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PURPOSE Chemotherapy has not demonstrated benefit over adjuvant endocrine therapy alone for postmenopausal patients with node-positive breast cancer with a 21-gene breast recurrence score (RS) of 25 or below (RS ≤ 25). We tested whether combined results from RS and the sensitivity to endocrine therapy (SET2,3) index of endocrine-related transcription (SETER/PR) adjusted for baseline prognostic index (BPI) improve prognostic assessment, and whether SET2,3 predicted benefit from anthracycline-based chemotherapy. METHODS A blinded retrospective clinical validation of SET2,3 in two randomized treatment arms from the SWOG S8814 trial comparing adjuvant anthracycline-based chemotherapy followed by tamoxifen endocrine therapy for 5 years, versus tamoxifen alone. SET2,3 assay was calibrated and measured using whole-transcriptome RNA sequence of tumor samples already tested for RS. The primary end point was disease-free survival (DFS). RESULTS There were 106 events in 283 patients over a median follow-up of 8.99 years. Proportional hazards assumptions were met during the first 5 years only. SET2,3 index and RS were not correlated (r = –0.04) and were independently prognostic (SET2,3: hazard ratio [HR], 0.48 per unit; 95% CI, 0.34 to 0.68; P < .001; RS: HR, 1.28 per 10 units; 95% CI, 1.14 to 1.44; P < .001). SET2,3 index did not predict chemotherapy benefit (interaction P = .77). SET2,3 was high in 93/175 (53%) patients with RS ≤ 25 (concordant low-risk), with 5-year DFS 97%. SET2,3 was low in 55/108 (51%) patients with RS > 25 (concordant high-risk), with 5-year DFS 53%. Both components of SET2,3 index were prognostic after adjustment for RS: SETER/PR (HR, 0.65; 95% CI, 0.46 to 0.92) and BPI (HR, 0.45; 95% CI, 0.31 to 0.64). CONCLUSION SET2,3 index was not correlated with RS, demonstrated additive prognostic performance, and was not chemopredictive in this subset of patients from S8814. The SETER/PR and BPI components of SET2,3 each added prognostic information to RS.

Objective Aromatase inhibitors (AI) are commonly used to treat hormone receptor positive (HR+) breast cancer. AI-induced musculoskeletal syndrome (AIMSS) is a common toxicity that causes AI treatment discontinuation. The objective of this genome-wide association study (GWAS) was to identify genetic variants associated with discontinuation of AI therapy due to AIMSS and attempt to replicate previously reported associations. Methods In the Exemestane and Letrozole Pharmacogenetics (ELPh) study, postmenopausal patients with HR+ non-metastatic breast cancer were randomized to letrozole or exemestane. Genome-wide genotyping of germline DNA was conducted followed by imputation. Each imputed variant was tested for association with time-to-treatment discontinuation due to AIMSS using a Cox proportional hazards model assuming additive genetic effects and adjusting for age, baseline pain score, prior taxane treatment, and AI arm. Secondary analyses were conducted within each AI arm and analyses of candidate variants previously reported to be associated with AIMSS risk. Results Four hundred ELPh participants were included in the combined analysis. Two variants surpassed the genome-wide significance level in the primary analysis (p-value−8), an intronic variant (rs79048288) within CCDC148 (HR=4.42, 95% CI: 2.67-7.33) and an intergenic variant (rs912571) upstream of PPP1R14C (HR=0.30, 95% CI: 0.20-0.47). In the secondary analysis, rs74418677, which is known to be associated with expression of SUPT20H, was significantly associated with discontinuation of letrozole therapy due to AIMSS (HR=5.91, 95% CI: 3.16-11.06). We were able to replicate associations for candidate variants previously reported to be associated with AIMSS in this cohort, but were not able to replicate associations for any other variants previously reported in other patient cohorts. Conclusions Our GWAS findings identify several candidate variants that may be associated with AIMSS risk from AI generally or letrozole specifically. Validation of these associations in independent cohorts is needed before translating these findings into clinical practice to improve treatment outcomes in patients with HR+ breast cancer.

Purpose Radiation therapy (RT) and hormone receptor (HR) inhibition are used for the treatment of HR-positive breast cancers; however, little is known about the interaction of the androgen receptor (AR) and estrogen receptor (ER) in response to RT in AR-positive, ER-positive (AR+/ER+) breast cancers. Here we assessed radiosensitisation of AR+/ER+ cell lines using pharmacologic or genetic inhibition/degradation of AR and/or ER. Methods Radiosensitisation was assessed with AR antagonists (enzalutamide, apalutamide, darolutamide, seviteronel, ARD-61), ER antagonists (tamoxifen, fulvestrant) or using knockout of AR. Results Treatment with AR antagonists or ER antagonists in combination with RT did not result in radiosensitisation changes (radiation enhancement ratios [rER]: 0.76–1.21). Fulvestrant treatment provided significant radiosensitisation of CAMA-1 and BT-474 cells (rER: 1.06–2.0) but not ZR-75-1 cells (rER: 0.9–1.11). Combining tamoxifen with enzalutamide did not alter radiosensitivity using a 1 h or 1-week pretreatment (rER: 0.95–1.14). Radiosensitivity was unchanged in AR knockout compared to Cas9 cells (rER: 1.07 ± 0.11), and no additional radiosensitisation was achieved with tamoxifen or fulvestrant compared to Cas9 cells (rER: 0.84–1.19). Conclusion While radiosensitising in AR + TNBC, AR inhibition does not modulate radiation sensitivity in AR+/ER+ breast cancer. The efficacy of ER antagonists in combination with RT may also be dependent on AR expression.

OBJECTIVE: Letrozole is a non-steroidal aromatase inhibitor (AI) used to treat hormone receptor positive (HR+) breast cancer. Variability in letrozole efficacy and toxicity may be partially attributable to variable systemic drug exposure, which may be influenced by germline variants in the enzymes responsible for letrozole metabolism including Cytochrome P450 2A6 (CYP2A6). The objective of this genome-wide association study (GWAS) was to identify polymorphisms associated with steady state letrozole concentrations. METHODS: The Exemestane and Letrozole Pharmacogenetics (ELPh) Study randomized post-menopausal patients with HR+ non-metastatic breast cancer to letrozole or exemestane treatment. Germline DNA was collected pre-treatment and blood samples were collected after 1 or 3 months of treatment to measure steady-state letrozole (and exemestane) plasma concentrations via HPLC/MS. Genome-wide genotyping was conducted on the Infinium Global Screening Array (>650,000 variants) followed by imputation. The association of each germline variant with age- and body mass index-adjusted letrozole concentrations was tested in self-reported white patients via linear regression assuming an additive genetic model. RESULTS: There were 228 patients who met the study specific inclusion criteria and had both DNA and letrozole concentration data for this GWAS. The association for one genotyped polymorphism (rs7937) with letrozole concentration surpassed genome-wide significance (p=5.26x10(−10)), explaining 13% of the variability in untransformed steady-state letrozole concentrations. Imputation around rs7937 and in silico analyses identified rs56113850, a variant in the CYP2A6 intron that may affect CYP2A6 expression and activity. rs7937 was associated with age- and body mass index-adjusted letrozole levels even after adjusting for genotype-predicted CYP2A6 metabolic phenotype (p=3.86x10(−10)). CONCLUSIONS: Our GWAS findings confirm that steady-state letrozole plasma concentrations are partially determined by germline polymorphisms that affect CYP2A6 activity, including variants near rs7937 such as the intronic rs56113850 variant. Further research is needed to confirm whether rs56113850 directly affects CYP2A6 activity and to integrate non-exonic variants into CYP2A6 phenotypic activity prediction systems.

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 A case-control study was performed to define clinical and genetic risk factors associated with osteonecrosis of the jaw in patients with metastatic cancer treated with bisphosphonates. Methods Clinical data and tissues were collected from patients treated with bisphosphonates for metastatic bone disease who were diagnosed with osteonecrosis of the jaw (cases) and matched controls. Clinical data included patient, behavioral, disease, and treatment information. Genetic polymorphisms in CYP2C8 (rs1934951) and other candidate genes were genotyped. Odds ratios from conditional logistic regression models were examined to identify clinical and genetic characteristics associated with case or control status. Results The study population consisted of 76 cases and 126 controls. In the final multivariable clinical model, patients with osteonecrosis of the jaw were less likely to have received pamidronate than zoledronic acid (odds ratio = 0.18, 95% Confidence interval: 0.03-0.97, p = .047) and more likely to have been exposed to bevacizumab (OR = 5.15, 95% CI: 1.67-15.95, p = .005). The exploratory genetic analyses suggested a protective effect for VEGFC rs2333496 and risk effects for VEGFC rs7664413 and PPARG rs1152003. Conclusions We observed patients with ONJ were more likely to have been exposed to bevacizumab and zoledronic and identified potential genetic predictors that require validation prior to clinical translation.

Endocrine therapy (ET) is an effective first-line therapy for women with estrogen receptor-positive (ER + ) breast cancers. While both ionizing radiation (RT) and ET are used for the treatment of women with ER+ breast cancer, the most effective sequencing of therapy and the effect of ET on tumor radiosensitization remains unclear. Here we sought to understand the effects of inhibiting estrogen receptor (ER) signaling in combination with RT in multiple preclinical ER+ breast cancer models. Clonogenic survival assays were performed using variable pre- and post-treatment conditions to assess radiosensitization with estradiol, estrogen deprivation, tamoxifen, fulvestrant, or AZD9496 in ER+ breast cancer cell lines. Estrogen stimulation was radioprotective (radiation enhancement ratios [rER]: 0.51–0.82). Conversely, when given one hour prior to RT, ER inhibition or estrogen depletion radiosensitized ER+ MCF-7 and T47D cells (tamoxifen rER: 1.50–1.60, fulvestrant rER: 1.76–2.81, AZD9496 rER: 1.33–1.48, estrogen depletion rER: 1.47–1.51). Combination treatment resulted in an increase in double-strand DNA (dsDNA) breaks as a result of inhibition of non-homologous end joining-mediated dsDNA break repair with no effect on homologous recombination. Treatment with tamoxifen or fulvestrant in combination with RT also increased the number of senescent cells but did not affect apoptosis or cell cycle distribution. Using an MCF-7 xenograft model, concurrent treatment with tamoxifen and RT was synergistic and resulted in a significant decrease in tumor volume and a delay in time to tumor doubling without significant toxicity. These findings provide preclinical evidence that concurrent treatment with ET and RT may be an effective radiosensitization strategy.

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.

BackgroundAromatase inhibitors (AI) reduce recurrence and death in patients with early-stage hormone receptor-positive (HR +) breast cancer. Treatment-related toxicities, including AI-induced musculoskeletal symptoms (AIMSS), are common and may lead to early AI discontinuation. The objective of this study was to replicate previously reported associations for candidate germline genetic polymorphisms with AIMSS.MethodsWomen with stage 0-III HR + breast cancer initiating adjuvant AI were enrolled in a prospective clinic-based observational cohort. AIMSS were assessed by patient-reported outcomes (PRO) including the PROMIS pain interference and physical function measures plus the FACT-ES joint pain question at baseline and after 3 and 6 months. For the primary analysis, AIMSS were defined as ≥ 4-point increase in the pain interference T-score from baseline. Secondary AIMSS endpoints were defined as ≥ 4-point decrease in the physical function T-score from baseline and as ≥ 1-point increase on the FACT-ES joint pain question from baseline. The primary hypothesis was that TCL1A rs11849538 would be associated with AIMSS. Twelve other germline variants in CYP19A1, VDR, PIRC66, OPG, ESR1, CYP27B1, CYP17A1, and RANKL were also analyzed assuming a dominant genetic effect and prespecified direction of effect on AIMSS using univariate logistic regression with an unadjusted α = 0.05. Significant univariate associations in the expected direction were adjusted for age, race, body mass index (BMI), prior taxane, and the type of AI using multivariable logistic regression.ResultsA total of 143 participants with PRO and genetic data were included in this analysis, most of whom were treated with anastrozole (78%) or letrozole (20%). On primary analysis, participants carrying TCL1A rs11849538 were not more likely to develop AIMSS (odds ratio = 1.29, 95% confidence interval: 0.55–3.07, p = 0.56). In the statistically uncorrected secondary analysis, OPG rs2073618 was associated with AIMSS defined by worsening on the FACT-ES joint pain question (OR = 3.33, p = 0.004), and this association maintained significance after covariate adjustment (OR = 3.98, p = 0.003).ConclusionCarriers of OPG rs2073618 may be at increased risk of AIMSS. If confirmed in other cohorts, OPG genotyping can be used to identify individuals with HR + early breast cancer in whom alternate endocrine therapy or interventions to enhance symptom detection and implement strategies to reduce musculoskeletal symptoms may be needed.

Breast cancer is the most diagnosed malignancy among women in the United States. Approximately 70% of breast tumors express estrogen receptor alpha and are deemed ER-positive. ER-positive breast tumors depend upon endogenous estrogens to promote ER-mediated cellular proliferation. Decades of research have led to a fundamental understanding of the role ER signaling in this disease and this knowledge has led to significant advancements in the clinical use of antiestrogens for breast cancer treatment. However, adjuvant breast cancer recurrence and metastatic disease progression due to endocrine therapy resistance are prominent and unresolved issues. The established role that estrogens play in breast cancer pathogenesis explains why some patients initially respond to endocrine therapy but also why a significant number of patients become refractory to antiestrogen treatment. It is been hypothesized that exposure to environmental steroid hormone mimics and/or acquired mechanisms of resistance may explain why endocrine therapy fails in a subset of breast cancer patients. This review will highlight: 1) the relationship between ER signaling and breast cancer pathogenesis, 2) the implication of environmental exposures on steroid hormone regulated processes including breast cancer, and 3) the unresolved issue of endocrine therapy resistance.

PURPOSE: UGT2B17 gene deletion (UGT2B17*2) has been reported to affect bone health as well as the pharmacokinetics of aromatase inhibitor (AI) drugs such as exemestane. The goal of this study was to assess associations between UGT2B17 gene deletion and bone health prior to and after 24 months of AI treatment in postmenopausal women with hormone receptor positive (HR+) breast cancer. METHODS: Bone health in women with HR+ breast cancer enrolled on the prospective randomized Exemestane and Letrozole Pharmacogenetics (ELPh) trial was determined by measuring bone turnover markers (BTM) and bone mineral density (BMD) pre-treatment and after 3 (BTM) and 24 (BMD) months of treatment with either the steroidal AI exemestane or the nonsteroidal AI letrozole. DNA samples were genotyped for UGT2B17*2. RESULTS: Of the 455 subjects included in the analyses, 244 (53.6%) carried at least one copy of UGT2B17*2. UGT2B17*2 was associated with lower pre-treatment BMD at the hip (P = 0.01) and spine (P = 0.0076). Letrozole treatment was associated with a greater decrease in BMD of the hip (P = 0.03) and spine (P = 0.03) than exemestane. UGT2B17 genotype was not associated with changes in BMD from 24 months of AI treatment, though in UGT2B17*2 homozygous patients, there was a trend toward greater decreases in BMD of the spine from treatment with letrozole compared with exemestane (P = 0.05). CONCLUSION: UGT2B17*2 may be associated with lower baseline BMD in women with HR+ breast cancer. Exemestane is less detrimental to bone health than letrozole in postmenopausal women treated with AI, and this effect may be confined to patients carrying UGT2B17*2, though this finding requires independent validation.

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.

Purpose/objective(s) Locoregional control remains an issue in women with multi-node positive estrogen receptor-positive (ER+) breast cancer and many women with triple-negative breast cancer (TNBC). There is growing evidence that CDK4/6 inhibition (CDK4/6i) sensitizes breast cancer cells to ionizing radiation (RT) by suppressing the DNA damage response, but the role of RB is currently unclear. We sought to understand the role of RB and the implications of RB loss in ER+ and TNBC. Materials/methods Clonogenic survival assays were used to calculate radiosensitivity and calculate enhancement ratios (rER). Cellular viability was quantified 72 hours after drug addition to calculate half-maximal inhibitory concentrations (IC50s). Phospho- and total RB expression levels were assessed by immunoblotting. DNA repair was assessed with γH2AX and RAD51 immunofluorescence. GFP-based plasmid reporter systems were used to assess homologous recombination (HR) and non-homologous end joining (NHEJ) competency. Isogenic RB1 knockout cells were generated with CRISPR-Cas9, and siRNA was used for transient RB1 knockdown. G1 cell cycle arrest was quantified using propidium iodide-based flow cytometry. In vivo efficacy of CDK4/6 inhibition + RT was assessed using MDA-MB-231 xenografts. Results Four TNBC cell lines with intact RB expression were radiosensitized (rER 1.08 - 2.22) after CDK4/6i with palbociclib, ribociclib, or abemaciclib, but two RB null TNBC models were not radiosensitized with combination treatment (rER: 0.84 - 1.00). In ER+ and TNBC cell lines, response to CDK4/6i + RT was accurately predicted by the presence or absence of RB protein, and higher RB expression led to increased radiosensitization of breast cancer cell lines at lower doses. In addition, pRB expression decreased in wild type TNBC cell lines treated with CDK4/6i + RT. HR was suppressed with CDK4/6i in RB wild type - but not mutant - cell lines. NHEJ efficiency in RB wild type TNBC was unchanged with CDK4/6i. Genetic knockdown of RB1 led to the loss of radiosensitization in ER+ and TNBC cell lines (rER: 0.84 - 1.13) as well as a decrease in sensitivity to CDK4/6i monotherapy. Parental and Cas9 control TNBC cells arrested in G1 24 hours after CDK4/6i and remained arrested at 48 hours, but RB1 CRISPR TNBC cells did not arrest after drug treatment. In a TNBC xenograft model with wild type RB expression (MDA-MB-231), CDK4/6i + RT led to significant radiosensitization in vivo and delayed time to tumor doubling. Conclusion RB loss mitigates CDK4/6 inhibitor-mediated radiosensitization of both ER+ and TNBC cells. Loss of RB expression prevents CDK4/6i-mediated radiosensitization, suggesting that RB expression may be a valuable clinical biomarker to predict efficacy of CDK4/6i + RT in future clinical trials. Our data also provide the preclinical rationale for CDK4/6i with RT not just in ER+ BC, but also in women with TNBC that express RB.

Background Immune checkpoint inhibition is effective in several cancers. Expression of programmed death-ligand 1 (PD-L1) on circulating tumor or immune effector cells could provide insights into selection of patients for immune checkpoint inhibition. Methods Whole blood was collected at serial timepoints from metastatic breast cancer patients and healthy donors for circulating tumor cell (CTC) and platelet PD-L1 analysis with a phycoerythrin-labeled anti-human PD-L1 monoclonal antibody (Biolegend clone 29E.2A3) using the CellSearch® assay. CTC PD-L1 was considered positive if detected on at least 1% of the cells; platelet PD-L1 was considered positive if ≥100 platelets per CellSearch frame expressed PD-L1. Results A total of 207 specimens from 124 metastatic breast cancer patients were collected. 52/124 (42%) samples at timepoint-1 (at or close to time of progressive disease) had ≥5 CTC/7.5ml whole blood. Of those, 21 (40%) had positive CTC PD-L1. In addition, platelet PD-L1 expression was observed in 35/124 (28%) at timepoint-1. Platelet PD-L1 was not detected in more than 70 specimens from 12 healthy donors. Platelet PD-L1 was associated with ≥5 CTC/7.5ml whole blood (p = 0.0002), less likely in patients with higher red blood cell counts (OR = 0.72, p Conclusion PD-L1 expression was found in metastatic breast cancer patients on both CTC and platelets in an independent fashion. Inter-patient platelet PD-L1 expression was highly heterogeneous suggesting that it is a biological event associated with cancer in some but not all patients. Taken together, our data suggest that CTC and platelet PD-L1 expression could play a role in predicting which patients should receive immune checkpoint inhibition and as a pharmacodynamics biomarker during treatment.

Purpose: Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors have improved progression-free survival for metastatic, estrogen receptor–positive (ER+) breast cancers, but their role in the nonmetastatic setting remains unclear. We sought to understand the effects of CDK4/6 inhibition (CDK4/6i) and radiotherapy in multiple preclinical breast cancer models. Experimental Design: Transcriptomic and proteomic analyses were used to identify significantly altered pathways after CDK4/6i. Clonogenic assays were used to quantify the radiotherapy enhancement ratio (rER). DNA damage was quantified using γH2AX staining and the neutral comet assay. DNA repair was assessed using RAD51 foci formation and nonhomologous end joining (NHEJ) reporter assays. Orthotopic xenografts were used to assess the efficacy of combination therapy. Results: Palbociclib significantly radiosensitized multiple ER+ cell lines at low nanomolar, sub IC50 concentrations (rER: 1.21–1.52) and led to a decrease in the surviving fraction of cells at 2 Gy (P < 0.001). Similar results were observed in ribociclib-treated (rER: 1.08–1.68) and abemaciclib-treated (rER: 1.19–2.05) cells. Combination treatment decreased RAD51 foci formation (P < 0.001), leading to a suppression of homologous recombination activity, but did not affect NHEJ efficiency (P > 0.05). Immortalized breast epithelial cells and cells with acquired resistance to CDK4/6i did not demonstrate radiosensitization (rER: 0.94–1.11) or changes in RAD51 foci. In xenograft models, concurrent palbociclib and radiotherapy led to a significant decrease in tumor growth. Conclusions: These studies provide preclinical rationale to test CDK4/6i and radiotherapy in women with locally advanced ER+ breast cancer at high risk for locoregional recurrence.

Aim: This study explored whether inherited variants in genes causing the hereditary neuropathy condition Charcot–Marie–Tooth disease are associated with sensitivity to paclitaxel-induced peripheral neuropathy (PN). Patients & methods: Hereditary neuropathy genes previously associated with risk of paclitaxel-induced PN were sequenced in paclitaxel-treated patients. Eight putative genetic predictors in five hereditary neuropathy genes ( ARHGEF10, SBF2, FGD4, FZD3 and NXN) were tested for association with PN sensitivity after accounting for systemic exposure and clinical variables. Results: FZD3 rs7833751, a proxy for rs7001034, decreased PN sensitivity (additive model, β = -0.41; 95% CI: -0.66 to -0.17; p = 0.0011). None of the other genetic predictors were associated with PN sensitivity. Conclusion: Our results support prior evidence that FZD3 rs7001034 is protective of PN and may be useful for individualizing paclitaxel treatment to prevent PN.

In the original publication of the article, the spelling of the sixth author's given name was incorrect. The corrected author name should read as "Wadie David". The original article has been corrected.

Aims Chemotherapy-induced peripheral neuropathy (PN) is a treatment limiting toxicity of paclitaxel. We evaluated if EPHA genetic variation (EPHA4, EPHA5, EPHA6, and EPHA8) is associated with PN sensitivity by accounting for variability in systemic paclitaxel exposure (time above threshold). Methods Germline DNA from 60 patients with breast cancer was sequenced. PN was measured using the 8-item sensory subscale (CIPN8) of the patient-reported CIPN20. Associations for 3 genetic models were tested by incorporating genetics into previously published PN prediction models integrating measured paclitaxel exposure and cumulative treatment. Significant associations were then tested for association with PN-related treatment disruption. Results EPHA5 rs7349683 (minor allele frequency = 0.32) was associated with increased PN sensitivity (β-coefficient = 0.39, 95% confidence interval 0.11-0.67, p = 0.007). Setting a maximum tolerable threshold of CIPN8 = 30, optimal paclitaxel exposure target is shorter for rs7349683 homozygous (11.6 h) than heterozygous (12.6 h) or wild-type (13.6 h) patients. Total number of missense variants (median = 0, range 0-2) was associated with decreased PN sensitivity (β-coefficient: -0.42, 95% confidence interval -0.72 to -0.12, P = .006). No association with treatment disruption was detected for the total number of missense variants or rs7349683. Conclusion Isolating toxicity sensitivity by accounting for exposure is a novel approach, and rs7349683 represents a promising marker for PN sensitivity that may be used to individualize paclitaxel treatment.

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.

PURPOSE: In patients taking tamoxifen, the CYP2D6 genotype causes different exposure of active metabolite endoxifen. The objective of this randomized, open-label, multicenter, phase II study was to prospectively evaluate whether CYP2D6 genotype–guided tamoxifen dosing in patients with hormone receptor–positive metastatic breast cancer could have an impact on the clinical outcome. METHODS: Patients who needed first-line tamoxifen therapy were enrolled. Based on individual CYP2D6 genotype, patients heterozygous (wild type [wt]/variant [V]) or homozygous (V/V) for variant alleles of decreased or no function were randomly assigned to receive tamoxifen at an increased dose (ID arm; 40 mg daily) or regular dose (RD arm; 20 mg daily), and patients homozygous for wild-type alleles (wt/wt) received tamoxifen at 20 mg daily. The primary endpoint was the progression-free survival (PFS) rate at 6 months. The secondary endpoints included PFS and correlation of Z-endoxifen concentration with clinical outcomes. RESULTS: Between December 2012 and July 2016, 186 patients were enrolled in Japan. Of 184 evaluable patients, 136 carried wt/V or V/V (ID arm, 70; RD arm, 66), and 48 carried wt/wt. PFS rates at 6 months were not significantly different between the ID and RD arms (67.6% v 66.7%). The serum trough concentrations of Z-endoxifen in the ID arm were significantly higher than those in the RD arm (median, 89.2 nM v 51.1 nM; P < .0001) and were also higher compared with wt/wt patients (72.0 nM; P = .045). No significant difference in Z-endoxifen concentrations was observed between patients with disease progression and those who were progression free at 6 months (P = .43). CONCLUSION: In patients with CYP2D6-variant alleles, increasing tamoxifen dosing did not achieve a higher PFS rate at 6 months. The CYP2D6 genotype solely cannot explain individual variability in the efficacy of tamoxifen.

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/Objective(s) Estrogen receptor (ER) expression is present in over 80% of breast tumors and has been shown to be a significant driver of tumor initiation and progression and therefore a target of first-line therapies for ER-positive (ER+) breast cancer (BC) patients. While both ionizing radiation (RT) and endocrine therapies (ET) are used for the treatment of women with ER+ BC, the sequencing of therapy and the effect of ET on tumor radiosensitization remains unclear. Here we assessed the efficacy and mechanism of ER inhibition in ER+ BC in combination with RT in multiple preclinical models. Materials/Methods Clonogenic survival assays were used to assess radiosensitization. ER inhibition was accomplished with tamoxifen and the selective ER degrader (SERD), fulvestrant, in ER+ MCF-7 and T47D cells or ER-negative (ER-) SUM-159 cells. DNA damage was assessed by yH2AX foci. Efficiency of homologous recombination (HR) or non-homologous end joining (NHEJ) was measured by RAD51 foci or using a pYFP reporter, respectively. Cell cycle effects were measured using flow cytometry with propidium iodide (PI) staining. Apoptosis was assessed by annexin V/PI via flow cytometry. Western blotting was used to quantify protein expression. An MCF-7 xenograft model was used to assess the efficacy of tamoxifen with RT in vivo where mice were pretreated with tamoxifen for one or six days prior to starting RT. Synergy was determined using the fractional tumor volume method. Results One-hour pretreatment with tamoxifen prior to RT resulted in radiosensitization of ER+ MCF-7 (enhR: 1.14-1.50) and T47D (enhR: 1.33-1.60) cells but not ER- SUM-159 cells (enhR: 0.99-1.02). MCF-7 and T47D cells treated with tamoxifen and RT did not alter the kinetics of dsDNA break repair as measured by yH2AX foci (P > 0.05) but demonstrated a decrease in NHEJ-mediated repair (P 0.05). While cell cycle arrest was induced at 24 hours after RT, no changes were observed with tamoxifen treatment in combination with RT. In addition, there were no significant changes in apoptosis in MCF-7 or T47D cells with treatment of tamoxifen, RT, or the combination (P > 0.05). In vivo, an MCF-7 xenograft study demonstrated a significant delay in time to tumor doubling (17 days in control, 40 days with tamoxifen, 32 days with RT, and undefined with combination; P Conclusion Our data suggest that endocrine therapies may be effectively used to radiosensitize ER+ breast tumors. This work also supports further clinical investigation of the timing of RT for patients receiving ET as concurrent use may be more effective than sequential, especially as ET prior to RT is increasingly used as a bridging therapy during the COVID pandemic.

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.

A number of studies have tested the hypothesis that breast cancer patients with low-activity CYP2D6 genotypes achieve inferior benefit from tamoxifen treatment, putatively due to lack of metabolic activation to endoxifen. Studies have provided conflicting data, and meta-analyses suggest a small but significant increase in cancer recurrence, necessitating additional studies to allow for accurate effect assessment. We conducted a retrospective pharmacogenomic analysis of a prospectively collected community-based cohort of patients with estrogen receptor-positive breast cancer to test for associations between low-activity CYP2D6 genotype and disease outcome in 500 patients treated with adjuvant tamoxifen monotherapy and 500 who did not receive any systemic adjuvant therapy. Tumor-derived DNA was genotyped for common, functionally consequential CYP2D6 polymorphisms (*2, *3, *4, *6, *10, *41, and copy number variants) and assigned a CYP2D6 activity score (AS) ranging from none (0) to full (2). Patients with poor metabolizer (AS = 0) phenotype were compared to patients with AS > 0 and in secondary analyses AS was analyzed quantitatively. Clinical outcome of interest was recurrence free survival (RFS) and analyses using long-rank test were adjusted for relevant clinical covariates (nodal status, tumor size, etc.). CYP2D6 AS was not associated with RFS in tamoxifen treated patients in univariate analyses (p > 0.2). In adjusted analyses, increasing AS was associated with inferior RFS (Hazard ratio 1.43, 95% confidence interval 1.00–2.04, p = 0.05). In patients that did not receive tamoxifen treatment, increasing CYP2D6 AS, and AS > 0, were associated with superior RFS (each p = 0.0015). This population-based study does not support the hypothesis that patients with diminished CYP2D6 activity achieve inferior tamoxifen benefit. These contradictory findings suggest that the association between CYP2D6 genotype and tamoxifen treatment efficacy is null or near null, and unlikely to be useful in clinical practice.

The aromatase inhibitors (AI) exemestane (EXE), letrozole (LET), and anastrozole suppress estrogen biosynthesis, and are effective treatments for estrogen receptor (ER)-positive breast cancer. Prior work suggests that anastrozole blood concentrations are associated with the magnitude of estrogen suppression. The objective of this study was to determine whether the magnitude of estrogen suppression, as determined by plasma estradiol (E2) concentrations, in EXE or LET treated patients is associated with plasma AI concentrations. Five hundred post-menopausal women with ER-positive breast cancer were enrolled in the prospective Exemestane and Letrozole Pharmacogenetic (ELPh) Study conducted by the COnsortium on BReast cancer phArmacogomics (COBRA) and randomly assigned to either drug. Estrogen concentrations were measured at baseline and after 3 months of AI treatment and drug concentrations were measured after 1 or 3 months. EXE or LET concentrations were compared with 3-month E2 concentration or the change from baseline to 3 months using several complementary statistical procedures. Four-hundred patients with on-treatment E2 and AI concentrations were evaluable (EXE n = 200, LET n = 200). Thirty (7.6%) patients (EXE n = 13, LET n = 17) had 3-month E2 concentrations above the lower limit of quantification (LLOQ) (median: 4.75; range: 1.42–63.8 pg/mL). EXE and LET concentrations were not associated with on-treatment E2 concentrations or changes in E2 concentrations from baseline (all p > 0.05). Steady-state plasma AI concentrations do not explain variability in E2 suppression in post-menopausal women receiving EXE or LET therapy, in contrast with prior evidence in anastrozole treated patients.

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.

// Martina S.J. McDermott 1 , Alexander A. Chumanevich 1 , Chang-uk Lim 1 , Jiaxin Liang 1 , Mengqian Chen 1 , Serena Altilia 1 , David Oliver 1 , James M. Rae 2 , Michael Shtutman 1 , Hippokratis Kiaris 1 , Balazs Győrffy 3 , Igor B. Roninson 1 and Eugenia V. Broude 1 1 Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, Columbia, SC, USA 2 University of Michigan Medical School, Ann Arbor, MI, USA 3 MTA TTK Lendulet Cancer Biomarker Research Group, Semmelweis University 2nd Department of Pediatrics, Budapest, Hungary Correspondence to: Eugenia V. Broude, email: // Keywords : CDK8, estrogen receptor, breast cancer, transcription, estrogen independence Received : January 04, 2017 Accepted : January 17, 2017 Published : January 29, 2017 Abstract Hormone therapy targeting estrogen receptor (ER) is the principal treatment for ER-positive breast cancers. However, many cancers develop resistance to hormone therapy while retaining ER expression. Identifying new druggable mediators of ER function can help to increase the efficacy of ER-targeting drugs. Cyclin-dependent kinase 8 (CDK8) is a Mediator complex-associated transcriptional regulator with oncogenic activities. Expression of CDK8, its paralog CDK19 and their binding partner Cyclin C are negative prognostic markers in breast cancer. Meta-analysis of transcriptome databases revealed an inverse correlation between CDK8 and ERα expression, suggesting that CDK8 could be functionally associated with ER. We have found that CDK8 inhibition by CDK8/19-selective small-molecule kinase inhibitors, by shRNA knockdown or by CRISPR/CAS9 knockout suppresses estrogen-induced transcription in ER-positive breast cancer cells; this effect was exerted downstream of ER. Estrogen addition stimulated the binding of CDK8 to the ER-responsive GREB1 gene promoter and CDK8/19 inhibition reduced estrogen-stimulated association of an elongation-competent phosphorylated form of RNA Polymerase II with GREB1. CDK8/19 inhibitors abrogated the mitogenic effect of estrogen on ER-positive cells and potentiated the growth-inhibitory effects of ER antagonist fulvestrant. Treatment of estrogen-deprived ER-positive breast cancer cells with CDK8/19 inhibitors strongly impeded the development of estrogen independence. In vivo treatment with a CDK8/19 inhibitor Senexin B suppressed tumor growth and augmented the effects of fulvestrant in ER-positive breast cancer xenografts. These results identify CDK8 as a novel downstream mediator of ER and suggest the utility of CDK8 inhibitors for ER-positive breast cancer therapy.

Tamoxifen therapy for estrogen receptor-positive breast cancer reduces the risk of recurrence by approximately one-half. Cytochrome P-450 2D6, encoded by the polymorphic cytochrome P-450 2D6 gene (CYP2D6), oxidizes tamoxifen to its most active metabolites. Steady-state concentrations of endoxifen (4-hydroxy-N-desmethyltamoxifen), the most potent antiestrogenic metabolite, are reduced in women whose CYP2D6 genotypes confer poor enzyme function. Thirty-one studies of the association of CYP2D6 genotype with breast cancer survival have yielded heterogeneous results. Some influential studies genotyped DNA from tumor-infiltrated tissues, and their results may have been susceptible to germline genotype misclassification from loss of heterozygosity at the CYP2D6 locus. We systematically reviewed 6 studies of concordance between genotypes obtained from paired nonneoplastic and breast tumor-infiltrated tissues, all of which showed excellent CYP2D6 genotype agreement. We applied these concordance data to a quantitative bias analysis of the subset of the 31 studies that were based on genotypes from tumor-infiltrated tissue to examine whether genotyping errors substantially biased estimates of association. The bias analysis showed negligible bias by discordant genotypes. Summary estimates of association, with or without bias adjustment, indicated no clinically important association between CYP2D6 genotype and breast cancer survival in tamoxifen-treated women.

Studies have identified several estrogen receptor α (ERα) ligand-binding domain (LBD) somatic mutations in endocrine therapy resistant, metastatic ER-positive breast cancers. The most common mutations, Tyr537Ser (Y537S) and Asp538Gly (D538G), are detected in ~ 30% of endocrine resistant metastatic breast cancer patients. These ESR1 mutations induce the agonist conformation of ERα, confer an estrogen-independent phenotype, and promote drug resistance to antiestrogens. ER-positive, estrogen-dependent MCF-7 cells were engineered to express either the Y537S or D538G mutants using CRISPR knock-in (cY537S and cD538G). These cells were used to screen several estrogen receptor degrader (ERD) compounds synthesized using the Proteolysis Targeting Chimeras (PROTAC) method to induce degradation of ERα via the ubiquitin–proteasome pathway. Wild-type MCF-7 and ERα LBD mutant cells were treated with ERD-148 (10 pM–1 µM) and assayed for cellular proliferation using the PrestoBlue cell viability assay. ERD-148 attenuated ER-dependent growth with IC50 values of 0.8, 10.5, and 6.1 nM in MCF-7, cY537S, and cD538G cells, respectively. Western blot analysis showed that MCF-7 cells treated with 1 nM ERD-148 for 24 h exhibited reduced ERα protein expression as compared to the mutants. The ER-regulated gene, GREB1, demonstrated significant downregulation in parental and mutant cells after 24 h of ERD-148 treatment at 10 nM. Growth of the ER-negative, estrogen-independent MDA-MB-231 breast cancer cells was not inhibited by ERD-148 at the ~ IC90 observed in the ER-positive cells. ERD-148 inhibits the growth of ER-positive breast cancer cells via downregulating ERα with comparable potency to Fulvestrant with marginal non-specific toxicity.

10527 Background: Among patients with early-stage breast cancer, approximately 6-10% have a pathogenic germline variant (PGV) conferring inherited cancer predisposition. In contrast, few studies have explored the frequency and types of PGVs identified in patients with metastatic breast cancer (MBC); therefore, additional data is needed. Methods: From 2011-2020, 278 patients with MBC underwent fresh tumor biopsy and blood sample collection for paired tumor/normal DNA (targeted exome capture with analysis of 1700 genes) and RNA (tumor transcriptome) sequencing through the Michigan Oncology Sequencing (Mi-ONCOSEQ) program. Somatic and germline alterations were annotated and classified according to degree of clinical actionability with results returned to treating oncologists. Retrospective chart review was performed to determine if: 1) a PGV was identified prior to Mi-ONCOSEQ testing, 2) patients met National Comprehensive Cancer Network (NCCN) guideline criteria for genetic testing on the basis of personal or family cancer history and 3) patients received subsequent therapy informed by a PGV. Results: Forty-eight of the 278 patients (17.3%) had at least one PGV identified, with a total of 50 PGVs identified in this cohort. Only twelve of these PGVs (24%) had been identified prior to Mi-ONCOSEQ testing. The most frequent PGVs identified were in CHEK 2 (n = 9, 18%), MUTYH (n = 6, 12%), BRCA 1 (n = 5, 10%), BRCA2 (n = 5, 10%), ATM (n = 4, 8%) and PALB2 (n = 4, 8%). Somatic loss of heterozygosity events (LOH) occurred in 30 of the 50 cases with PGVs identified (60%). LOH events were observed in 83.3% of BRCA1, BRCA2, ATM and PALB2 PGVs, but were less frequently observed with CHEK2 (33.3%) and MUTYH (66.7%). Two hundred sixteen out of 278 patients (77.7%) in this cohort met NCCN criteria for genetic testing, although six patients with a PGV identified (CHEK2: n = 5; MUTYH: n = 1) did not meet NCCN criteria. Twenty-nine PGVs identified (58%) had potential therapeutic relevance and 11 patients (22.9%) received targeted therapy based on the PGV. Conclusions: The frequency of PGVs identified in this cohort is nearly double the frequency reported for patients with early-stage disease, suggesting that certain PGVs may confer worse prognosis. CHEK2, the most frequently identified PGV, was less likely to have an identifiable LOH event. The direct role of CHEK2 PGVs in tumor pathogenesis is uncertain, but other mechanisms of silencing the wild type allele must be considered. Despite the majority of patients meeting NCCN criteria for genetic testing, those with PGVs in CHEK2 were less reliably identified by this mechanism. The majority of PGVs identified were of potential therapeutic relevance, supporting the recommendation for genetic testing in all patients with MBC.

Background: Epidemiologic studies and secondary analyses of randomized trials supported the hypothesis that selenium and vitamin E lower prostate cancer risk. However, the Selenium and Vitamin E Cancer Prevention Trial (SELECT) showed no benefit of either supplement. Genetic variants involved in selenium or vitamin E metabolism or transport may underlie the complex associations of selenium and vitamin E. Methods: We undertook a case–cohort study of SELECT participants randomized to placebo, selenium, or vitamin E. The subcohort included 1,434 men; our primary outcome was high-grade prostate cancer (N = 278 cases, Gleason 7 or higher cancer). We used weighted Cox regression to examine the association between SNPs and high-grade prostate cancer risk. To assess effect modification, we created interaction terms between randomization arm and genotype and calculated log likelihood statistics. Results: We noted statistically significant (P < 0.05) interactions between selenium assignment, SNPs in CAT, SOD2, PRDX6, SOD3, and TXNRD2, and high-grade prostate cancer risk. Statistically significant SNPs that modified the association of vitamin E assignment and high-grade prostate cancer included SEC14L2, SOD1, and TTPA. In the placebo arm, several SNPs, hypothesized to interact with supplement assignment and risk of high-grade prostate cancer, were also directly associated with outcome. Conclusion: Variants in selenium and vitamin E metabolism/transport genes may influence risk of overall and high-grade prostate cancer, and may modify an individual man's response to vitamin E or selenium supplementation with regards to these risks. Impact: The effect of selenium or vitamin E supplementation on high-grade prostate cancer risk may vary by genotype. Cancer Epidemiol Biomarkers Prev; 25(7); 1050–8. ©2016 AACR.

Cytochrome P450 17A1 (CYP17A1) is the requisite enzyme for synthesis of sex steroids, including estrogens and androgens. As such, inhibition of CYP17A1 is a target for inhibiting the growth of hormone-dependent cancers including prostate and breast cancer. Abiraterone, is a first in class potent and selective CYP17A1 inhibitor that has been approved for the treatment of castration-resistant prostate cancer. Given that, androgens are the precursors for estrogen production, it has been proposed that abiraterone could be an effective form of treatment for estrogen receptor (ER)-positive breast cancer, though its utility in this context has yet to be established. Abiraterone has a core steroid-like chemical structure, and so we hypothesized that it may bind to nuclear steroid receptors including ER and have estrogenic activity. We tested this hypothesis by investigating abiraterone's ability to directly modulate ER signaling in breast cancer cell line models. We show that abiraterone directly activates ER, induces ER-target gene expression, and elicits estrogen-response-element reporter activity in the ER-positive cell lines MCF-7 and T47D. Abiraterone also induced cell proliferation by ~2.5-fold over vehicle in both MCF-7 and T47D cells. Importantly, abiraterone-induced cell proliferation and ER-activity was blocked by the selective estrogen receptor downregulator (SERD) fulvestrant, confirming that abiraterone directly acts at the ER. These data suggest that abiraterone should be combined with other ER antagonists when used for the clinical management of ER-positive breast cancer.

AIMS In the SWOG S0226 trial the combination of anastrozole plus fulvestrant (n = 349) was superior to anastrozole alone (n = 345) in hormone receptor (HR)-positive metastatic breast cancer. Here we report a pharmacokinetic subset analysis investigating a possible drug interaction between anastrozole and fulvestrant. METHODS Post-menopausal patients with HR-positive metastatic breast cancer were randomized to anastrozole with or without concurrent fulvestrant. Blood samples were collected at 2, 4, 6 and 8 months, just prior to receiving the next dose of anastrozole and fulvestrant. Drug concentrations were measured via LC/MS-MS. Anastrozole concentration was compared in patients on anastrozole alone vs. patients on concomitant fulvestrant. Comparisons were made at each time point using parametric tests and over time using a linear mixed effects model. RESULTS A total of 483 anastrozole concentration measurements were included, 224 samples from 64 patients on the anastrozole alone arm and 259 from 73 patients on the combination arm. The mean anastrozole concentration in the combination arm was significantly lower than that in the anastrozole alone arm at each sample collection time (all P

In the prospective Exemestane and Letrozole Pharmacogenetics trial of adjuvant aromatase inhibitor (AI) therapy for early-stage breast cancer, worsening of multiple treatment-related symptoms during AI therapy predicted AI early discontinuation. If these findings are confirmed in independent trials, early detection of changes in PRO measures could be used clinically to target interventions in patients at high risk for early discontinuation., Background. Early discontinuation of aromatase inhibitors (AIs) is common and leads to poor outcomes but is challenging to predict. In the Exemestane and Letrozole Pharmacogenetics trial, a high rate of early discontinuation due to intolerance was observed. We hypothesized that early changes in patient-reported outcomes (PROs) predict AI discontinuation and that biochemical factors are associated with changes in PROs. Patients and Methods. Postmenopausal women with early-stage breast cancer enrolled in a prospective randomized trial of exemestane versus letrozole completed questionnaires at baseline and serially over 24 months to assess overall quality of life (EuroQOL Visual Analog Scale [VAS]); mood; and multiple symptoms, including a musculoskeletal symptom cluster. A joint mixed-effects/survival model was used to estimate the effect of the change in PROs on AI discontinuation. Associations between biochemical factors and change in PROs were examined. Results. A total of 490 patients were analyzed. Worsening of EuroQOL VAS and the musculoskeletal cluster were associated with the highest risk for early discontinuation (hazard ratio [HR], 2.77 [95% confidence interval (CI), 2.72–2.81; p = .015]; HR, 4.39 [95% CI, 2.40–8.02; p < .0001], respectively). Pharmacokinetics and estrogen metabolism were not consistently associated with change in PRO measures. No clinically significant differences in any PRO between AIs were observed. Conclusion. Changes in PROs early during AI therapy were associated with treatment discontinuation. Identification of these changes could be used to target interventions in patients at high risk for early discontinuation. Implications for Practice: Early changes in patient-reported outcomes (PROs) can predict nonpersistence to aromatase inhibitor therapy. If used in clinical practice, PROs might identify women at highest risk for early discontinuation and allow for interventions to improve tolerance before significant toxicities develop. Further research is needed to improve capturing PROs in routine clinical practice.

Purpose: Aromatase inhibitors can exert unfavorable effects on lipid profiles; however, previous studies have reported inconsistent results. We describe the association of single-nucleotide polymorphisms (SNP) in candidate genes with lipid profiles in women treated with adjuvant aromatase inhibitors. Experimental Design: We conducted a prospective observational study to test the associations between SNPs in candidate genes in estrogen signaling and aromatase inhibitor metabolism pathways with fasting lipid profiles during the first 3 months of aromatase inhibitor therapy in postmenopausal women with early breast cancer randomized to adjuvant letrozole or exemestane. We performed genetic association analysis and multivariable linear regressions using dominant, recessive, and additive models. Results: A total of 303 women had complete genetic and lipid data and were evaluable for analysis. In letrozole-treated patients, SNPs in CYP19A1, including rs4646, rs10046, rs700518, rs749292, rs2289106, rs3759811, and rs4775936 were significantly associated with decreases in triglycerides by 20.2 mg/dL and 39.3 mg/dL (P < 0.00053), respectively, and with variable changes in high-density lipoprotein (HDL-C) from decreases by 4.2 mg/dL to increases by 9.8 mg/dL (P < 0.00053). Conclusions: Variants in CYP19A1 are associated with decreases in triglycerides and variable changes in HDL-C in postmenopausal women on adjuvant aromatase inhibitors. Future studies are needed to validate these findings, and to identify breast cancer survivors who are at higher risk for cardiovascular disease with aromatase inhibitor therapy. Clin Cancer Res; 22(6); 1395–402. ©2015 AACR.

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.