side effects of chemotherapy. In many countries, scalp cooling has been introduced to prevent or reduce chemotherapy-induced alopecia. The theory is that scalp cooling causes cutaneous vasoconstriction, which reduces blood flow to the hair follicles during peak plasma concentrations of the chemotherapeutic agents and therefore reduces cellular uptake of these agents. It also results in reduced biochemical activity, which makes hair follicles less susceptible to the damage of the chemotherapy agents. Historically success rates are have been variable, but based on non-randomized studies, scalp cooling appears to be effective in preventing chemotherapy-induced alopecia especially in more recent studies. Methods We are conducting a prospective multi-center randomized controlled non-blinded trial to evaluate the safety and efficacy of the Orbis Paxman Hair Loss Prevention System in reducing the incidence of chemotherapy-induced alopecia. Women with stage I-II breast cancer who will receive neoadjuvant or adjuvant anthracycline- or taxane-based chemotherapy, for at least four cycles are eligible. Participants are randomized in a 2:1 ratio to scalp-cooling or no cooling. Scalp-cooling is done using the Orbis Paxman Hair Loss Prevention System prior to, during and after each chemotherapy administration. The primary efficacy endpoints are hair preservation, defined as CTCAE v4 alopecia <2, and device safety. Two hundred and thirty five (235) patients are planned to be enrolled which will provide 85% power to detect a 20% difference in hair preservation, 15% in control group and 35% in scalp-cooling group . Secondary endpoints include: wig/scarf use and quality of life assessed by the EORTC QLQ-30, HADS and BIS. Study participants will be followed for 5 years post-study for time to first recurrence, overall survival, site of first recurrence, and incidence of isolated scalp metastasis. Citation Format: Nangia JR, Wang T, Rude M, Osborne C, Papish S, Abraham J, Holmes F, Savin M, Paxman R, Hilsenbeck SG, Osborne CK, Rimawi M. Scalp cooling alopecia prevention trial (SCALP) for patients with early stage breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr OT3-02-08.

Abstract S3-02: Molecular predictors of outcome on adjuvant CAF plus tamoxifen (T) vs T in postmenopausal patients (pts) with ER+, node+ breast cancer – Transcriptome expression analysis of the phase III trial SWOG-8814

Abstract BACKGROUND: In SWOG-8814A, pts with ER+ node+ breast cancer and low 21 gene recurrence scores (RS) had good prognosis and no CAF benefit, but high RS predicted longer survival from CAF followed by T (CAF-T) vs T (Albain, Lancet Oncol 2010). The aims of SWOG-8814B were to identify novel genes and networks for 1) prognosis of early and late relapse and 2) prediction of CAF benefit, using whole transcriptome expression analysis with next generation RNA sequencing (NGS). METHODS: Stored RNA previously extracted for SWOG-8814A (T, CAF-T arms; T, 5 yrs) was analyzed for RNA/library yield (see companion abstract Cherbavaz et al. for methods). Genes were sequenced and expression of mRNA species was related to disease-free survival (DFS) using Cox proportional hazards. Discovery analyses controlled false discovery rate (FDR) at 10%. Genes were identified for prognosis on T and prediction on CAF-T vs T. Networks of genes/pathways were explored. Early (0-5 yrs) and late (5-13+ yrs) time periods were studied. Gene Ontology, Cytoscape, pathway and hierarchical clustering were used for functional gene and metagene analyses. RESULTS: Of 367 samples, 354 (96%; 142 T, 212 CAF-T; 141 DFS events) had sufficient RNA/library yield, with 20,101 genes sequenced. For prognosis on T, there were 2327 and 568 genes discovered in early and all-yrs follow-up, with only 9 genes prognostic after 5 yrs. Prognosis analyses for residual risk after CAF-T were uninformative. Functional mapping found that genes prognostic for worse DFS were enriched for proliferation (G2M, M-phase), cellular metabolism, DNA repair, stress response and EMT; whereas, those with better DFS involved transcription regulation/repression via zinc finger proteins. Hierarchical clustering (T arm) found significant DFS prognostic metagene signatures for ER-related genes, immune response, ECM/stroma, chromatin remodeling-transcription factor activity and TGFb pathway. All varied for early vs late DFS events. For example, low ER/high stroma expression signatures correlated with high proliferation gene expression and were strongly associated with early events (standardized [st] HR 2.94, p<0.001). Late recurrence was associated with high proliferation, both individually (stHR 1.51, p=.035) and in combination with higher ER expression (stHR 1.51, p=0.09). Fifteen genes predicted CAF benefit (9 better DFS, 6 worse), or 129 genes if FDR relaxed to 20%. Cluster analysis for CAF prediction is ongoing. CONCLUSIONS: Unique genes, clusters and pathways were identified by NGS of archival material in ER+ N+ breast cancer, including previously unreported signatures. While ER, stroma and proliferation-related signatures were associated with early prognosis, proliferation best predicted worse DFS after 5 yrs. NGS of the primary tumor is most informative for early events in pts with only 5 years of T, with few genes selecting only for late relapse. If validated, these signatures may identify pts with excellent DFS despite positive nodes for endocrine therapy alone as well as others for whom chemotherapy and/or biologics are also required . SUPPORT: NCI CA 180888, 180819, 180821, 180820, 180863; in part, Genomic Health, Inc. Citation Format: Albain KS, Crager MR, Barlow WE, Baehner FL, Bergamaschi A, Rae JM, Ravdin PM, Tripathy D, Gralow JR, Livingston RB, Osborne CK, Ingle JN, Pritchard KI, Davidson NE, Carey LA, Cherbavaz DB, Sing AP, Shak S, Hortobagyi GN, Hayes DF. Molecular predictors of outcome on adjuvant CAF plus tamoxifen (T) vs T in postmenopausal patients (pts) with ER+, node+ breast cancer – Transcriptome expression analysis of the phase III trial SWOG-8814. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr S3-02.

The effect of trastuzumab therapy on clinical benefit from fulvestrant treatment for metastatic estrogen receptor-positive breast cancer patients.

155 Background: Overexpression of HER2/neu is associated with tamoxifen resistance in breast cancer (Osborne CK et al. J Natl Canc Inst 2003; 95:353-361). However pts may present with both estrogen receptor (ER) and HER2/neu + tumors. The benefit of adding fulvestrant to trastuzumab is unclear. The objective of the study was to determine the effect of trastuzumab on fulvestrant therapy. Methods: This was an IRB approved record review of patients (pts) from three medical oncologists with biopsy-proven ER+ metastatic breast cancer treated with fulvestrant who also had their primary tumor tested for HER2/neu. Demographic data collected included age at diagnosis, type and stage of cancer, original and metastatic ER, progesterone receptor (PR), and HER-2/neu biomarkers, and site(s) of metastasis, and primary local and systemic treatment. All pts with HER-2/neu + primary tumors received trastuzumab. The duration of fulvestrant therapy was calculated. Time to clinical disease progression on fulvestrant was measured as a surrogate for duration of clinical benefit. Results: Eighty-five metastatic ER+ fulvestrant treated breast cancer pts with known primary tumor HER2/neu status were identified and the duration of therapy calculated. All eleven (13%) pts with documented HER2/neu + primary tumors received trastuzumab. The duration of therapy for HER2/neu + pts (772 (51-1911) days (median (range)) was longer than HER2/neu negative pts (360 (60-2,739) days, p=0.059). The median duration of fulvestrant therapy was 425 days. Pts with HER2/neu + tumors were more likely to be treated beyond the median fulvestrant therapy with an odds ratio of 6.2 (1.26 to 30.92 95% confidence interval, p=0.0249). Conclusions: Trastuzumab plus fulvestrant therapy was associated with a more prolonged clinical response than fulvestrant alone in pts with metastatic breast cancer. This synergism may be due to the effect of trastuzumab inhibiting the activation of transcriptional coactivator MED1, a recently discovered key crosstalk point between HER2/neu and ER signaling pathways in mediating endocrine resistance (Cancer Res 2012;72(21):5625;PLoS One 2013; 8:e70641).

Abstract 4409: The characterization of isomerically stable fixed ring derivatives of tamoxifen metabolites 4-hydroxytamoxifen and 4-hydroxy-N-desmethyltamoxifen (endoxifen) in vitro.

Abstract Tamoxifen is the most widely used agent in the world for antihormonal therapy of Estrogen Receptor (ER)-positive breast cancer for both pre- and postmenopausal patients. Tamoxifen is a pro-drug and needs to be metabolically activated to a more potent antiestrogen with a higher affinity for the ER. Initially 4-hydroxytamoxifen (4OHT) was the active metabolite of interest, but more recently 4-hydroxy-N-desmethyltamoxifen (endoxifen) is also of significant interest. The role of endoxifen in therapy remains controversial, however both 4OHT and endoxifen exist in two isomeric forms- cis and trans. Previously, it was shown that cis-isomers of tamoxifen metabolites have weak anti-estrogenic properties using in vitro models available (Jordan VC et al, Endocrinology, 1987, 122(4); 1449-54). However, studies show that an increase of cis-isomers of 4OHT in animals and patients may contribute to tamoxifen resistance (Osborne CK et al, JNCI, 1991, 83; 1477-82; Osborne CK et al, J Clin Oncol, 1992, 10; 304-10). Thus, it is important to know the estrogenic properties of clinically relevant tamoxifen metabolites. To document the anti-estrogenic profiles of both geometric isomers of two major hydroxylated metabolites of tamoxifen, isomerically stable fixed-ring derivatives of 4OHT and endoxifen were synthesized. We have assessed the anti-estrogenic properties of these compounds using cell proliferation assays in selectively cloned hypersensitive ER-positive human breast cancer cell lines MCF-7:WS8 and T47D:A18. Also to assess to estrogenic/anti-estrogenic properties of synthesized compounds on transcriptional level, we have used real-time PCR of rat Prolactin (Prl) mRNA isolated from GH3 rat pituitary cell line. Our results indicate that the fixed-ring trans-4OHT and trans-Endoxifen are potent antiestrogens and equivalent to each other, and to their non-fixed-ring counterparts in both cell proliferation assays and Prl gene activity, as they do not possess any agonistic activities by themselves and are able to inhibit the estrogenic actions of 0,1nM 17β-estradiol (E2) completely (IC50 1nM). However, cis-isomers of the fixed-ring compounds are very weakly estrogenic by themselves but are very weak anti-estrogens in combination with 0,1nM E2 in all assays. Conclusions: the weak estrogen-like activity and low potency of the cis-isomers is unlikely to influence the growth of breast cancer during long-term therapy. The high potency of trans-endoxifen and 4OHT will dominate the control of the tumor growth. Support: This study (PYM) was supported by Susan G. Komen for the Cure International Postdoctoral Fellowship under award number SAC100009 (VCJ). Citation Format: Philipp Y. Maximov, Cynthia Myers, Daphne J. Fernandes, V. Craig Jordan. The characterization of isomerically stable fixed ring derivatives of tamoxifen metabolites 4-hydroxytamoxifen and 4-hydroxy-N-desmethyltamoxifen (endoxifen) in vitro. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4409. doi:10.1158/1538-7445.AM2013-4409