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Purpose: Triple negative breast cancer (TNBC) is an aggressive breast cancer subset with poor outcomes. Since TNBC is resistant to hormone therapies, there are few effective therapies available for TNBC patients. One potential therapeutic strategy exists in targeting specific molecular components of an individual patient’s cancer. Prior work in our group has nominated monopolar spindle kinase I (TTK) as a gene upregulated in breast cancer patients. Specifically, TTK expression was found to be increased in cancerous breast tissue compared to healthy tissue and correlated with cancer recurrence following radiotherapy. Importantly, the implications of TTK inhibition and radiotherapy on the immune system is not well understood. In this study, we aimed to elucidate the role of combined TTK inhibition and radiotherapy in syngeneic murine mouse models. We hypothesize that TTK inhibition will radiosensitize murine TNBC models to radiotherapy both in vitro and in vivo and modulate the immune tumor microenvironment. Methods: Cell viability assays were implemented to determine the half-maximal inhibitory concentrations (IC50) of TTK inhibitor. Clonogenic survival assays were used to determine the radiation enhancement ratios (rERs) of TTK inhibition in vitro. Syngeneic murine mouse models were used to assess therapeutic effects of TTK inhibition and RT in vivo. 4T1 TNBC cells were injected bilaterally into the flanks of BALB/c mice and treated with combinations of radiotherapy and TTK inhibition. Tumor growth was monitored and, following the completion of the study, final tumor weights were recorded and tumor tissue was collected to perform immunofluorescent microscopy. Results: Single-agent TTK inhibition via treatment with the ATP-competitive inhibitor empesertib inhibits the growth of murine TNBC cell lines with IC50 values up to 30nM. Sub-IC50 values of TTK inhibitor induced radiosensitization in the murine TNBC cells 4T1 (rERs ≤ 2.4) and Py8119 (rERs ≤ 1.6). TTK knockdown also resulted in changes in radiosensitization in vitro. Furthermore, we also observed a similar phenotype in vivo. In our 4T1 model system, mice receiving combined treatment had significantly decreased tumor growth compared to mice that receiving single-agent therapies or vehicle control alone. Quantities of monocyte derived suppressor cells and CD8+ T cells were altered with radiotherapy and TTK inhibition. Conclusion: Our data suggests that TTK inhibition and radiotherapy is synergistic in murine TNBC and alters the tumor immune microenvironment. This combined therapy suggests that changes in the underlying immune mechanisms as a result of the synergistic treatment efficacy are important in TNBC. Future work will examine the underlying mechanisms of TTK inhibition and radiotherapy on systemic and tumoral immune changes. Citation Format: Kassidy M. Jungles, Zhuwen Wang, Caroline R. Bishop, Kalli R. Jungles, Cydnee Wilson, Meilan Liu, Ashley N. Pearson, Erin A. Holcomb, Ben Chandler, Jadyn James, Amanda Huber, Lori J. Pierce, Corey Speers, James M. Rae, Michael D. Green. Targeting monopolar spindle kinase I (TTK) as a radiosensitizing strategy in syngeneic murine models of triple negative breast cancer (TNBC) and its implications on the tumor immune microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2823.

Background: Adjuvant aromatase inhibitors (AI) reduce recurrence and mortality after early stage hormone receptor-positive (HR+) breast cancer (BC). Treatment-related toxicities, including AI-induced musculoskeletal symptoms (AIMSS), are common during adjuvant AI therapy. Prior work suggests the risk of AIMSS is associated with inherited germline genetic polymorphisms in several genes, such as TCL1A, CYP19A1, OPG, and VDR. These pharmacogenetic associations require replication in independent cohorts prior to clinical translation to identify patients at risk for AIMSS. The objective of this retrospective pharmacogenetic analysis was to replicate previously reported associations for candidate germline genetic polymorphisms with AIMSS. Methods: Women with stage 0-III HR+ BC initiating adjuvant endocrine therapy (ET) with tamoxifen or an AI were enrolled in a prospective clinic-based observational cohort. The type of ET was selected by the provider. A baseline (BL) blood sample was collected for isolation of germline DNA for pharmacogenetic analysis. AIMSS were assessed by patient-reported outcomes (PRO). Participants completed PROMIS pain interference (PI), PROMIS physical function (PF) and Functional Assessment of Cancer Therapy - Endocrine Symptoms (FACT-ES) measures at BL and after 3, 6, 9, 12, 24, 48 and 60 months (mo). The FACT-ES includes one question about joint pain, rated on a 5-point scale (“not at all” to “very much”). This secondary retrospective pharmacogenetic analysis was conducted in participants receiving AI therapy for whom blood samples and PRO scores at BL and 3 and/or 6 mo were available. For the primary analysis, we defined AIMSS as a ≥4 point increase in PI T score from BL to 3 and/or 6 mo. For secondary analyses, we evaluated alternate definitions of AIMSS including a ≥4 point decrease in PF T score and a ≥1 category increase on the FACT-ES joint pain question from BL to 3 and/or 6 mo. The primary hypothesis was that TCL1A rs11849538 is associated with AIMSS. Twelve other germline variants in CYP19A1, VDR, PIRC66, OPG, ESR1, CYP27B1, CYP17A1, and RANKL previously reported to be associated with AIMSS were also analyzed. We assumed a dominant genetic effect and pre-specified the direction of effect on AIMSS for each variant. We conducted univariate logistic regression to evaluate associations between each definition of AIMSS and candidate polymorphism using an unadjusted α=0.05. Significant univariate associations in the expected direction were adjusted for age, race, body mass index (BMI), prior taxane and type of AI using multivariable logistic regression. Results: Of 182 participants on AI, 143 with PRO and genetic data available were included in this analysis. Median age was 67, 85% were white, median BMI was 27.8 and 27% had prior taxane. 78% received anastrozole, 20% letrozole and 2% exemestane. On primary analysis, participants carrying TCL1A rs11849538 were not more likely to develop AIMSS as defined by increase in PI T score by ≥4 (OR=1.29, 95% CI: 0.55-3.07, p=0.56). None of the other polymorphisms was associated with increase in PI T score by ≥4. On secondary analysis, OPG rs2073618 was associated with AIMSS, as defined by an increase on the FACT-ES joint pain question ≥1 (OR=3.33, 95% CI: 1.48-7.49, p=0.004) and this association maintained significance after covariate adjustment (OR=3.98, 95% CI: 1.61-9.84, p=0.003). Age, race, BMI, prior taxane and type of AI were not associated with AIMSS on multivariate analysis. No other polymorphisms were associated with AIMSS on secondary analyses. Conclusions: Carriers of OPG rs2073618 may be at increased risk of AIMSS. If confirmed in other cohorts, OPG genotyping may be able to identify individuals with HR+ early BC at increased risk for AIMSS during AI therapy. Alternate ET or interventions to reduce musculoskeletal symptoms may be needed for this population. Citation Format: Daniel L Hertz, Karen Lisa Smith, Yuhua Zong, Christina L Gersch, Andrea Pesch, Arti Patel, Jennifer Lehman, N. Lynn Henry, Kelley M Kidwell, James M Rae, Vered Stearns. Association of OPG rs2073618 and aromatase inhibitor induced musculoskeletal symptoms [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS5-25.

Background: Endocrine therapies (ETs) are effective in hormone receptor positive metastatic breast cancer (MBC), but resistance is a major clinical problem. Thymidine Kinase 1 (TK1) plays a key role in DNA synthesis and is a marker of cellular proliferation. Serum (sTK1) activity is associated with poor prognosis in MBC patients treated with chemotherapy. SWOG S0226 trial demonstrated that anastrazole (A) + fulvestrant (F) is more effective than A alone in post-menopausal women with hormone receptor positive MBC, specifically those without prior adjuvant tamoxifen (Mehta et al NEJM 2019). We hypothesized that baseline (BL) and serial sTK1 levels are prognostic and demonstrate differential activity of A+F vs. A. Methods: sTK1 activity was assessed in 1,726 archived S0226 serum samples (BL, cycle 2, 3, 4, and 7) using DiviTum® assay (100% evaluation rate). Pre-specified cutoff of ≥200 DiviTum Unit per liter (Du/L) was considered high. Progression-free survival (PFS) and overall survival (OS) were analyzed by Kaplan-Meier, log-rank tests and Cox regression. Results: Serum samples at BL were available in 432/694 (62%) patients. Outcomes on this subset were comparable to those on the full trial. Median sTK1 at BL was 135 Du/L. sTK1 was elevated in 171 (40%) patients. Overall, patients with high vs. low BL sTK1 had significantly worse PFS [hazard ratio (HR)=1.76; 95% CI (1.43-2.16); P Funding: NIH/NCI grants U10CA180888, U10CA180819, U24CA196175; and in part by AstraZeneca. Biovica has funded the TK1 testing. Citation Format: Costanza Paoletti, William E. Barlow, Mattias Bergqvist, Rita S. Mehta, Julie R. Gralow, Gabriel N. Hortobagyi, Kathy S. Albain, Lajos Pusztai, Priyanka Sharma, Andrew K. Godwin, Alastair M. Thompson, Daniel F. Hayes, James Rae. Evaluating serum thymidine kinase 1 in hormone receptor positive metastatic breast cancer patients receiving first line endocrine therapy in the SWOG S0226 trial [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS2-04.

Purpose: CDK4/6 inhibitors (CDK4/6i) are standard of care for the treatment of locally advanced and metastatic estrogen receptor-positive (ER+), HER2-negative metastatic breast cancer (BC). CDK4/6 inhibition + radiation therapy (RT) is synergistic in both ER+ and triple negative breast cancers (TNBC), but the underlying mechanism is not entirely understood. In this study, we evaluated how pre-existing or genetically engineered deficits in DNA damage response genes (BRCA1/2, RAD51, RB1, XRCC6, TP53) influence radiosensitization. We hypothesized that inhibition of homologous recombination (HR) would prevent CDK4/6i-mediated radiosensitization and blocking non-homologous end joining (NHEJ) would be synergistic. Methods: Cellular proliferation assays determined the half-maximal inhibitory concentrations (IC50) of the 3 approved CDK4/6i palbociclib, ribociclib, and abemaciclib. Clonogenic survival assays determined the radiation enhancement ratios (rERs) and evaluated the efficacy of CDK4/6i + RT. Immunofluorescence assays measured RAD51 foci formation and quantified micronuclei formation following RT and/or CDK4/6 inhibition. Immunoprecipitation with myc-RAD51 and GFP-RB assessed potential protein-protein interactions. Results: While ER+ and TNBC cell lines with wild type BRCA1 expression are radiosensitized by CDK4/6i, BRCA1-deficient SUM-149 cells are not radiosensitized by CDK4/6i at concentrations up to 1µM (rER: 0.92-1.01). In an MCF-7 isogenic model of BRCA2 knockout, CDK4/6i-mediated radiosensitization was abolished compared to Cas9 control or parental cell lines. In ER+ BC cell lines (MCF-7-p53 wt, T47D-p53 mutant), transient or genetic knockdown of RAD51 prevented CDK4/6i-induced radiosensitization. The total quantity of RT-induced RAD51 foci increased in vitro following overexpression of RB-a tumor suppressor and downstream target of CDK4/6. RB overexpression also rescued CDK4/6i-mediated radiosensitization in RB-deficient cell lines through changes in HR efficiency but not via NHEJ or altered micronuclei formation. Moreover, immunoprecipitation of RAD51 in ER+ (MCF-7) and TNBC (MDA-MB-231) cells exhibited an interaction with RB. Conversely, loss of the NHEJ-associated protein Ku70 (XRCC6) was synergistic with palbociclib + RT in MCF7 (rER: 1.76-2.44) and T47D (rER: 1.61-3.88) cells. Finally, CRISPR Cas9-mediated loss of the tumor suppressor p53 (TP53) did not affect radiosensitization induced by CDK4/6i in isogenic p53 wt ER+ (MCF-7, rER: 1.19-1.33) and p53 wt TNBC (CAL-51, rER: 1.23-1.52) cell lines with TP53 loss. Conclusions: Taken together, our results in multiple non-overlapping isogenic models of ER+ and TNBC suggest that CDK4/6i-mediated radiosensitization of BC cell lines occurs through impaired HR activity and RB signaling, and not through the actions of p53 or NHEJ-mediated DNA repair. Citation Format: Kassidy M. Jungles, Andrea M. Pesch, Nicole Hirsh, Anna R. Michmerhuizen, Kari Wilder-Romans, Benjamin C. Chandler, Meilan Liu, Lynn Lerner, Lori J. Pierce, James M. Rae, Corey W. Speers. Expression of DNA damage response proteins modifies the efficacy of CDK4/6 inhibitor-mediated radiosensitization in breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 216.

Clinical decisions for precision and immuno-oncology therapies are based on predictive biomarkers commonly obtained from a single metastatic biopsy, or from archived primary tumor material. Circulating genomic biomarkers present a minimally invasive way to monitor the intra-patient tumor heterogeneity and its fluctuations in order to provide a real-time evaluation of the changing clonal architecture with potential therapeutic implications. Single-cell DNA next generation sequencing (scNGS) of circulating tumor cells (CTC) is a particularly well-suited method of unraveling and monitoring that heterogeneity to complement biomarker information obtained from tissue and cell-free circulating tumor DNA (ctDNA). In this proof-of-concept study we analyzed 123 CTC, 15 white blood cells (WBC), and ctDNA from 15 CTC-positive lobular breast cancer patients, five of whom had CTC available at both metastatic baseline and after progression on a variety of therapies chosen at their physician’s discretion. CTC were enriched with the CellSearch® system and isolated as single cells with the DEPArray™ system. Whole genome amplified CTC DNA underwent scNGS with the Oncomine Comprehensive Assay covering ~500 genes and 1.1Mb of genomic space to detect mutations, copy number alterations, tumor mutation burden (TMB) and microsatellite instability (MSI). 99.1% of cells were informative, with a mean sequencing depth of 664x. Using our previously developed, CTC-based precision medicine reporting platform, MI-CTCSeq, multiple CTC in seven of 15 patients (47%) had mutations that were actionable by FDA-approved targeted therapies including in the oncogenes PIK3CA (alpelisib) and FGFR2 (erdafitinib). 13 patients (87%) displayed intra-patient, inter-CTC genomic heterogeneity of putative driver mutations. Two of five (40%) patients with CTC at both baseline and progression displayed fluctuations in their CTC subclonal makeup between timepoints. One of the two harbored a baseline ESR1 (estrogen receptor α) p.D538G activating mutation that largely disappeared at progression and was replaced by a CTC subclone with a different ESR1 activating mutation, p.Y537S. Intriguingly, this patient’s CTC also harbored an FGFR2 p.K659M mutation in an actionable “hotspot” at progression, which was absent at baseline, suggesting potential utility of serial monitoring by CTC scNGS. TMB scores and MSI status in CTC were highly concordant with those measured in clinical tissue biopsies. Taken together, these data suggest the non-invasive interrogation of the CTC genomic landscape and its serial monitoring to inform precision and immuno-oncology treatments in real time. Citation Format: Andi K. Cani, Emily M. Dolce, Elizabeth P. Darga, Kevin Hu, Chia-Jen Liu, James M. Rae, Daffyd G. Thomas, Scott A. Tomlins, Arul M. Chinnaiyan, Aaron M. Udager, Costanza Paoletti, Erin F. Cobain, Daniel F. Hayes. Serial monitoring of single-cell circulating tumor cell genomics in metastatic lobular breast cancer to identify precision and immuno-oncology biomarkers with therapeutic implications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1700.

Purpose: The estrogen receptor (ER) is expressed in over 80% of breast tumors and has been shown to be a significant driver of breast cancer (BC) pathogenesis and therefore a target of effective first-line therapies. While both ionizing radiation (RT) and endocrine therapies (ET) are used for the treatment of ER+ BC, the effect of ET on tumor radiosensitization remains unclear, with concerns it may be radioprotective based on G1 cell arrest with ET treatment. Here we assessed the efficacy and mechanism of ER-mediated radiosensitization using various pharmacologic approaches in ER+ BC. Methods: Radiosensitization with ER inhibitors (tamoxifen [TAM], fulvestrant [FULV], AZD9496) was assessed using clonogenic survival assays. DNA damage was assessed by the neutral comet assay. Efficiency of homologous recombination (HR) or non-homologous end joining (NHEJ) as well as changes in cell cycle, apoptosis, and senescence were assessed. The efficacy of TAM with RT in vivo was assessed with an MCF-7 xenograft model. Results: The selective estrogen receptor modulator TAM radiosensitized ER+ MCF-7 (enhancement ratio [enhR]: 1.14-1.50) and T47D (enhR: 1.33-1.60) cells but not ER-negative SUM-159 cells (enhR: 0.99-1.02). The selective estrogen receptor degrader (SERD) FULV had similar radiosensitization effects in MCF-7 (enhR: 1.33-1.76) and T47D cells (enhR: 0.97-2.81) with no radiosensitization observed in SUM-159 cells (enhR: 1.01-1.03). The novel oral SERD AZD9496 radiosensitized MCF-7 cells (enhR: 1.36-1.56). MCF-7 cells treated with TAM and RT had an increase in dsDNA breaks compared to RT alone as measured by the comet assay (p Citation Format: Anna R. Michmerhuizen, Lynn M. Lerner, Andrea M. Pesch, Connor Ward, Rachel Schwartz, Kari Wilder-Romans, Meilan Liu, Charles Nino, Kassidy Jungles, Ruth Azaria, Alexa Jelley, Nicole Zambrana Garcia, Alexis Harold, Amanda Zhang, Bryan Wharram, Daniel F. Hayes, James M. Rae, Lori J. Pierce, Corey W. Speers. Inhibition of estrogen receptor signaling as a strategy for radiosensitization of ER+ breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2697.

Background. SWOG trial S8814 randomized postmenopausal patients with pathologic lymph node-positive (N+) breast cancer that was hormone receptor-positive to receive adjuvant anthracycline-based chemotherapy (cyclophosphamide, doxorubicin, fluorouracil) followed by tamoxifen endocrine therapy for 5 years (CAF-T), versus tamoxifen alone (TAM). The 21-gene Breast Recurrence Score® assay was prognostic in S8814 and predicted chemotherapy benefit in patients with higher Recurrence Score® (RS) (Albain et al, Lancet Oncol 2009). Other prognostic signatures have yet to be evaluated in this cohort. The sensitivity to endocrine therapy index (SET2,3) measures non-proliferative hormone receptor-related transcription (SETER/PR) adjusted for a baseline prognosis index derived from tumor size, nodes involved and a 4-gene molecular subtype (RNA4) (Du et al, Ann Oncol 2021). SET2,3 has been shown to provide prognostic information independent from neoadjuvant chemotherapy response. We sought to evaluate the predictive and prognostic value of SET2,3 in SWOG 8814. Methods. Independently, the SET2,3 index and cut point were calibrated from their diagnostic platform to the whole transcriptome RNA sequencing (RNAseq) platform in 85 sample pairs. Expression of the 31 transcripts used for SET2,3 were provided from RNAseq data of 283 tumors in S8814 (all previously tested for RS). Blinded calculated results of SET2,3 were then merged with outcome data. The planned analysis tested whether SET2,3 (continuous index, dichotomized high/low) provided additional prognostic information to RS (overall and in pts. with RS≤25) by treatment arm, and whether low SET2,3 was associated with chemo benefit. Cox proportional hazards models of disease-free survival (DFS) included SET2,3; RS; treatment arm; and (where relevant) interaction term and reported hazard ratios (HR) and 95% confidence intervals (95%CI). Results. There were 106 events over median follow-up of 9.1 years in 283 patients. 175 patients had RS ≤25, 108 had RS >25. Distribution of the SET2,3 low was similar in both RS high (51%) and low groups (47%), reflecting minimal correlation between the two. As proportional hazards assumptions were met during the first 5 years only the analysis was restricted to 5 years. Adjusting for treatment arm, high SET2,3 category was highly prognostic in this randomized trial (HR 0.27, 95% CI 0.15-0.49, p Table: Multivariable Cox models in the overall population and subset with RS≤25 by treatment arm.CohortTreatment ArmContinuous Recurrence ScoreContinuous SET2,3HR (95%CI) per 10 unitsp-valueHR (95%CI)per 1 unitp-valueAll RS(N=283)CAF-TAM(N=166)1.21 (1.04-1.40)0.0120.48 (0.31-0.76)0.002TAM(N=117)1.44 (1.18-1.76)< 0.0010.48 (0.27-0.88)0.017RS≤25 (N=175)CAF-TAM(N=99)1.43 (0.58-3.49)0.440.34 (0.15-0.73)0.006TAM(N=76)1.66 (0.46-5.93)0.440.38 (0.14-1.05)0.062 Citation Format: Corey W. Speers, W. Fraser Symmans, William E. Barlow, Alex Trevarton, Stephanie The, Lili Du, James M. Rae, Steven Shak, Frederick L. Baehner, Priyanka Sharma, Lajos Pusztai, Gabriel N. Hortobagyi, Daneil F Hayes, Kathy S. Albain, Andrew Godwin, Alastair Thompson. Evaluation of the predicted sensitivity to endocrine therapy (SET2,3 index) and the 21-gene Breast Recurrence Score® assay in node-positive postmenopausal breast cancer: Results from an analysis in the SWOG S8814 trial [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD9-06.

Purpose: Estrogen receptor (ER) expression is present in over 80% of breast tumors and has been shown to be a significant driver of breast cancer (BC) pathogenesis and therefore a target of first-line therapies for ER-positive (ER+) BC patients. While both ionizing radiation (RT) and endocrine therapies (ET) are used for the treatment of ER+ BC, the sequencing of therapy and the effect of ET on tumor radiosensitization remain unclear. Recently, this question has become much more clinically relevant when many physicians started offering ET as a bridging strategy to surgery and RT during the COVID-19 pandemic. Here we assessed the efficacy and mechanism of ER inhibition in ER+ BC in combination with RT in preclinical models. Methods: Clonogenic survival assays were used to assess radiosensitization. Inhibition of ER signaling was accomplished by treating ER+ MCF-7 and T47D cells with the selective ER modulator (SERM), tamoxifen, or the selective ER degrader (SERD), fulvestrant. The ER-negative SUM-159 cells were used as a negative control. DNA damage was assessed by the neutral comet assay. Efficiency of homologous recombination (HR) was measured by Rad51 foci or a GFP reporter system. Non-homologous end joining (NHEJ) efficiency was assessed with a pEYFP reporter. Cell cycle effects were measured using flow cytometry with propidium iodide (PI) staining. Apoptosis was assessed by annexin V/PI via flow cytometry. Senescence was measured using β-galactosidase staining. Western blotting was used to quantify expression of proteins and phospho-proteins involved in cell cycle and apoptosis. An MCF-7 xenograft model was used to assess the efficacy of tamoxifen with RT in vivo. Synergy was determined using the fractional tumor volume (FTV) method. Results: ER inhibition with tamoxifen radiosensitized ER+ MCF-7 (10-250 nM, enhR: 1.14-1.50) and T47D (500 nM-2.0 µM, enhR: 1.33-1.60) cells but not ER-negative SUM-159 cells (500 nM-2.0 µM, enhR: 0.99-1.02). ER degradation with fulvestrant had similar radiosensitization effects in MCF-7 (1-25 nM, enhR: 1.33-1.76) and T47D cells (0.5-5 nM, enhR: 0.97-2.81) with no radiosensitization observed in SUM-159 cells (1-25 nM, enhR: 1.01-1.03). MCF-7 cells treated with 500 nM tamoxifen and 4 Gy RT had an increase in dsDNA breaks compared to RT alone as measured by the comet assay (p Citation Format: Anna R Michmerhuizen, Lynn Lerner, Andrea M Pesch, Connor Ward, Rachel Schwartz, Kari Wilder-Romans, Meilan Liu, Bryan Wharram, Alexis Harold, Ruth Azaria, Nicole Zambrana Garcia, Daniel F Hayes, James M Rae, Lori J Pierce, Corey W Speers. Endocrine therapy treatment radiosensitizes estrogen receptor-positive breast cancers [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-02-04.

Background: Increased rates of locoregional recurrence (LR) have been observed in TNBC despite chemotherapy and radiation (RT). A novel radiosensitizer screen nominated the AR as a promising target for treatment of radioresistant breast cancer, including TNBC. We assessed the activity of seviteronel (Sevi), a selective CYP17 lyase and AR inhibitor in Phase 2 clinical development for advanced breast and prostate cancer, as a potential radiosensitizer in AR+ TNBC model. Methods: Clonogenic survival assays were used to determine the intrinsic RT sensitivity of 21 breast cancer cell (BCC) lines. IC50 values were determined for 130 clinically available compounds and correlation coefficients were calculated using IC50 values and SF-2Gy. Gene expression was measured using RNA Seq or qRT-PCR and protein expression was measured using RPPA arrays. AR function was assessed using functional inhibition with Sevi in MDA-MB-453, ACC-422, ACC-460, SUM-185 (all four AR+ TNBC), MDA-MB-231 (AR- TNBC), and T47D (AR- ER+) BCC lines. Double-stranded DNA (dsDNA) break repair was assessed with γH2AX foci counting. In vivo tumor growth was measured with varying control and treatment groups (16-20 tumors/group). Kaplan-Meier analysis was performed to estimate local control. A Cox proportional hazards model and multi-variate analysis (MVA) were used to determine variables associated with LRF survival. Results: Our novel radiosensitizer screen identified the activity of anti-androgen therapy as a potentially effective strategy for radiosensitization in RT-resistant BCC lines (R2 =0.46, p-value < 0.01) (Speers et al, J Clin Oncol 35, 2017 (suppl; abstr e12102). Heterogeneity in AR expression was identified in human BCC lines and TNBC samples from patients (N=2098). There was a strong correlation between AR RNA expression and protein expression across all BC intrinsic subtypes. AR inhibition using Sevi induced radiation sensitivity in vitro with an enhancement ratio (ER) of 1.24-1.69 in four different AR+ TNBC lines. No such radiosensitization was seen in AR(-) TNBC or ER+, AR(-) BCC lines. Radiosensitization was at least partially dependent on impaired dsDNA break repair with significant delays in dsDNA break repair at 16 and 24 hours in all AR+ TNBC lines examined (p-value < 0.01). AR inhibition with Sevi significantly radiosensitized AR+ TNBC xenografts in mouse models and markedly delayed tumor-volume tripling time (TTT) and tumor growth (MDA-MB-453: median TTT 16.1 days for RT alone vs. not reached after 45 days for Sevi+RT, p-value Conclusions: Our results implicate the AR as a mediator of radioresistance in breast cancer and support the rationale for developing Sevi as a novel radiosensitizing agent in AR+ TNBC. Citation Format: Speers CW, Chandler B, Olsen E, Wilder-Romans K, Moubadder L, Nyati S, Rae J, Hayes DF, Spratt DE, Wahl DR, Eisner J, Feng FY, Pierce LJ. Radiosensitization of androgen receptor (AR)-positive triple-negative breast cancer (TNBC) cells using seviteronel (INO-464), a selective CYP17 lyase and AR inhibitor [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 P1-09-05.

Addressing drug resistance is a core challenge in cancer research, but the degree of heterogeneity in resistance mechanisms in cancer is unclear. In this study, we conducted next-generation sequencing (NGS) of circulating tumor cells (CTC) from patients with advanced cancer to assess mechanisms of resistance to targeted therapy and reveal opportunities for precision medicine. Comparison of the genomic landscapes of CTCs and tissue metastases is complicated by challenges in comprehensive CTC genomic profiling and paired tissue acquisition, particularly in patients who progress after targeted therapy. Thus, we assessed by NGS somatic mutations and copy number alterations (CNA) in archived CTCs isolated from patients with metastatic breast cancer who were enrolled in concurrent clinical trials that collected and analyzed CTCs and metastatic tissues. In 76 individual and pooled informative CTCs from 12 patients, we observed 85% concordance in at least one or more prioritized somatic mutations and CNA between paired CTCs and tissue metastases. Potentially actionable genomic alterations were identified in tissue but not CTCs, and vice versa. CTC profiling identified diverse intra- and interpatient molecular mechanisms of endocrine therapy resistance, including loss of heterozygosity in individual CTCs. For example, in one patient, we observed CTCs that were either wild type for ESR1 (n = 5/32), harbored the known activating ESR1 p.Y537S mutation (n = 26/32), or harbored a novel ESR1 p.A569S (n = 1/32). ESR1 p.A569S was modestly activating in vitro, consistent with its presence as a minority circulating subclone. Our results demonstrate the feasibility and potential clinical utility of comprehensive profiling of archived fixed CTCs. Tissue and CTC genomic assessment are complementary, and precise combination therapies will likely be required for effective targeting in advanced breast cancer patients. Significance: These findings demonstrate the complementary nature of genomic profiling from paired tissue metastasis and circulating tumor cells from patients with metastatic breast cancer. Cancer Res; 78(4); 1110–22. ©2017 AACR.

Purpose: Compared to other breast cancer subtypes, triple negative breast cancers (TNBC) derive the least benefit from adjuvant radiation (RT) which contributes to higher rates of locoregional recurrence. Thus, there is a critical need to identify clinical strategies to increase the effectiveness of RT therapy in TNBC. Methods: Alamar blue proliferation assays were used to calculate half maximal inhibitory concentration (IC50) values for each Bcl-2 family inhibitor 72 hours after drug treatment. Clonogenic survival assays were used to evaluate radiosensitivity and to calculate the radiation enhancement ratio (rER) after combination treatment. Apoptosis was assessed through formation of cleaved PARP and annexin V/PI-based flow cytometry. Xenograft models with MDA-MB-231 cells and TNBC patient-derived xenografts (PDX4664) were used to assess radiosensitization in vivo. Results: A novel radiosensitizer screen identified Bcl-2 family inhibition as a potentially effective treatment strategy in radioresistant breast cancer models. Single-agent response to pan Bcl-2 family inhibition (ABT-263) or Bcl-xL inhibition (WEHI-539, A-1331852) was more effective in PIK3CA wild type (wt) TNBC (IC50 < 1µM) compared to PIK3CA mutant TNBC. Inhibition of apoptosis with ABT-263 led to radiosensitization of PIK3CA/PTEN wild-type TNBC cell lines (rER: 1.09-1.74), but had no effect on PIK3CA/PTEN mutant TNBC (rER: 0.87-1.18). Radiosensitization was observed to be Bcl-xL-dependent, with Bcl-xL inhibitor-specific radiosensitization (rER: 1.12-2.38) but a lack of Bcl-2 inhibitor (ABT-199, rER: 0.94 - 1.21) or MCL-1 inhibitor-mediated radiosensitization (S63845, rER: 0.91 - 1.06). In PIK3CA wt TNBC, combination treatment of Bcl-2 family inhibition and RT significantly increased the percent of apoptotic cells (p < 0.001) and led to increased formation of cleaved PARP 48 hours after RT. Sensitivity to RT was dependent on expression of MCL-1, an anti-apoptotic protein that is overexpressed in PIK3CA/PTEN mutant TNBC. Overexpression of MCL-1 in PIK3CA/PTEN wild type TNBC rescued radioresistance (rER: 0.99-1.09), whereas co-inhibition of MCL-1 and Bcl-xL in PIK3CA/PTEN mutant TNBC was sufficient to overcome radioresistance (rER: 2.32 - 2.35). In vivo, nonspecific Bcl-2 family inhibition or specific Bcl-xL inhibition in combination with RT decreased tumor growth and increased time to tumor tripling (p < 0.0001) in PIK3CA wt models of TNBC. Conclusions: In this study, we demonstrated that inhibition of Bcl-2 family proteins in combination with RT led to increased levels of apoptosis and cell death in PIK3CA/PTEN wt - but not PIK3CA/PTEN mutant - TNBC and we identified MCL-1 as a critical mediator of this radiosensitIvity. Together, these results indicate that Bcl-xL inhibition may be a feasible clinical strategy for the radiosensitization of PIK3CA/PTEN wild-type TNBC. Citation Format: Andrea M. Pesch, Benjamin C. Chandler, Anna R. Michmerhuizen, Nicole Hirsh, Kari Wilder-Romans, Meilan Liu, Tanner Ward, Dana Messinger, Charles Nino, Cassandra Ritter, James M. Rae, Corey W. Speers. Radiosensitization of PIK3CA wild type triple negative breast cancers with Bcl-family inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1943.

Purpose: Cyclin-dependent kinase 4&6 (CDK4/6) inhibitors have been approved for the treatment of metastatic, estrogen receptor positive (ER+) breast cancers, but there is growing interesting in CDK4/6 inhibition as a therapeutic strategy in other breast cancer subtypes, including triple negative breast cancer (TNBC). Previous studies have shown that CDK4/6 inhibition radiosensitizes ER+ breast cancers, but the interaction between CDK4/6 inhibition and radiation (RT) in TNBC is incompletely understood. Methods: Cellular viability was quantified 72 hours after drug treatment (in the absence of RT) to calculate a half maximal inhibitory concentration (IC50) value of proliferation. Radiation enhancement ratios (rER) and surviving fractions of cells after RT were calculated using clonogenic survival assays in RB1 wild type and mutant TNBC cell lines. Homologous recombination (HR) was assessed using RAD51 foci formation and a stable HR reporter system. G1 cell cycle arrest was quantified using propidium iodide-based flow cytometry. CRISPR-induced knockout of RB1 and transient siRNA-mediated knockdown of RB1 in TNBC cell lines was used in both clonogenic survival assays and immunofluorescence experiments. In vivo efficacy of CDK4/6 inhibition + RT was assessed using TNBC patient-derived xenograft models (PDX4664). Results: Although most TNBC cell lines are resistant to CDK4/6 inhibitor monotherapy (IC50 > 250nM) compared to ER+ cells, treatment with 250nM-1μM palbociclib radiosensitized RB1 wild type TNBC (MDA-MB-231, CAL-51, SUM-159, CAL-120; rER 1.08 – 2.22) but failed to radiosensitize RB1 mutant TNBC (CAL-851, MDA-MB-468; rER: 0.84 – 1.00). Radiosensitization of TNBC cell lines also occurred with short term ribociclib or abemaciclib pretreatment. At 6 and 16 hours following RT, significant suppression of RT-induced homologous recombination (HR) activity (RAD51 foci) was observed in RB1 wild type (p < 0.001) but not RB1 mutant (p > 0.05) TNBC cell lines. Cell cycle arrest after short term CDK4/6 inhibition was dependent on the presence of RB1. In addition, genetic knockdown of RB1 in RB1 wild type TNBC lead to a loss of CDK4/6 inhibitor-mediated HR suppression (p > 0.05) and diminished radiosensitization. Conclusions: In TNBC, CDK4/6 inhibition and RT leads to suppression of HR activity in an RB1-dependent manner. While ongoing studies seek to elucidate the role of RB1 in HR suppression in the context of CDK4/6 inhibitor-mediated radiosensitization of TNBC, our data suggests that CDK4/6 inhibition + RT could be a valuable clinical strategy to radiosensitize a wide range of breast cancer subtypes, including RB1 wild type TNBC. Citation Format: Andrea M. Pesch, Nicole Hirsh, Anna R. Michmerhuizen, Benjamin C. Chandler, Kari Wilder-Romans, Meilan Liu, Lori J. Pierce, James M. Rae, Corey W. Speers. CDK4/6 inhibition radiosensitizes RB1 wild type triple negative breast cancers through impaired homologous recombination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1952.

Background: S100A8 and S100A9 are members of a family of calcium binding proteins that regulate inflammatory response, and are biomarkers of inflammatory diseases, S100A8/A9 preferentially form heterodimers that interact with their receptor, RAGE, to activate signaling pathways (ERK1/2 MAPK, JNK, and NF-κB) and stimulate tumor cells. Elevated expression of S100A8/A9 has been observed in cancers of the bladder, esophagus, colon, ovary, and breast. S100A8/A9 are expressed intratumorally by cancer cells and in the stroma by infiltrating immune and myeloid cells as well. We investigated the associations of elevated expression of intratumoral and stromal S100A8 with survival outcomes in breast cancer. Methods: Tissue microarrays (TMA) were constructed from breast cancer specimens from patients with stage I-III breast cancer treated at the University of Michigan Comprehensive Cancer Center between 2004-2006, ensuring a minimum of 10-year follow-up. Each patient was represented on the TMA by representative regions of non-necrotic tumor and distant normal tissue. Automative Quantitative Immunofluorescence (AQUA) was performed for S100A8 protein, and samples were scored for intratumoral and stromal S100A8 expression. S100A8 staining was assessed as a continuous value and by exploratory dichotomous cutoffs. Associations with disease-free survival (DFS) or overall survival (OS) and S100A8 expression, either as continuous value or based on the exploratory cutoffs, were determined using the Kaplan-Meier method and Cox proportional hazards models. Results: In the entire patient cohort, high intratumoral S100A8 expression, as a continuous measure, was a significant prognostic factor for OS (univariable hazard ratio [HR] 1.26, 95% confidence interval [CI] 1.02-1.56, p=0.036), and for DFS (multivariable HR [95%CI] = 1.24 [1.01-1.53], p = 0.043). Exploratory analyses demonstrated optimal cutoffs of intratumoral and intrastromal staining that greatly separated survival curves. We evaluated whether the prognostic significance of S100A8 expression is different in breast cancer patients based on hormone receptor status and determined that neither intratumoral nor stromal S100A8 expression were significantly associated with outcomes. Conclusions: Elevated intratumoral and stromal expression of S100A8 are significant indicators of poor outcome in breast cancer patients. These data further support a biological role for S100A8 signaling in mammary carcinogenesis and aggressive tumor behavior. Evaluation of S100A8 protein expression might provide additional prognostic information beyond traditional breast cancer prognostic biomarkers. Further validation is necessary to investigate these findings. Citation Format: Miller P, Kidwell K, Thomas D, Sabel M, Rae J, Hayes DF, Lippman ME, El-Ashry D. High intratumoral and stromal S100A8 expression is prognostic of poor outcome in 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 P4-12-13.

Background: Exemestane is a third generation steroidal aromatase inhibitor (AI) used for the treatment of estrogen receptor (ER) positive breast cancer in postmenopausal women. Differences in AI treatment efficacy and side effects may be due, in part, to variability in drug exposure. We previously reported that patients who self-report as white and those who carry the low-activity CYP3A4*22 single nucleotide polymorphism (SNP) have increased exemestane steady-state concentrations. Additional SNPs in CYP3A may contribute to pharmacokinetic variability and explain this inter-race difference. CYP3A5*3 (rs776746) is a non-expresser genotype that is far more common in European (minor allele frequency (MAF)∼0.94) than African (MAF∼0.18) individuals. CYP3A7*1C (rs45446698) is believed to tag adult expression of the fetal CYP3A7 enzyme and is relatively uncommon in tested cohorts (European MAF=0.04, African MAF Methods: 500 patients were randomly assigned to either drug on the Exemestane and Letrozole Pharmacogenetics (ELPh) Study. Clinical data and DNA were collected at baseline and blood samples were collected after 1 or 3 months of treatment to measure steady-state exemestane concentration via HPLC/MS. Genotyping for CYP3A5*3 and CYP3A7*1C was performed via Taqman Allelic Discrimination. Pharmacogenetic association with log-transformed concentrations were tested for each variant by inclusion in a multivariable model with CYP3A4*22 and self-reported race, assuming additive genetic effect, using Tobit regression to censor concentrations below the lower limit of quantification. SNPs with suggestive p-values 40, body mass index (BMI), and prior chemotherapy) to assess their independent contribution. Results: In 231 evaluable patients there was a suggestive trend toward lower steady-state exemestane concentrations for CYP3A7*1C carriers (6.3 vs. 8.0 ng/mL) in the model including CYP3A4*22 and race (p=0.083). In the final multivariable model each CYP3A7*1C allele decreased exemestane concentration 31.5% (p=0.035, Table 1). CYP3A5*3 was not associated with exemestane concentration (p>0.2). Multivariable Model of Exemestane Concentration % change in concentration (95% CI)p-valueCYP3A4*22 (rs35599367)64.5% (23%, 120%)0.0008CYP3A7*1C (rs45446698)-31.5% (-52%, -2.6%)0.035Self-Reported White47.2% (9.0%, 99%)0.012AST or ALT>4041.3% (1.0%, 98%)0.044BMI-0.9% (-2.4%, 0.55%)0.22Prior Chemotherapy-23.5% (-37%, -7.6%)0.006CI: Confidence Interval Conclusions: Patients with breast cancer who carry CYP3A7*1C have lower steady-state exemestane concentrations but this association does not explain the greater concentrations in self-reported white patients. Ongoing analyses will determine whether exemestane concentration predicts treatment efficacy or toxicity, and if so, whether genetic and clinical factors can be useful for individualizing dosing to optimize outcomes. CYP3A7*1C should be prioritized for analyses of pharmacokinetic variability of other CYP3A substrates. Citation Format: Hertz DL, Kidwell KM, Gersch CL, Desta Z, Storniolo AM, Stearns V, Skaar TC, Hayes DF, Henry NL, Rae JM. Genetic variation in CYP3A affects steady-state exemestane concentrations but does not explain inter-race difference [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 P6-09-11.

This abstract was withdrawn by the authors.

Background: The prognostic significance of increased levels of CD8+ tumor infiltrating lymphocytes(TILs) in ER- breast cancer has been described. We sought to identify possible immune-related biomarkers for predicting benefit from letrozol(LET) or tamoxifen(TAM) for recurrence in ER+ breast cancer. Patient and Methods: We used Illumina DASL Assay to measure gene expression in FFPE primary breast cancers from a subset of postmenopausal patients enrolled in the BIG 1-98 randomized phase 3 trial comparing 5 years LET (n=344) vs TAM (n=381) as adjuvant endocrine therapy. Gene sets (n=1910) that represent cell states and perturbations within the immune system from the Human Immunology Project Consortium were used in an exploratory analysis to identify possible predictive signatures. Results: We identified five distinct gene signatures from previously reported laboratory experiments associated with immune cell differentiation that are highly predictive of benefit (reduced breast cancer recurrence risk) of LET over TAM, each with gene signature p-values Conclusion: The role of selective estrogen receptor modulators on immune response has been well described, where TAM has been shown to prevent differentiation and activation of dendritic cells (Naibandian 2005). Similarly, it has been shown that MET inhibitors negatively regulate neutrophils suggesting that anti-MET drugs in cancer could impact immune response (Finisquerra 2015). These findings suggest that if TAM is a negative regulator of immune response why in the ATAC clinical trial, the combination therapy of anastrozole plus TAM were not significantly different from TAM alone were anastrozole was superior. With the increasing importance of understanding the role of immune response on outcome and the use of combination therapies the assessment of TILs in the neoadjuvant setting will be critical for guiding therapy. Citation Format: Willis S, Gray KP, Regan MM, Rae JM, Kammler R, Young B, Ditzel HJ, Lyng MB, Colleoni M, Viale G, Leyland-Jones B. Immune related gene expression signatures predict benefit of letrozole over tamoxifen in BIG 1-98. [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 P3-07-36.

Background: Anastrozole (A) blocks estrogen production by inhibiting the activity of CYP19 aromatase. Fulvestrant (F) blocks estrogen receptor (ER) signaling by competitive binding, leading to ER degradation by ubiquitination. SWOG S0226 ("Phase III Randomized Trial of Anastrozole versus Anastrozole and Fulvestrant (250mg LD) as First Line Therapy for Post Menopausal Women with Metastatic Breast Cancer," ClinicalTrials.gov Identifier:NCT00075764) demonstrated that combination of A+F is superior to A alone as first-line therapy for patients with ER positive metastatic breast cancer (Mehta et al, NEJM, 2012). Our functional preclinical studies have shown that single nucleotide polymorphisms (SNPs) in SULT1A1 and UGT1A4, drug conjugation enzymes that inactivate A and F, result in decreased enzyme activity toward these drugs (Edavana et al, DMD, 2013; Edavana et al Pharmgenomics Pers Med 2013). We therefore hypothesized that these SNPs will be associated with disease outcomes in S0226 patients due to altered drug levels. Methods: Germline DNA was available for 295 (43.5%) patients enrolled in S0226 overall (157 on A and 138 on A+F). SNPs in SULT1A1 and UGT1A4 were determined either by direct sequencing or allele-specific PCR (TaqMan) assays. Results: There was no difference in progression-free survival (PFS) or overall survival (OS) comparing patients with or without available germline DNA (p = 0.86 and 0.36, respectively). The SULT1A1 G902A allele (rs6839), which confers decreased mRNA and enzymatic activity, was associated with improved PFS (GG/GA vs. AA; HR 0.74, 95% CI 0.56-0.98, p=0.033) and OS (HR 0.70, 95% 0.50-0.98, p=0.039). In exploratory subset analyses of PFS, the SULT1A1 G902A association was similar across both treatment arms (A HR=0.75; 95% CI 0.51-1.10; A+F HR=0.73; 95% CI 0.48-1.11). For OS there was some evidence of a difference by treatment (A HR=0.60; 95% CI 0.38-0.96; A+F HR=0.82; 95% CI 0.50-1.32), though no significant interaction was evident (p=0.30). The UGT1A4 G-163A promoter variant, which leads to decreased protein expression, was not associated with PFS (AA/AG vs. GG HR 0.88, 95% CI 0.68-1.14, p=0.33); however, this variant was associated with OS (HR 0.71, 95% CI 0.52-0.96, p=0.027). In subset analyses with OS, the difference was marginally stronger in the A arm (HR 0.63, 95% CI 0.42-0.97, p=0.035) compared to the A+F arm (HR 0.77, 95% CI 0.49-1.21, p=0.25), though the interaction was not significant (p=0.40). Conclusion: SULT1A1 and UGT1A4 gene variants resulting in decreased enzyme activity were associated with better PFS, OS or both in patients enrolled in SWOG S0226. Planned validation studies correlating these SNPs with drug levels and disease outcomes in additional patient cohorts will establish their clinical utility in identifying patients who benefit from A and F alone or in combination. Funding: Supported by NIH/NCI CA118981; NIH/NCI/NCTN grants CA180888, CA180819, and CA180863; and in part by AstraZeneca. Citation Format: Kadlubar SA, Barlow WE, Mehta RS, Daniels JR, Albain KS, Vandengerg TA, Dakhil SR, Tirumali NR, Lew DL, Gralow JR, Livingston RB, Hortobagiyi GN, Hayes DF, Rae JM. Association between gene variants in SULT1A1 and UGT1A4 and disease outcomes in patients enrolled in SWOG S0226 and treated with anastrozole alone or in combination with fulvestrant for metastatic 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 P3-07-64.

Background: Tamoxifen therapy for estrogen receptor positive (ER+) breast cancer reduces recurrence risk by about half. Steady-state concentrations of endoxifen, a potent anti-estrogenic tamoxifen metabolite, are reduced in women whose CYP2D6 genotypes confer poor enzyme function. Many studies have measured associations between genetically impaired CYP2D6 function and tamoxifen resistance. It has been suggested that the subset of studies using DNA extracted from tumor-infiltrated tissue may have been susceptible to genotyping error induced by loss of heterozygosity (LOH); the putative non-differential genotype misclassification may have biased these studies' estimates toward the null. We reviewed the clinical epidemiology studies conducted to date to assess the importance of loss-of-heterozygosity (LOH) at the CYP2D6 locus and its implications for assessing tamoxifen effectiveness. Methods: We searched for the terms "tamoxifen" and "CYP2D6" in PubMed, including all papers and abstracts through 31 May 2015 on the association of CYP2D6 gene variants and the risk of breast cancer recurrence or mortality. We used a quantitative bias analysis (QBA) to evaluate the importance of genotype misclassification in studies that extracted DNA from tumor-infiltrated tissue. We conducted a random effects meta-analysis to evaluate all studies simultaneously, and within groups according to whether DNA was derived from tumor-infiltrated tissue or non-neoplastic tissue. Results: Thirty-one studies investigated CYP2D6 genotype and breast cancer recurrence, yielding relative effect estimates ranging from 0.08 to 14. DNA was extracted from blood or non-neoplastic tissue in 21 of these 31 studies (68%), and from tumor-infiltrated tissue in the remaining 10 (32%). Our analysis of the association between variant/variant genotype compared with wildtype/wildtype genotype included 21 of the 31 studies. Sixteen (76%) of these 21 studies extracted DNA from blood or non-neoplastic tissue and five (24%) extracted DNA from tumor-infiltrated tissue. Genotype misclassification parameters for the QBA were estimated from six concordance studies. There was little difference between the effect estimates (EE) and 95% confidence/simulation intervals (95% CI/SI) before and after QBA (EE=1.71, 95%CI=1.24, 2.36, and 1.80 95%SI=1.28, 2.54, respectively). Studies using non-neoplastic DNA had higher variance than those based on tumor-infiltrated tissue DNA, half reported implausibly high EE, and many were susceptible to design and analysis errors that would bias estimates of association away from the null. Conclusions: We found little relative bias in the summary estimates of association, either overall or when limited to the tumor-infiltrated tissue DNA studies. Three guideline panels, based on robust evidence, recommend against CYP2D6 genotype-guided tamoxifen therapy. Alternatives for optimizing the effectiveness of tamoxifen therapy, such as assuring adherence and persistence, are more likely to achieve clinically important benefits. Citation Format: Ahern TP, Hertz DL, Damkier P, Ejlertsen B, Hamilton-Dutoit SJ, Rae JM, Regan MM, Thompson AM, Lash TL, Cronin-Fenton DP. CYP2D6 genotype and breast cancer recurrence in tamoxifen treated patients: An evaluation of the importance of loss-of-heterozygosity. [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 P3-07-23.

Background: Increased rates of locoregional recurrence have been observed in TNBC despite chemotherapy and radiation (RT). Thus, approaches that result in radiosensitizaton in TNBC are critically needed. We characterized the RT response of 21 breast cancer cell (BCC) lines using clonogenic survival assays. We paired this with high-throughput drug screen data to identify AR as a top target for radiosensitization and assess AR inhibition as a radiosensitization strategy for TNBC. Methods: Clonogenic survival assays were used to determine the intrinsic RT sensitivity of 21 BCC lines. IC50 values were determined for 130 clinically available compounds and correlation coefficients were calculated using IC50 values and SF-2Gy. Gene expression was measured using RNA Seq and protein expression was measured using RPPA arrays in human tumor samples (n=2,061) and BCC lines (n=51). AR function was assessed using siRNA knockdown or functional inhibition with MDV3100 (enzalutamide). We measured in vivo tumor growth with varying control and treatment groups (16-20 tumors/group). Kaplan-Meier analysis was performed to estimate local control and survival. A Cox proportional hazards model was used to identify factors of survival, and MVA was used to determine variables associated with LRF survival. Results: Our radiosensitizer screen nominated bicalutamide as a most effective drug in treating RT-resistant BCC lines (R2= 0.46, p-value 2000 human breast tumor samples and found significant heterogeneity in AR expression with enrichment of expression at the protein and RNA level in TNBC. This same heterogeneity was also identified in human BCC lines. There was a strong correlation between AR RNA expression and protein expression (R2= 0.72, p Conclusion: Our results implicate AR as a mediator of radioresistance in breast cancer and support the rationale for developing clinical strategies, including clinical trials, to inhibit AR as a novel radiosensitizing target in TNBC. Citation Format: Speers C, Zhao SG, Liu M, Rae JM, Hayes DF, Feng FY, Pierce LJ. Androgen receptor (AR): A novel target and mechanism for radiosensitization and treatment in triple-negative breast cancers (TNBC). [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 P6-13-05.

Background: Exemestane is a second generation steroidal aromatase inhibitor (AI) used for the treatment of estrogen receptor (ER) positive breast cancer in postmenopausal women. Variability in AI treatment efficacy and side effects seen across patients may be due, in part, to inter-patient differences in drug exposure. This exposure variability is likely caused by patient genetics factors, such as single nucleotide polymorphisms (SNPs) in drug metabolizing enzymes, or clinical factors such as patient body size, organ function, and comorbidities. The objective of this secondary correlative analysis was to identify genetic and clinical characteristics that affect steady state exemestane concentration, with a specific focus on the influence of inherited genetic variants and baseline hepatic function. Methods: 500 patients were enrolled on the Exemestane and Letrozole Pharmacogenetics (ELPh) Study and randomized to either drug. Clinical data and DNA were collected at baseline and blood samples were collected after 1 or 3 months of treatment to measure steady-state exemestane concentration via HPLC/MS. Genotyping was performed on a custom Sequenom MassARRAY iPLEX that included the recently discovered low activity CYP3A4*22 (rs35599367) SNP and several other SNPs with putative functional consequence in enzymes thought to be involved in exemestane metabolism (CYP1A1/2, CYP1B1, CYP3A4, CYP4A11, AKR1C3/4, AKR7A2). Our primary hypothesis was that patients carrying CYP3A4*22 variants would have higher serum exemestane concentrations. Other SNPs and clinical characteristics (hepatic and renal function, age, body mass index (BMI), time of sample collection, prior chemotherapy) were assessed for independent association, and then adjusted for in a multivariable tobit regression model for CYP3A4*22 on log-transformed censored exemestane concentration. Results: 246 (225 randomized to exemestane arm, 21 crossed-over from letrozole arm) patients had exemestane steady state levels and were evaluable in this analysis. As hypothesized, the CYP3A4*22 polymorphism (minor allele frequency=0.06) was associated with a 54% increase in exemestane concentration (95% CI: 14% - 109%, p40) at baseline (95% CI: 2% - 104%, p=0.02), 1% lower per unit increase in BMI (95% CI: 0% - 3%, p=0.05), and 20% lower in patients who received prior chemotherapy (95% CI: 4% - 34%, p=0.03). Age, renal impairment, and other SNPs were not associated with exemestane concentration. After adjustment for significant clinical covariates the CYP3A4*22 SNP remained significant (p Conclusions: Genetic and clinical predictors of exemestane concentration were discovered in a large cohort of prospectively enrolled estrogen responsive breast cancer patients. Ongoing analyses will determine whether the variability in exemestane concentration was associated with downstream effects on estrogen depletion or treatment-related toxicity. If so, these genetic and clinical characteristics could be useful for individualizing dosing of exemestane to ensure that all patients are receiving maximal benefit with minimal toxicity. Citation Format: Hertz DL, Kidwell KM, Seewald NJ, Gersch CL, Desta Z, Flockhart DA, Storniolo AM, Stearns V, Skaar TC, Hayes DF, Henry NL, Rae JM. CYP3A4*22 polymorphism is associated with increased exemestane concentrations in postmenopausal breast cancer patients. [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 P5-12-05.

Background: The anti-estrogen tamoxifen is metabolized into the more potent anti-estrogen, endoxifen, primarily by polymorphic CYP2D6. An association between CYP2D6 genotype and tamoxifen efficacy was assessed in a meta-analysis of 4,973 breast cancer patients by the ITPC. A subgroup analysis in 1,996 estrogen receptor (ER)-positive postmenopausal patients receiving 20[thinsp]mg/day tamoxifen for 5 years (criterion 1) found CYP2D6 poor metabolizer genotype was associated with worse invasive disease free survival (IDFS; hazard ratio (HR) 1.25, 95% confidence interval (CI) 1.06-1.47, P=0.009). This meta-analysis did not include data from two prospective trials (Anastrozole, Tamoxifen, Alone or In Combination (ATAC) and Breast Intergroup (BIG) 1-98) both of which meet the patient criteria but failed to replicate the ITPC findings. Some ITPC investigators criticized ATAC and BIG 1-98 for genotyping CYP2D6 from tumor-derived DNA, positing this leads to genotyping errors detected by Hardy-Weinberg Equilibrium (HWE). However, ITPC analyses did not exclude tumor-derived CYP2D6 genotypes or report HWE. Therefore, we re-analyzed the ITPC data to investigate claims that tumor-derived genotyping causes HWE departure and masks the association between CYP2D6 genotype and tamoxifen efficacy. Methods: The ITPC dataset was filtered to patients fulfilling criterion 1. HWE for CYP2D6*4 was analyzed in Caucasian patients (n=1,619) by study, by DNA source (tumor or blood), and in the entire subgroup. The ITPC meta-analysis was rerun stratified by DNA source using the same specifications (patients, phenotype, genotype, statistical model, etc.) as in ITPC. Results: Significant HWE deviation for CYP2D6*4, the most common variant (MAF =0.2), was not observed in any study genotyped from tumor but was observed in one study genotyped from blood. Combining studies led to significant HWE deviations in studies genotyped from both blood and tumor, and for the entire subcohort (Table 1). Associations between CYP2D6 genotype and IDFS stratified by DNA source yielded similar, non-statistically significant, results (blood: n=933, HR=1.19, 95% CI 0.91-1.57, P=0.21; tumor: n=997, HR=1.19, 95% CI 0.99-1.44, P=0.07). Conclusions: HWE deviations for CYP2D6*4 are not uniformly or exclusively found in studies using tumor DNA, but can occur as a statistical consequence of combining genotypes from heterogeneous populations like in the multi-institution BIG 1-98 and ATAC studies. Re-analysis of the ITPC dataset stratified by DNA source refutes the hypothesis that genotyping tumor DNA masks a pharmacogenetic association. These findings reaffirm the validity of the BIG 1-98 and ATAC analyses and support inclusion of these studies in the ITPC meta-analysis to rigorously assess the association between CYP2D6 genotype and tamoxifen efficacy. CYP2D6*4 HWE Test in Caucasian Patients from Each ITPC Study, by DNA Source, and CombinedDNA SourceStudy NumberNHWEBlood2700.68 4530.03 6130.28 84640.12 930.56 102220.0002 Total8250.0004Tumor51970.41 62170.04 83800.04 Total7940.0037CombinedTotal16190.000006 Citation Format: Kidwell KM, Hertz DL, Leyland-Jones B, Regan MM, Dowsett M, Rae JM. Analysis of the International tamoxifen pharmacogenomics consortium (ITPC) dataset shows that genotyping DNA derived from tumor does not introduce CYP2D6 genotyping error or mask an association with tamoxifen efficacy. [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 P6-09-02.

Purpose: Although cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors like palbociclib, ribociclib, and abemaciclib have improved progression free survival in patients with metastatic, estrogen receptor positive (ER+) breast cancer, acquired resistance to these drugs limits their efficacy. Despite promising new studies defining the utility of CDK4/6 inhibitors in the upfront, non-metastatic setting, there is limited data available on the effects of concurrent CDK4/6 inhibition and radiation (RT). Methods: Transcriptomic and proteomic expression data was used to quantify changes in RNA and protein expression in ER+ breast cancer cell lines (MCF-7, T47D) after short term (16 hour) CDK4/6 inhibition. Proliferation assays were used to determine the half-maximal inhibitory concentration (IC50) of palbociclib, ribociclib, and abemaciclib. Clonogenic survival assays were performed to calculate the radiation enhancement ratio (rER) and the surviving fraction at 2 Gy for each treatment. Homologous recombination (HR) proficiency was assessed using RAD51 and γH2AX foci formation and a pYFP reporter was used to assess non-homologous end joining (NHEJ) efficiency. Western blots were used to quantify protein expression. MCF-7 xenografts were used to study the efficacy of combination (palbociclib + RT) therapy in vivo. MCF-7 and T47D cell lines with acquired resistance to CDK4/6 inhibition (IC50 >1uM) were used for comparison in all assays. Results: Transcriptomic and proteomic analyses identified changes in expression of DNA damage response mediators and cell cycle machinery with short term CDK4/6 inhibition. Palbociclib significantly radiosensitized ER+ cell lines at concentrations at or below the IC50 value in clonogenic survival assays (MCF-7 rER: 1.22-1.52, T47D rER: 1.23-1.50) and led to a decrease in the surviving fraction of cells at 2 Gy (p < 0.001). Similar results were observed in ribociclib- (rER: 1.08 - 1.68) and abemaciclib-treated (rER: 1.19 - 2.05) cells. MCF-7 and T47D cells treated with CDK4/6 inhibition and RT showed a decrease in RAD51 foci formation, suggesting a decrease in HR efficiency (p < 0.001). However, CDK4/6 inhibition did not affect NHEJ efficiency (p > 0.05). CDK4/6 inhibition + RT led to a decrease in expression of protein expression of HR meditators like p-CHK1 but did not affect phosphorylation of NHEJ proteins like pKu80/pKu70. Cells with acquired resistance to CDK4/6 inhibition did not demonstrate radiosensitization (MCF-7 rER: 0.93 - 1.03, T47D rER: 0.96 - 1.11) or changes in RAD51 foci formation with combination treatment. Conclusions: Our data suggests that CDK4/6 inhibitor-mediated radiosensitization may be effective in ER+ breast cancers prior to the development of CDK4/6 inhibitor resistance. These studies provide preclinical rationale to test CDK4/6 inhibition + RT in women with locally-advanced ER+ breast cancer at high risk for locoregional recurrence. Citation Format: Andrea M. Pesch, Nicole Hirsh, Benjamin C. Chandler, Anna R. Michmerhuizen, Cassandra L. Ritter, Marlie Androsiglio, Kari Wilder-Romans, Meilan Liu, Christina L. Gersch, Jose M. Larios, James M. Rae, Corey W. Speers. CDK4/6 inhibitor-mediatated radiosensitization of estrogen receptor positive breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6280.

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 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.

Introduction: Fifty percent of ER positive MBC patients do not benefit from ET. Potential mechanisms of resistance to ET in this patient population include absence of ER expression by deletion or suppression, alteration in ER signaling pathway genes, or upregulation of multiple growth factor receptor pathways. We hypothesized that genotyping and phenotyping of CTC combined with genomic analysis of ptDNA will provide important insights into the multiple mechanisms of ET resistance and that a set of blood tests might serve as a "liquid biopsy" abrogating the need for tissue specimens. Methods: Twenty-four patients providing informed consent were enrolled into the Mi CTC-ONCOSEQ study, a companion trial to Mi-ONCOSEQ (the Michigan Oncology Sequencing Program). Seven of these patients (5 with ER immunohistochemistry (IHC) positive and 2 with ER negative cancers) who had available archived primary and metastatic cancer tissue, a research metastatic biopsy for genomic analysis, and who had ≥5CTC/7.5 ml whole blood (WB) characterized for ER protein (CTC-ER) are the focus of this report. All the patients were ET refractory. None of them was progressing on fulvestrant at the time of study entry. CTC enumeration and phenotyping was performed with CellSearch©. Circulating ptDNA was analyzed by droplet digital polymerase chain reaction (ddPCR). ER status from archived tissue was obtained from chart review. ER mRNA expression was determined in the research biopsy of metastatic tissue by using quantitative RNA sequencing. Mutational status of ER gene, ESR1, was determined by Next-gen Sequencing using the Illumina HiSeq 2500 platform. Results: The 2 control patients with triple negative breast cancer had negative CTC-ER. Discordance between CTC-ER and tissue ER by IHC was observed (Table). Two of the 5 ER positive patients retained CTC-ER positivity (39% and 19% of the CTC). One of them (#7) harbored an ESR1 mutation in the research biopsy tissue and in ptDNA, whereas the other (#14) had wild type (WT) ESR1. CTC-ER protein levels in patients #12, 17 and 24 were negative. All had WT ESR1 in the research biopsy tissue. Of note, patient #12 had WT ESR1 in the research biopsy, but an ESR1 mutation was detected in her ptDNA. Pt#CTC-ER Tissue-ER ESR1 status in research biopsyESR1 status in ptDNA N[deg]CTC/7.5ml WB% CTC-ER +Primary by IHCMet by IHCMet research biopsy by mRNA 71839%+++Y537SY537S141619%+NA+WTWT12130%+++WTD538G17160%++weakly+WTWT242750%+weakly+weakly+WTWT Conclusions: These exploratory data suggest heterogeneous mechanisms of resistance to ET in patients with previously determined ER-positive MBC, including ESR1 mutations in ER positive cases (seen in 2 patients) and loss of ER expression (seen in CTC of 3 patients). In contrast, other cancers continue to express WT ESR1, and therefore must have developed alternative mechanisms of resistance. At least 2 of these mechanisms can be detected and monitored with complementary circulating assays: CTC and ptDNA. Further investigations are needed to understand the heterogeneous mechanisms of resistance to ET. Citation Format: Paoletti C, Aung K, Cannell EM, Darga EP, Chu D, Kidwell KM, Thomas DG, Tokudome N, Brown ME, McNutt LM, Gersch C, Schott AF, Park BH, Robinson DR, Chinnaiyan AM, Rae JM, Hayes DF. Molecular analysis of cancer tissue, circulating tumor cells (CTC) and cell-free plasma tumor DNA (ptDNA) suggests variable mechanisms of resistance to endocrine therapy (ET) in estrogen receptor (ER) positive metastatic breast cancer (MBC). [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 P3-05-01.

Background Aromatase inhibitors (AI) can exert unfavorable effects on lipid profiles, but previous studies have reported inconsistent results. Given the intricate biological relationship between estrogen and lipid profiles, these mixed results may be explained in part by variation in genes encoding proteins involved in the drug's target and in estrogen metabolism and signaling. The purpose of this study was to investigate associations of single-nucleotide polymorphisms (SNP) in candidate genes with AI-mediated changes in lipid profiles. Methods We completed a prospective multicenter randomized observational open-label study to test the association of SNPs in candidate genes on biomarkers of estrogenic and anti-estrogenic activity in post-menopausal women with early breast cancer who were recommended adjuvant AI therapy. Eligible women were randomly assigned to exemestane or letrozole, and were followed for 2 years. We genotyped 137 SNPs from germ line DNA in the following candidate genes: ARVCF, COMT, CYP19A1, ESR1, ESR2, PGR, EP300, EZH2, NCOA1-3, NCOR1-2, NRIP, and PELP1. Lipid profiles including total cholesterol (TC), low-density lipoprotein (LDL), high-density lipoprotein (HDL), and triglycerides (TG) were measured at baseline and 3 months after initiating AI. We conducted genetic association data analysis and multivariate linear regressions to analyze the genetic effects using dominant, recessive, and additive models. Multivariate analysis included age, body mass index, prior hormone replacement therapy, and prior tamoxifen. To adjust for multiple comparisons, only SNPs with a p Results We enrolled 502 women in to the study, but for this analysis we excluded women who did not have genetic data (n = 33), had incomplete data (n = 23), discontinued or crossed over AI therapy (n = 48), women not fasting at both time points (n = 89), or those on lipid-lowering medications (n = 162). A total of 200 women were evaluable (letrozole 107, exemestane 93). Lipid profiles in all patients (n = 200) at baseline and 3 months after initiating AI, respectively, were as follows: TC 204.9 and 203.3 (unchanged, p = 0.43); HDL 61.3 and 56.8 (decreased, p = 6.3E-10); LDL 122.2 and 124.6 (unchanged, p = 0.22); and TG 107.1 and 103.6 (unchanged, p = 0.26). Genetic association and multivariate analysis revealed that SNPs in ESR1 and NCOR1 are significantly associated with additional changes in lipid parameters as summarized in Table 1. Table 1.Significant findings of multivariate linear regressions analyzing genetic associations between candidate gene SNPs and lipid profiles of AI-treated women.CohortNumberSNP (gene)Minor Allele FrequencyLipid ParameterModel UsedMean Absolute Change (mg/dL)P-valueAll patients184rs9340958 (ESR1)0.07TCRecessive-2.250.0003Letrozole96rs9340958 (ESR1)0.07TCRecessive5.280.00009 101rs3020368 (ESR1)0.09TCRecessive6.350.00007Exemestane93rs3798758 (ESR1)0.03HDLDominant, additive-7.970.00001 88rs926848 (ESR1)0.03HDLDominant, additive-7.970.00002 93rs61753150 (NCOR1)0.01TGDominant, additive-11.630.00003 Conclusions Variants in genes involved in estrogen metabolism and signaling are associated with changes in lipid profiles in AI-treated women and should be validated in other studies. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-08-11.

Introduction: Only ∼ 50% of patients (pts) with estrogen receptor (ER) positive metastatic breast cancer (MBC) benefit from endocrine therapy (ET). Currently only clinical judgment can be used to identify pts with endocrine-refractory MBC, who are better palliated with chemotherapy. Circulating Tumor Cells (CTC) are reliably enumerated using an automated immunomagnetic system (CellSearch®; Veridex LLC). High CTC levels predict rapid progression in pts with MBC. We have developed a multi-parameter assay, the CTC-Endocrine Therapy Index (CTC-ETI) using CellSearch® that may identify pts with ER positive MBC who are unlikely to benefit from ET and may be better served with chemotherapy. CTC-ETI scores are assigned based on CTC levels coupled with the relative percent and degree of marker positivity on the CTC. We report preliminary results from a pilot single institutional study. Methods: CellSearch® has 4 fluorescence channels. Three distinguish CTC from WBC (DAPI, anti-cytokeratin, anti-CD45). The 4th “empty” channel was used to measure ER, BCL-2, HER-2, and Ki-67 expression with fluorescent-labeled antibodies. These 4 markers reflect sensitivity (ER, BCL-2) or resistance (HER-2, Ki-67) to ET. Forty ml of blood was drawn into 4 CellSave® tubes from pts with progressive MBC. Whole blood from 4 tubes was pooled and divided into 4 different 7.5 ml aliquots of blood, which were processed and characterized for CTC counts and each of the four molecular markers using the CXC CellSearch® kit. Results: 21 pts have been accrued to the feasibility study. One patient was ineligible. Five of 20 pts had low CTC counts ( Conclusions: ER, BCL-2, HER-2, and Ki-67 can be accurately determined on CTC using the 4th channel in the CellSearch® system to calculate CTC-ETI. We predict that lower CTC-ETI scores (low or no CTC, or CTC with high CTC ER and BCL-2 and low CTC HER-2 and Ki-67) could be associated with favorable response to ET. Successful completion of the feasibility study will lead to a prospective trial to determine if high CTC-ETI at baseline predicts resistance and rapid progression on ET in women starting a new endocrine therapy for MBC. Supported by Veridex, LLC, Fashion Footwear Charitable Foundation of New York/QVC Presents Shoes on Sale ™ (DFH), Associazione Sandro Pitigliani and by a studentship from FIRC (CP). Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-07-16.

Aim: To assess whether candidate polymorphisms in CYP19 (aromatase) and TCL1A were associated with recurrence, musculoskeletal symptoms (MSKs) or hot-flushes in primary estrogen receptor-positive breast cancer in postmenopausal women treated with tamoxifen or anastrozole. Background. Recently, variants rs10459592 and rs4775936 in CYP19 (aromatase) have been reported to be associated with aromatase inhibitor (AI) response in patients with metastatic breast cancer (Park, et al, CCP, 2011) and variants in CYP19 and at the 3’ end of TCL1A (rs11849538) with AI-induced arthralgia (Ingle, et al, JCO, 2010). These findings require validation before clinical application. The ATAC trial was a prospective randomized double-blind placebo-controlled trial that compared the adjuvant use of anastrozole versus tamoxifen for 5 years. The trial's detailed efficacy and safety data, long term (10-year) follow-up and high event rate make it an ideal setting for pharmacogenetic single nucleotide polymorphism (SNP) validation studies. Here we report our findings testing for correlations with 3 SNPs in CYP19 and TCL1A with clinical outcomes including any disease recurrence, distant recurrence, MSKs, and hot-flushes. Methods: Genotypes were determined using ABI Taqman assays without knowledge of the patient's treatment assignment or outcomes. Hazard ratios (HR) and corresponding 95% confidence intervals (CI) were estimated by the proportional hazards regression model and were adjusted for: age, tumor size, grade, and nodal status (for survival analyses) and age, hormone replacement therapy, body mass index, and smoking (for toxicity analyses). Results: 807 patients were genotyped for CYP19 SNPs rs10459592 and rs4775936 and 842 for TCL1A SNP rs11849538. The rs10459592 genotype frequencies were 16.6% Wt/Wt, 51.5% Wt/Vt, and 32% Vt/Vt. There was a trend towards decreased recurrence in Vt/Vt and Vt/Wt compared to Wt/Wt (HR=0.77 [0.50−1.22] and 0.68 [0.42−1.09], p[trend]=0.1), similar for both anastrozole- and tamoxifen-treated patients and also similar for distant recurrence. For the same SNP, there was a trend toward more MSKs in Vt/Vt and Vt/Wt compared to Wt/Wt (HR=1.22 [0.80−1.88] and 1.47 [0.94−2.30], p[trend]=0.08), primarily due to the effect in the tamoxifen cohort (HR=1.88 [0.89−3.99], p[trend]=0.1). The rs4775936 genotype frequencies were 26.0% Wt/Wt, 52.1% Wt/Vt and 21.9% Vt/Vt. Trends in the same direction as those for rs10459592 were found with rs4775936, albeit less significant. The TCL1A genotype frequencies were 77.5% Wt/Wt, 20% Wt/Vt and 2.5% Vt/Vt. No association was observed between TCL1A genotypes and recurrences or MSKs, however, the small number of Vt/Vt patients on tamoxifen had greater incidence of hot flushes (HR=1.99 [0.9−3.91], p=0.09). Conclusion: These data do not support an association between the TCL1A 3’ variant and AI-induced MSKs independent of severity. Our data do support the CYP19 SNP rs10459592 and the loosely linked rs4775936, which previously were associated with letrozole efficacy, as probably being associated with better disease outcome on anastrozole and tamoxifen and with worse drug side effects. The degree of association requires definition in further data sets. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-06-02.

Background. A number of studies have provided widely heterogeneous results for and against the hypothesis that genetic variants in drug metabolizing enzymes can influence patient response to tamoxifen. Most of these studies are confounded by relatively small numbers of patients, lack of comprehensive genotype information, lack of detailed clinical outcome data, and patient selection biases. The ATAC trial was a prospective randomized double-blind placebo-controlled trial designed to compare the adjuvant use of anastrozole versus tamoxifen for 5 years. The trial's detailed efficacy and safety data, long term (10-year) follow-up and high number of events make it an ideal setting for pharmacogenetic analyses. Here we report our initial findings testing for correlations with SNPs in CYP2D6 and UGT2B7, the primary enzymes responsible for the presumed activation and inactivation of tamoxifen, respectively, with clinical outcomes including any disease recurrence, distant recurrence, and overall survival. Methods: CYP2D6 genotype data for the 7 most common alleles were used to assign each patient a ‘score’ based on predicted allele activities from 0 (no activity) to 2 (high activity). Patients were also tested for the UGT2B7*2 variant, which has lower enzymatic activity and has been shown to correlate with higher serum endoxifen concentrations. All genotype determinations were made without knowledge of the patient's treatment assignment or outcomes. Results: Comprehensive CYP2D6 genotype data were obtained on 588 and 615 patients randomized to receive tamoxifen, and anastrozole, respectively. After median 10 years of follow-up, there were 115 recurrences in the tamoxifen cohort. We found no associations between any of the CYP2D6 scores and rates of recurrence in tamoxifen treated patients. A test for trend across CYP2D6 scores was not significant. In the anastrozole cohort, there were 92 recurrences, and there was no evidence of an association between CYP2D6 score with rates of recurrence. Likewise, there was no detectable association in either the tamoxifen or anastrozole group with UGT2B7 genotype alone or in combination with CYP2D6 score. Concomitant SSRI usage was recorded in these patients and is being assessed for possible impact on these results. Conclusion: These data from a large prospective clinical trial, with detailed outcome data and long-term follow-up do not support the hypothesis that patient with decreased CYP2D6 enzymatic activity receive less benefit from tamoxifen therapy compared to wild-type CYP2D6 patients. Variants in drug targets and estrogen signaling pathways may be the genetic basis for patient variability in anti-estrogen risk versus benefit profiles. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr S1-7.

Circulating tumor cells (CTC) are emerging as a powerful prognostic and predictive biomarker in several types of cancer, including breast, colon, and prostate. Studies of CTC in metastasis and further development of CTC as a biomarker in cancer have been limited by the inability to repetitively monitor CTC in mouse models of cancer. We have validated a method to enumerate CTC in blood samples obtained from living mice using a modified version of an in vitro diagnostic system for quantifying CTC in patients. Different routes of blood collection were tested to identify a method to reproducibly recover CTC from tumor-bearing mice without interference from contaminating normal murine epithelial cells. CTC are present in blood samples from mice bearing orthotopic xenografts of several different breast cancer cell lines and primary breast cancer cells from patient biopsies. We also show that this technology can be used for serial monitoring of CTC in mouse xenograft models of human breast cancer. These results establish a new method for studying CTC in mouse models of epithelial cancer, providing the foundation for studies of molecular regulation of CTC in cancer and CTC as biomarker for therapeutic efficacy. [Cancer Res 2008;68(14):5529–32]

Background: We previously performed an informatics-based analysis on RNA sequencing libraries from 7,256 tumor and normal tissue specimens to delineate the landscape of long noncoding RNAs (lncRNAs) in the human transcriptome. This analysis identified 58,648 lncRNAs, including over 45,000 novel transcripts (Iyer MK et al, Nature Genetics, 2015). We now interrogate this lncRNA compendium to identify top candidate estrogen receptor (ER)-associated lncRNAs in breast cancer and characterize their association with disease progression. Methods: To prioritize differentially expressed lncRNAs in cancer vs normal tissue, and in ER+ vs ER- disease, we performed Sample Set Enrichment Analysis (SSEA) on >1000 RNA Seq libraries, from breast cancer and normal tissue samples from The Cancer Genome Atlas project. The effect of the top prioritized lncRNA on cancer phenotypes was studied via in vitro proliferation, colony formation, invasion and tamoxifen resistance assays in MCF7 and T47D cells, and via in vivo mouse xenograft studies and chick chorioallantoic membrane (CAM) assays. To study the mechanism by which this lncRNA promotes tumor progression, we identified its top protein interactors and subdomains responsible for function, and then studied the effects of disrupting function of this lncRNA on cancer phenotypes. Finally, in a "guilt-by-association" study, we developed a signature of 150 protein coding genes most strongly associated with our lncRNA of interest, and investigated the association of this signature with clinical outcomes using Oncomine analyses. Results: SSEA analysis on over 1000 TCGA samples nominated Breast Cancer Associated Transcript (BRCAT 431) as the top overexpressed ER-regulated lncRNA in breast cancer. In vitro experiments demonstrate that siRNA-mediated knockdown of BRCAT431 resulted in significantly decreased proliferation, colony formation, and invasion (by >50% in most assays). Tamoxifen resistance was associated with significantly increased BRCAT431 levels in both MCF7 and T47D cells, and knockdown of BRCAT431 reversed tamoxifen resistance. In vivo xenograft and CAM studies demonstrate that knockdown of BRCAT431 also significantly decreased xenograft growth and tumor invasion by >50%. RNA pulldown followed by mass spectrometry identified the RNA binding protein hnRNPL as a key protein interacting with BRCAT431. Deletion studies identified a 27 base region of BRCAT431 necessary for its interaction with hnRNPL, and loss of this region abrogated BRCAT431- induced invasion. Finally, guilt-by-association studies demonstrate a strong association between BRCAT431 overexpression and tumor grade, recurrence, and metastases. Conclusion: In this study, we develop the largest reported compendia of breast cancer lncRNAs. We prioritize BRCAT431 as the top lncRNA upregulated in ER-positive breast cancers, and demonstrate that it confers aggressive oncogenic phenotypes in vitro and in vivo. We identify a novel mechanism by which this lncRNA functions. Our results suggest that by promoting tamoxifen resistance, BRCAT431 increases the clinical risk of recurrence and metastases in breast cancer. Overall, this study supports the rationale for investigating lncRNAs as novel biomarkers and therapeutic targets in breast cancer. Citation Format: Feng FY, Niknafs Y, Han S, Ma T, Speers C, Malik R, Evans J, Zhang C, Pierce LJ, Hayes DF, Rae JM, Chinnaiyan AM. Interrogating the landscape of long noncoding RNAs in breast cancer to identify predictors of tamoxifen resistance. [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 S4-05.

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.

Purpose: GATA3 is involved in estrogen signaling and mammary cell differentiation, and is frequently mutated in breast cancer. Germline variations of GATA3 are prognostic in childhood acute lymphoblastic leukemia (ALL). Thus, we sought to examine their prognostic and predictive role in breast cancer. Methods: SWOG S8897 comprised two groups of breast cancer patients. In the first high-risk group, women were randomly assigned to CAF vs. CMF, and secondarily randomized to tamoxifen or not. In the second low risk group, women did not receive any adjuvant therapy after surgery. Germline DNA was extracted from uninvolved axillary lymph nodes and 12 candidate GATA3 SNPs were genotyped. Associations of genotypes with treatment outcomes were evaluated in 441 women in the treated and 799 in the untreated groups separately. Hazard ratio (HR) for disease free (DFS) and overall survival (OS) for each SNP was estimated using multivariate Cox hazard regression. Further stratified analyses by tamoxifen were performed in the treated group. Results: After correcting for multiple testing, we identified significant associations of two variants (rs3802604 and rs568727) with DFS and OS for patients who received adjuvant chemotherapy. Women carrying the variant GG genotype at rs3802604 had significantly poorer DFS (HR = 1.95, 95% CI: 1.27-2.99, p = 0.002) and OS (HR = 2.45, 95% CI: 1.48-4.05, p = 0.0005), compared to women carrying the common AA genotype. Associations of similar magnitude were found for rs568727. In contrast, no association with either SNP was found in the untreated group. Subgroup analysis suggested that these two SNPs more strongly influenced treatment outcomes in the patients who also received tamoxifen. Seven additional variants were also associated with OS and DFS in the tamoxifen treated group. Only 3 of the 12 SNPs analyzed in GATA3 were not associated with survival in this subgroup; however, these three SNPs (rs3781093, rs3824662, and rs3802600) associated with OS in the group not treated with tamoxifen. Functional annotation revealed that several GATA3 SNPs, e.g., rs3802604, rs369421, and rs3824662, are likely to function by affecting transcription factor binding and/or altering regulatory chromatin states at this locus. Conclusions: GATA3 harbors common germline genetic variants that are related to survival following adjuvant chemotherapy and endocrine therapy in breast cancer patients. Citation Format: Victoria L. Larsen, William E. Barlow, Jun J. Yang, Qianqian Zhu, Laura F. Hutchins, Susan A. Kadlubar, Kathy S. Albain, Robert B. Livingston, James M. Rae, I-Tien Yeh, Peter M. Ravdin, Silvana Martino, Alan P. Lyss, C. Kent Osborne, Gabriel N. Hortobagyi, Daniel F. Hayes, Christine B. Ambrosone, Song Yao. Germline genetic variants in GATA3 and breast cancer treatment outcomes in SWOG 8897 trial. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2032.

Approximately 70% of all breast cancers express the Estrogen Receptor (ER) and inhibition of the ER signaling remains the mainstay of systemic therapy in such cancers. The advent of endocrine therapies (ET) have contributed significantly to reduction in the incidence, recurrence and mortality associated with breast cancer. However, approximately one-third of patients with ER-positive breast cancer do not benefit from adjuvant ET, and nearly all patients with ER-positive metastatic breast cancer (MBC) ultimately become refractory to all known ETs. While several mechanisms of resistance to ET have been proposed, there are currently no clinically effective treatments for ET resistance. Thus, ET resistance remains a major obstacle to the effective treatment of a significant subset of ER-positive breast cancers. We have recently reported recurrent somatic mutations in the ligand binding domain (LBD) of the Estrogen Receptor gene (ESR1) in metastatic breast cancer patients with a history of prior treatment with ET. The ESR1 mutations result in constitutive activation of the ER and confer ET resistance when ectopically expressed in ER-positive cell lines. In an attempt to develop novel therapeutic strategies to treat ET-resistant breast cancer, we have evaluated OTX015, a clinical bromodomain and extraterminal (BET) domain inhibitor, in pre-clinical models of ET resistance due to ESR1 mutations. Our studies suggest that BET inhibition may serve as an attractive strategy for treatment of ET resistance due to ESR1 mutations. Citation Format: Prasanna G. Alluri, Jose Larios, Rohit Malik, James Rae, Arul M. Chiinaiyan. Targeting estrogen receptor mutations for treatment of endocrine therapy resistance in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2156.

Background: We have previously performed a bioinformatics-based analysis on 7,256 RNA sequencing libraries from tumors, normal tissues, and cell lines to delineate the landscape of long noncoding RNAs (lncRNAs) in the human transcriptome. This analysis identified 58,648 lncRNAs, including over 45,000 novel transcripts (Iyer and Niknafs et al, Nature Genetics, 2015). We now interrogate this lncRNA compendium to identify top candidate estrogen receptor (ER)-associated lncRNAs in breast cancer and characterize their association with disease progression. Methods: To identify and prioritize lncRNAs that were differentially expressed in cancer vs normal tissue, and in ER-positive vs ER-negative disease, we performed differential expression testing using a previously described non-parametric tool for RNA-seq count data called Sample Set Enrichment Analysis (SSEA) on over 1000 RNA sequencing libraries including breast cancer and normal tissue samples from the The Cancer Genome Atlas (TCGA) project. The effect of the top prioritized lncRNA on cancer phenotypes was studied via proliferation, colony formation, invasion and tamoxifen resistance assays in MCF7 and T47D cell lines, and via mouse xenograft studies in vivo. The mechanism by which this lncRNA promotes tumor progression was investigated by identifying its top protein interactors and its subdomains responsible for function, and then studying the effects of disrupting function of this lncRNA on cancer phenotypes. Finally, in a “guilt-by-association” study, we developed a signature of 150 protein coding genes most strongly associated with our lncRNA of interest, and investigated the association of this signature with tumor grade, recurrence, and metastases using Oncomine analyses. Results: Differential expression analyses on over 1000 TCGA RNA-seq samples nominated Breast Cancer Associated Transcript (BRCAT431) as the top overexpressed ER-regulated lncRNA in breast cancer. In vitro experiments demonstrate that shRNA-mediated knockdown of BRCAT431 resulted in significantly decreased proliferation, colony formation, and invasion (by >50% in most assays). Tamoxifen resistance was associated with significantly increased BRCAT431 levels in both MCF7 and T47D cells, and knockdown of BRCAT431 reversed tamoxifen resistance. In vivo xenograft studies demonstrate that knockdown of BRCAT431 also significantly decreased xenograft growth and tumor invasion by >50%. RNA pulldown followed by mass spectrometry identified the RNA binding protein hnRNPL as a key protein interacting with BRCAT431. Deletion studies identified a 27 base-pair region of BRCAT431 necessary for its interaction with hnRNPL, and loss of this region abrogated BRCAT431-induced invasion. Finally, guilt-by-association studies demonstrate a strong association between BRCAT431 overexpression and tumor grade, recurrence, and metastases. Conclusion: In this study, we develop the largest reported compendium of breast cancer lncRNAs. We prioritize BRCAT431 as the top lncRNA upregulated in ER-positive breast cancers, and demonstrate that it confers aggressive oncogenic phenotypes in vitro and in vivo. We identify a novel mechanism by which this lncRNA functions through interaction with hnRNPL. Our results suggest that by promoting tamoxifen resistance, this lncRNA increases the clinical risk of recurrence and metastases in breast cancer. Citation Format: Yashar S. Niknafs, Sumin Han, Teng Ma, Chao Zhang, Matthew K. Iyer, Rohit Malik, Anton Poliakov, Corey Speers, Daniel Hayes, James Rae, Felix Y. Feng, Arul M. Chinnaiyan. Interrogation of the landscape of long noncoding RNAs in breast cancer to identify an ER-regulated predictor of tamoxifen resistance. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr B19.

Introduction: Somatic mutations, including those in TP53, PIK3CA, and estrogen receptor alpha (ESR1), are key to the biology of cancer and response to therapy. Recently, somatic cancer-associated mutations have been identified in circulating cell free plasma tumor DNA (ptDNA). Less is known about the mutation profile of DNA extracted from CTC (CTC-DNA). Since CTC-DNA provides mutational information of single cells, we hypothesize CTC-DNA will complement ptDNA to give greater insight into tumor heterogeneity. Methods: Patients with ER positive MBC who were enrolled in the Mi CTC-ONCOSEQ, a companion trial to Mi-ONCOSEQ (the Michigan Oncology Sequencing Program), and who had ≥5CTC/7.5 ml whole blood were included. CTC were enriched from white blood cells (WBC) with CellSearch© (CXC kit). CTC and WBC were then purified using DEPArrayTM. DNA from individual CTC and WBC was isolated and subjected to whole genomic amplification (Ampli 1TM WGA). Genetic analysis was performed on individual CTC, pooled CTC and pooled WBC DNA by multiplexed PCR based targeted next generation sequencing (NGS) using the Oncomine Comprehensive Panel (targeting ∼130 onco- and tumor suppressor genes) and the Ion Torrent Proton. All patients had exome sequencing performed on research biopsies of metastases using an Illumina HiSeq 2500 platform. Results: This pilot study was conducted using high quality DNA from two patients assessed to date. Both patients had lobular carcinoma and as expected harbored somatic, deleterious CDH1 (E-cadherin) mutations (frameshift and non-sense) in both research biopsy and CTC-DNA. These data supported our approach. Patient #1 was TP53 wild type in her research biopsy, but multiple CTC harbored somatic TP53 frame-shift mutations (Table). Patient #2 harbored an ESR1 Y537S mutation in her research biopsy. However, only 4 of 7 CTC also harbored this somatic, heterozygous mutation. Prioritized mutations in CTCPt#Cell Type (CTC vs WBC), numberGeneMutationVariant fraction (expected 1=homozygous; 0.5=heterozygous)Found in research biopsy?1CTC_A2CDH1p.I584fs1YES CTC_A4 1 CTC_A7 0.54 CTC_pool* 0.74 WBC_pool 0 CTC_A2TP53p.152_156del1NO CTC_A4 1 CTC_A7 0.51 CTC_pool* 0.88 WBC_pool 0 2CTC_A9ESR1p.Y537S0.52YES CTC_D1 0.34 CTC_D2 0.46 CTC_D6 0.65 CTC_pool* 0.35 WBC_pool 0 CTC_A12 0 CTC_D3 0 CTC_D7 0 CTC_A12CDH1p.Q641X1YES CTC_A9 1 CTC_D1 1 CTC_D3 1 CTC_D6 1 CTC_pool* 1 WBC_pool 0 * pool of all CTC Conclusions: We demonstrate the ability to purify CTC, isolate, and amplify DNA of suitable quality for genetic analysis using a comprehensive targeted sequencing panel. Both known and novel alterations were identified in comparison to research biopsy specimens. This approach allows single cell analysis demonstrating heterogeneity of mutational status in different single cells. Studies of CTC-ESR1 and other genetic abnormalities in patients with known tissue mutations who participated in Mi CTC-ONCOSEQ are now underway. Citation Format: Paoletti C, Cani AK, Aung K, Darga EP, Cannell EM, Hovelson DH, Yazdani M, Blevins AR, Tokudome N, Larios JM, Thomas DG, Brown ME, Gersch C, Schott AF, Robinson DR, Chinnaiyan AM, Bischoff F, Hayes DF, Rae JM, Tomlins SA. Somatic genetic profiling of circulating tumor cells (CTC) in metastatic breast cancer (MBC) patients. [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 P2-02-19.

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 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.

Androgen and estrogen steroid hormones are potent growth stimuli for both prostate and breast cancer. Cytochrome P450 17A1 (CYP17A1), a P450 monooxygenase, is required for the synthesis of these hormones and is the target of abiraterone, a drug approved for the treatment of castration-resistant prostate cancer. CYP17A1 catalyzes the 17α hydroxylation of the C21 steroids pregnenolone and progesterone and has a lyase activity that cleaves the C17-20 bond to produce the sex steroid precursors, dehydroepiandrosterone (DHEA) and androstenedione. We identified twelve nonsynonymous single nucleotide polymorphisms (SNPs) in CYP17A1 and hypothesized that the resultant amino acid changes might alter hydroxylase and lyase activity and thereby impact steroid hormone synthesis. This hypothesis was examined by transiently transfecting HEK293T cells, with either wild-type or polymorphic CYP17A1 constructs. Cells were treated for 3 hours with tritiated ([3H]) progesterone and the production of hydroxylated (OH) metabolites analyzed by HPLC. In cells transfected with wild-type CYP17A1, 8.11% and 55.5% of the [3H]-progesterone was recovered as 16-OH progesterone, and 17-OH progesterone, respectively. Using the ratio of 17-OH progesterone to 16-OH progesterone as a quantitative measure of CYP17A1 enzyme activity, wild-type CYP17A1 yields a ratio of 6.9+/- 0.05. In contrast, the SNP (rs200063521) which causes an aspartic acid to histidine amino acid change at residue 216, results in a 2.2 fold decrease in 16OH-progesterone synthesis compared to wild-type suggesting altered hydroxylase function. CYP17A1 protein expression, as determined by western blot analysis, suggests that a glycine to arginine conversion at residue 162 (rs141821705) results in decreased protein levels. Studies to further characterize the CYP17A1 variants are underway. This work could lead to a better understanding of the role of CYP17A1 SNPs in cancer progression and response to CYP17A1 targeted therapies. Citation Format: Cameron P. Capper, Jiayan Liu, Lauren McIntosh, José M. Larios, Michael D. Johnson, Paul F. Hollenberg, Richard J. Auchus, James M. Rae. Using a cell based model system to characterize CYP17A1 genetic variants. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1863. doi:10.1158/1538-7445.AM2015-1863

Background: Long noncoding RNAs (lncRNAs) have recently been associated with the development and progression of a variety of human cancers. To date, the interplay between known oncogenic drivers, such as estrogen receptor (ER), and lncRNAs has not been well described. In this study, we identify M41 as the top outlier lncRNA in ER-positive vs ER-negative breast cancer and investigate its role in preclinical cancer phenotypes and clinical outcomes. Methods and Materials: RNA sequencing was performed on 89 breast cancer samples and cell lines, including 42 ER+ cases, and a modified cancer outlier analysis was used to identify lncRNAs enriched in ER-positive disease. To assess ER regulation of the top enriched lncRNA (M41), ChIP-Seq and ChIP-PCR was used to detect binding of ER to M41 promoter and qPCR was used to determine changes in M41 expression following 10 nM estradiol treatment in MCF7 and T47D cells. Following knockdown via siRNA, the impact of M41 expression was assessed on cell invasion, migration, proliferation, and anchorage-independent growth. The impact of M41 knockdown on tamoxifen sensitivity was assessed by cell proliferation studies in MCF7 cells with acquired tamoxifen resistance. Lastly, clinical associations between M41 expression and grade/node status, as well as event-free survival (EFS), was determined using ANOVA and Kaplan-Meier analyses of TCGA samples. Results: M41, an uncharacterized lncRNA located on chr21q22.2, was identified as the top outlier lncRNA in ER-positive vs ER-negative breast cancer. M41 demonstrated outlier expression (RPKM values>50) in 15% of ER-positive cancers, and was not significantly expressed in normal breast tissue. ChIP studies show that ER robustly binds to the M41 promoter. Estradiol stimulation significantly increased M41 expression in a time-dependent manner. Knockdown of M41 significantly inhibited all assessed oncogenic phenotypes in the ER-positive MCF7 and T47D cells, with a 60-80% decrease in both invasion and anchorage-independent growth, but had no effect in the ER-negative MDA-MB-231 cell line (which has minimal M41 expression). M41 expression was greater than 10-fold higher in tamoxifen-resistant MCF7 cells compared to parental controls (p Conclusion: We have identified M41 as an ER-associated oncogenic lncRNA that contributes to preclinical cancer phenotype, promotes tamoxifen resistance in cell line models, and associates with poor outcomes in clinical samples. We suggest that M41 represents a novel biomarker candidate for the prognosis of ER-positive breast cancers and provides new insight into the biological complexity of breast tumor biology. Citation Format: Felix Y Feng, Teng Ma, Corey Speers, Matthew K Iyer, Shuang Zhao, John R Prensner, James M Rae, Lori J Pierce, Arul M Chinnaiyan. The long noncoding RNA M41 promotes aggressiveness and tamoxifen resistance in ER-positive breast cancers [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr PD6-1.

Breast cancer presents as either estrogen receptor α (ERα) positive or negative, with ERα+ tumors responding to antiestrogen therapy and having a better prognosis. By themselves, mRNA expression signatures of estrogen regulation in ERα+ breast cancer cells do not account for the vast molecular differences observed between ERα+ and ERα− cancers. In ERα− tumors, overexpression of epidermal growth factor receptor (EGFR) or c-erbB-2, leading to increased growth factor signaling, is observed such that mitogen-activated protein (MAP) kinase (MAPK) is significantly hyperactivated compared with ERα+ breast cancer. In ERα+/progesterone receptor–positive, estrogen-dependent MCF-7 breast cancer cells, we stably overexpressed EGFR or constitutively active erbB-2, Raf, or MAP/extracellular signal-regulated kinase kinase, resulting in cell lines exhibiting hyperactivation of MAPK, estrogen-independent growth, and the reversible down-regulation of ERα expression. By global mRNA profiling, we found a “MAPK signature” of ∼400 genes consistently up-regulated or down-regulated in each of the MAPK+ cell lines. In several independent profile data sets of human breast tumors, the in vitro MAPK signature was able to accurately distinguish ER+ from ER− tumors. In addition, our in vitro mRNA profile data revealed distinct mRNA signatures specific to either erbB-2 or EGFR activation. A subset of breast tumor profiles was found to share extensive similarities with either the erbB-2-specific or the EGFR-specific signatures. Our results confirm that increased MAPK activation causes loss of ERα expression and suggest that hyperactivation of MAPK plays a role in the generation of the ERα− phenotype in breast cancer. These MAPK+ cell lines are excellent models for investigating the underlying mechanisms behind the ERα− phenotype. (Cancer Res 2006; 66(7): 3903-11)

The cytochrome P450 17A1 (CYP17A1) inhibitor abiraterone, which blocks synthesis of all steroid hormones including androgens and estrogens, is an effective treatment for castrate resistant prostate cancer. Its utility for treating hormone receptor-positive breast cancer has yet to be established. In prostate cancer, the anti-proliferative effects of abiraterone are mediated by inhibiting CYP17A1 activity resulting in a significant reduction in circulating concentrations of the sex steroids dehydroepiandrosterone (DHEA), androstenedione, testosterone, and dihydrotestosterone (DHT). Recently, it has been proposed that the reduction in aromatizable androgens by abiraterone may make this drug useful for the management of hormone-dependent breast cancer. However, data from our lab suggests that abiraterone has estrogenic properties and can induce proliferation in breast cancer cells expressing the estrogen receptor (ER). Crystal violet assays were used to assess abiraterone-induced proliferation in the ER-positive, estrogen-dependent breast cancer cell lines MCF-7 and T47D. We show that abiraterone induced a dose dependent increase in cell proliferation with an IC50 of 3.7 μM and maximal stimulation (200%) observed at 8μM, compared to vehicle treated cells. Abiraterone also induced the expression of the ER response gene, GREB1. Abiraterone-induced proliferation and gene expression was blocked by increasing doses of the selective estrogen receptor down-regulator (SERD) fulvestrant. Abiraterone induced the dose-dependent expression of luciferase in MCF-7 cells transfected with an estrogen responsive luciferase reporter construct. In conclusion, our data suggest that abiraterone can induce ER-positive breast cancer cell proliferation in vitro by acting as a weak ER agonist. Further studies are underway to determine whether estrogenic effects of abiraterone are also observed using in vivo models of ER response. If abiraterone exhibits estrogenic effects in vivo then these data would suggest that abiraterone should be combined with an ER antagonist if used for the clinical management of women with ER+ tumors. Citation Format: Cameron P. Capper, Michael D. Johnson, José M. Larios, James M. Rae. CYP17A1 and abiraterone: Implications for breast cancer endocrine therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5568. doi:10.1158/1538-7445.AM2014-5568

Introduction: Fulvestrant is a selective estrogen receptor down-regulator (SERD). Recent studies have shown that the efficacy of fulvestrant is dose-related. However, at the higher dose (500 mg/month) most cancers develop resistance and progress. We previously reported expression of several markers, including estrogen receptor (ER) and BCL-2, on breast cancer circulating tumor cells (CTC) using CellSearch®. We now report pilot data showing inter-patient heterogeneity of these markers on CTC in patients with known ER positive breast cancer whose disease is progressing on fulvestrant. Methods: We conducted a pilot trial to determine the analytical validity of measuring expression of markers of endocrine sensitivity (ER, BCL-2) or resistance (HER-2, Ki-67) with fluorescent-labeled antibodies using the CellSearch® system. Patients with ER positive metastatic breast cancer (MBC) whose disease was progressing on any type of therapy were eligible after signed informed consent. This report is limited to the subjects who were progressing on fulvestrant. Whole blood (WB) was characterized for CTC counts and each of the four molecular markers using the CXC CellSearch® kit. Results: Of 50 enrolled patients, seven were progressing on fulvestrant. Two patients had no detectable CTC, while five patients had an average of ≥5 CTC/7.5 mL WB. Results are shown in a table below: CTC-ERCTC-BCL-2Patient #Fulvestrant dose (mg/month)Days since last doseN CTC/7.5 mL of WB% of CTC-ER+N CTC/7.5 mL of WB% of CTC-BCL-2+295002880%110%4550028170%170%2250341010%714%850031812%1735%172507728%367% These exploratory data suggest widely different mechanisms of resistance to fulvestrant in different patients with ER positive MBC. In two of the patients (29, 45) treated with 500 mg/month, both CTC-ER and CTC-BCL-2 expression were absent, suggesting no signaling through the ER pathway. We hypothesize either that fulvestrant was actively down-regulating ER, but the cancers had adopted other growth and survival pathways, or that ER negative, hormone-independent clones had evolved. In the other three cases, ER was clearly present with evidence of signaling, based on BCL-2 expression. Two of these patients (2, 17) were on the lower dose of fulvestrant, now considered to be less effective. However, the third (8) was on the higher dose and yet still had evidence of ER signaling. This observation suggests that some patients may benefit from even higher doses of SERD therapy. Conclusions: These pilot results suggest heterogeneous biological or pharmacological mechanisms of resistance to SERD therapy. These data suggest that CTC-ER and CTC-BCL-2 expression could serve as pharmacodynamic monitoring tools for dose escalation of fulvestrant or other SERDs. Further molecular analysis might provide biological bases for resistance to fulvestrant. Supported by Veridex, LLC, Fashion Footwear Charitable Foundation of New York/QVC Presents Shoes on Sale™ (DFH), Associazione Sandro Pitigliani and by a studentship from FIRC (CP). Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr PD6-4.

Introduction. CTC can be reproducibly and reliably enumerated using a commercially-available, automated immunomagnetic system (CellSearch®; Veridex LLC). High levels of CTC predict rapid progression in patients with metastatic breast cancer (MBC). Only ∼ 50% of patients with estrogen receptor (ER) positive MBC benefit from endocrine therapy (ET). Patients with endocrine-refractory MBC are better palliated with chemotherapy. We have developed a multi-parameter assay using CellSearch® that may identify patients with ER positive MBC who are unlikely to benefit from ET and may be better served with chemotherapy. Methods. CellSearch® has four fluorescence channels. Three of these are used to distinguish CTC from WBC (DAPI, anti-cytokeratin, anti-CD45). The 4th “empty” channel was used to measure ER, Bcl-2, HER2, and Ki67 expression with antigen-specific fluorescent-labeled antibodies. These four markers were chosen because of their associations with sensitivity (ER, Bcl-2) or resistance (HER2, Ki-67) to ET. Cultured human breast cancer cells (MCF-7: ER +, BCL-2 +, HER2 -, Ki67 +; MDA-MB-231: ER -, Bcl-2 -, HER2 -, Ki67 +; BT-474: ER +, Bcl-2 +, HER2 +, Ki67 +) were spiked into 7.5 ml human whole blood from normal donors and separated and characterized using the CXC CellSearch® kit. Results. Each cell line stained appropriately for the respective markers, with appropriate negative controls, although staining was heterogeneous, even within a single cell line. Using these preliminary data, we have developed a CTC-ETI in which scores are assigned to the individual categories consisting of cell counts coupled with the relative percent and degree of cell positivity of each marker. We predict that lower scores (low or no CTC, or CTC with high % and intensity of ER and Bcl-2; low HER2 and Ki-67) will be associated with favorable response to ET. Conclusions. CellSearch® can be used to measure the expression of 4 clinically useful and proven breast cancer biomarkers. A clinical trial is underway at U. Michigan to permit calculation of CTC-ETI in patients with MBC, which will lead to a prospective trial to determine if high CTC-ETI predicts resistance and rapid progression on ET. Supported by Veridex, LLC, Fashion Footwear Charitable Foundation of New York/QVC Presents Shoes on Sale ™ (DFH), Associazione Sandro Pitigliani and by a studentship from FIRC (CP) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4154. doi:10.1158/1538-7445.AM2011-4154

Previously we have shown that androgens induce the growth of estrogen-dependent, estrogen receptor (ER)-positive breast cancer cell lines via metabolism to estrogen-like steroids, independent of CYP19 aromatase. One pathway involves the 3β-hydroxysteroid dehydrogenase (3β-HSD) mediated metabolism of 5α-dihydrotestosterone (DHT) to 5α-androstane-3β,17β-diol (3βAdiol). 3βAdiol induces the growth of ER+ breast cancer cells by directly activating ER. However, in MCF-7 cells, maximal growth induction by 3βAdiol was only ∼75% of maximal growth induced by 17β-estradiol (E2), suggesting that 3βAdiol acts as a weak or partial agonist of ERα. In this study, we characterize 3βAdiol as a partial agonist of ERα using cell growth assays and an in vitro receptor binding assay. We use cDNA microarrays to examine gene expression induced by the partial agonist 3βAdiol vs. the full agonist E2. We tested if 3βAdiol as a partial agonist could antagonize E2-induced breast cancer cell growth. MCF-7 cells in estrogen-free conditions were treated with 100pM E2 (∼EC90) and increasing concentrations of 3βAdiol. Increasing 3βAdiol suppressed growth induced by E2 to the maximal level induced by 3βAdiol alone. These data confirm that 3βAdiol acts as a partial agonist of ERα. Additionally, we performed an in vitro, florescence polarization-based receptor binding assay to determine the relative affinities of 3βAdiol and E2 for ERα. Based on their ability to compete with a labeled ligand for binding to ERα, 3βAdiol bound ERα with an approximately 10-fold lower affinity than E2. This decreased affinity of 3βAdiol for ERα is consistent with the reduced potency of 3βAdiol versus E2 observed in growth assays. To compare changes in gene expression induced by either the full agonist E2 or partial agonist 3βAdiol, samples were analyzed with Affymetrix HG-U133 Plus 2.0 microarrays. MCF-7 cells were treated for 24 hours with 10nM of steroid or vehicle control. To evaluate overall changes in gene expression induced by the steroids, probe sets were selected that had >2-fold change in expression versus the control array, with at least one sample having an expression value >26. 1273 probe sets were differentially expressed between the E2 and control arrays, and 1318 between the 3βAdiol and control arrays, however, 1030 of these were common to both steroids. Genes in canonical estrogen signaling pathways were similarly regulated by both E2 and 3βAdiol, including GREB1, Bcl-2, TFF1 and CXCL12. Only 45 probe sets were differentially expressed between the E2 and 3βAdiol arrays. These data demonstrate that 3βAdiol is a partial agonist of ERα in our breast cancer cell model. The majority of changes in gene expression are similar between the full and partial agonist. These data suggest that the differential growth induction by the full and partial agonist are likely due to differential levels or time courses of gene induction, rather than the induction of a novel set of genes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4593.

The two most common endocrine treatment modalities, anti-estrogen therapy and aromatase inhibitor therapy target estrogen receptor (ER) activation and estrogen (E2) biosynthesis, respectively. Although these therapies are effective in many patients with ER-positive tumors, prospective clinical trials have demonstrated that there exists a portion of patients with breast cancers that are initially responsive to endocrine therapy but subsequently relapse. Because of the lack of perfect correlation between ER and/or progesterone receptor (PR) status and patients who benefit from hormone therapy, there is a need to identify additional protein markers that would improve prediction of hormone response.Gene Regulated by Estrogen in Breast cancer 1 (GREB1) is regulated by estrogen (E2) in breast cancer and may serve as a candidate marker to predict response to endocrine therapy. The focus of this study was to define the significance of GREB1 protein expression in breast cancer and its correlation with estrogen receptor α status and epidermal growth factor receptor 2 (HER2) expression. Based on the query of 16 breast cancer expression array studies in the Oncomine Research database, GREB1 mRNA is expressed at greater levels in breast carcinomas than normal breast tissue (p value: 2.1E-5). In addition, GREB1 mRNA is significantly more commonly expressed in ER-positive breast cancer patients (n=1651) than ER-negative patients (n=670) (P value: 4.2E-5 ∼ 1.1E-34). Finally, GREB1 mRNA expression was observed to be lower in HER2+ breast cancer patients than in HER-2 – patients (p-value: 0.034). These mRNA expression data supported the hypothesis that GREB1 may have potential as a new biomarker for predicting E2-dependent and anti-E2 responsive breast cancers.Using a novel GREB1 monoclonal antibody generated by our laboratory, Immunohistochemical staining (IHC) was performed on a tissue microarrays containing invasive breast carcinomas from a cohort of 142 operable breast cancer patients (104 ER+ and 38 ER- cancers) with paired uninvolved tissue from the same patient. GREB1 protein was detected in both uninvolved normal tissue and ER+ breast cancer, but was absent in ER- tumors. Thus, IHC staining revealed GREB1 protein expression, predominantly localized in the cell nucleus, has a significant correlation with ER status (p-value Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2008.

It has been well established that GREB1 mRNA expression is induced by estrogen in breast cancers and that GREB1 is critically involved in the estrogen-induced growth of breast cancer. To date, all studies of GREB1 have focused on mRNA levels using PCR and array technologies with very little known about GREB1 protein expression in normal and breast cancer cells. The lack of a specific antibody to GREB1 has inhibited protein-based investigations in target cells. Our collaborative research group has generated a novel monoclonal GREB1 antibody (GREB1ab) for research use in Western blotting as well as immunohistochemical (IHC) applications.Our team created a hybridoma expressing a murine monoclonal antibody against a 119 amino acid peptide specific to human GREB1. By Western Blot Analysis, GREB1ab detects a 216 kD protein corresponding to GREB1a in ER+ breast cancer cells expressing GREB1 as well as cells transfected with a GREB1a expression plasmid. GREB1ab specificity was verified using ICI 182,780, an estrogen receptor antagonist to prevent GREB1 induction, as well as silencing siRNA targeting GREB1 mRNA expression. GREB1 protein expression was reduced in MCF-7 cells treated with estrogen plus ICI 182,780 compared with that of estrogen treatment. There was no detectable GREB1 protein expression when GREB1 mRNA is silenced by siRNA at 48 hours. In a time course study, inhibition of GREB1 protein occurred as early as 24 hours and lasted up to 72 hours. Thus, by Western Blot Analysis, the monoclonal GREB1ab is specific for GREB1 and can be employed to detect changes in GREB1 expression in breast cancer cell lines under various experimental conditions.GREB1ab was validated for detection of GREB1 by IHC in breast cancer cell lines and breast tissue microarrays (TMA). IHC staining with GREB1ab revealed GREB1 was strongly expressed in the ERα-positive breast cancer cell line, MCF-7, with weak GREB1 expression in ERα-negative MDA-231 cells. A panel of breast cancer cell lines was screened for endogenous expression of GREB1 protein in a TMA format using IHC. As expected, ER-positive cell lines (n=5) were observed to express GREB1 while ER-negative cell lines (n=11) did not express detectable levels. Using breast cancer tissue whole sections, IHC with the GREB1ab indicated protein expression in ERα positive breast cancer tissue as well as normal breast tissue, with little GREB1 expression in ERα negative breast cancer tissue.The monoclonal GREB1ab is specific for GREB1 protein. This antibody will serve as a tool for investigations focused on the expression, distribution and function of GREB1 in normal breast and breast cancer tissues. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2126.

Background. Several studies aimed at correlating genetic variants in the enzymes responsible for tamoxifen metabolism and disposition with therapeutic outcomes have provided equivocal and conflicting data. The IMPACT trial compared the preoperative used of tamoxifen with anastrozole alone or in combination in postmenopausal breast cancer patients and provided strong evidence that tamoxifen-induced changes in the nuclear proliferation marker Ki67 can act as a surrogate marker of tamoxifen response. An advantage of this study design is that all patients contribute data to the end-point analyses which included changes in Ki67 and objective clinical response as defined by the WHO criteria. We therefore genotyped patients from IMPACT for variants in the enzyme (cytochrome p450 2D6 (CYP2D6)) responsible for the conversion of tamoxifen to its potent anti-estrogenic metabolite endoxifen, as well as the enzyme responsible for the detoxification and inactivation of endoxifen via conjugation (UDP-glucuronosyltransferase 2B7 (UGT2B7).Results: Depending on their CYP2D6 variant genotype, patients were assigned an 'activity score' based on predicted allele activities from 0 (no activity) to 2 (high activity). Patients were also tested for the UGT2B7*2 variant, which has lower enzymatic activity and is therefore correlated with higher serum endoxifen concentrations. Of the 135 patient samples available for genotype determinations, 123 and 134 we evaluable for CYP2D6 and UGT2B7, respectively. There was no evidence of an association between CYP2D6 score, as determined by genotype alone, with suppression of Ki67 after 2 or 12 weeks, or with objective clinical response after 16 weeks of therapy in the tamoxifen alone, anastrozole alone or combination groups. Furthermore, this lack of association was not altered by adjustment of the CYP2D6 score for concomitant medications, known to inhibit CYP2D6 and to correlate with lower serum endoxifen concentrations. Lastly, while a statistical test for a potential interaction between CYP2D6 genotype and UGT2B7 genotype was negative, there was a marginally statistically significant association between UGT2B7 genotype alone with suppression of Ki67 after 2 weeks in the tamoxifen only group.Conclusion: Although this was a small study, these data do not support the hypothesis that patients must have active CYP2D6 in order to benefit from tamoxifen therapy. Larger more definitive studies are underway to test the effects of drug metabolizing enzymes on anti-estrogen therapies. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2011.

Abstract #1043 Approximately 1/3 of hormone dependent breast cancers do not respond to tamoxifen treatment. The fact that these tumors are intrinsically resistant suggests that underlying genetic factors are responsible for this phenotype. The two major types of genetic variation are single nucleotide polymorphisms (SNPs) and copy number variations (CNVs). SNPs are the most common form of genetic alteration with an estimated 10 million SNPs across the genome and likely influence both disease susceptibility and response to therapy. Traditionally, researchers have attempted to identify SNPs associated with a clinical outcome in a “top-down” approach by conducting linkage and SNP association studies. However, here we report on a “bottom-up” approach whereby SNPs are first tested in vitro for a functional effect. This awards two benefits over a “top-down” approach: 1) identification of functional SNPs and 2) detailed characterization of the SNP so that the clinical association studies can be designed accordingly. Tamoxifen acts by blocking estradiol (E2) from binding to the estrogen receptor (ERα) and by recruiting corepressors, which then actively repress gene transcription. The critical role of corepressors, especially SMRT, in the proper action of tamoxifen makes them likely to be involved in tamoxifen resistance. Thus, we used a “bottom-up” approach to identify SNPs in ERα corepressors which affect the ability of tamoxifen to suppress ERα function. Here we report the detailed in vitro characterization of a number of SNPs in SMRT (NCoR2) with respect to altered ERα function in the presence of tamoxifen. Further, these SNPs are currently being genotyped so that associations can be made in a population of women receiving tamoxifen as either preventative or adjuvant therapy in which extensive surrogate markers for ERα function were collected during the 1-year follow (hot flash rate, hot flash intensity, lipid profiles, and bone mineral density). Through the discovery of common polymorphisms affecting Tamoxifen response, we hope to identify new diagnostic criteria to predict which patients will respond to anti-estrogen therapy and which will be better candidates for another line of therapy, such as aromatase inhibitors. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1043.

Abstract #3049 Background: Estrogen receptors (ER) and associated factors, including progesterone receptors (PgR), modulate estrogenic effects both in normal tissue and in hormone receptor (HR)-positive breast cancers. ERα expression occurs in the majority of breast cancers, although the level of overexpression varies between tumors. Multiple single nucleotide polymorphisms (SNPs) in the genes that encode ER, termed ESR1 (ERα) and ESR2 (ERβ), have been shown to affect phenotypic outcomes, including breast cancer risk, bone mineral density, hot flashes, and lipid levels. We hypothesized that SNPs in genes that encode ER may affect level of ER overexpression. Methods: DNA was extracted from white blood cells from 100 subjects with HR-positive breast cancer enrolled in a tamoxifen observational study. SNPs in ESR1 (XbaI: rs#9340799, PvuII: rs#2234693) and ESR2 (01: rs#1256049; 02: rs#4986938) were determined from germline DNA using TaqMan. ER overexpression in paraffin-embedded formalin-fixed tumor specimens was quantified with immunohistochemistry (IHC) of tumor specimens on slides (Ventana Image Analysis System) and with RT-PCR using the Oncotype DX breast cancer assay (Genomic Health, Inc). Gene expression of other ER-associated genes (PgR, BCL2, and SCUBE2) was determined using RT-PCR (Oncotype DX). Expression of ER and associated genes across ER genotype was performed using the Kruskal-Wallis test. For all analyses, a p value of Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3049.