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Amy L Pasternak,1 Vincent D Marshall,1 Christina L Gersch,2 James M Rae,2 Michael Englesbe,3 Jeong M Park1 1Department of Clinical Pharmacy, University of Michigan College of Pharmacy, Ann Arbor, MI, 48109, USA; 2Department of Internal Medicine, Michigan Medicine, Ann Arbor, MI, 48109, USA; 3Department of Surgery, Michigan Medicine, Ann Arbor, MI, 48109, USACorrespondence: Amy L Pasternak Email amylp@med.umich.eduPurpose: CYP3A5 genotype is a significant contributor to inter-individual tacrolimus exposure and may impact the time required to achieve therapeutic concentrations and number of tacrolimus dose adjustments in transplant patients. Increased modifications to tacrolimus therapy may indicate a higher burden on healthcare resources. The purpose of this study was to evaluate whether CYP3A5 genotype was predictive of healthcare resource utilization in pediatric renal and heart transplant recipients.Patients and Methods: Patients < 18 years of age with a renal or heart transplant between 6/1/2014– 12/31/2018 and tacrolimus-based immunosuppression were included. Secondary use samples were obtained for CYP3A5 genotyping. Clinical data was retrospectively collected from the electronic medical record. Healthcare resource utilization measures included the number of dose changes, number of tacrolimus concentrations, length of stay, number of clinical encounters, and total charges within the first year post-transplant. Rejection and donor-specific antibody (DSA) formation within the first year were also collected. The impact of CYP3A5 genotype was evaluated via univariate analysis for the first year and multivariable analysis at 30, 90, 180, 270, and 365 days post-transplant.Results: Eighty-five subjects were included, 48 renal transplant recipients and 37 heart transplant recipients. CYP3A5 genotype was not associated with any outcomes in renal transplant, however, a CYP3A5 expresser phenotype was a predictor of more dose changes, more tacrolimus concentrations, longer length of stay, and higher total charges in heart transplant recipients. CYP3A5 genotype was not associated with rejection or DSA formation. Age and induction therapy were associated with higher total charges.Conclusion: CYP3A5 genotype may predict healthcare resource utilization in the first year post-transplant, although this may be mitigated by differences in tacrolimus management. Future studies should evaluate the impact of genotype-guided dosing strategies for tacrolimus on healthcare utilization resources.Keywords: pharmacogenetics, cost of care, pediatric transplant

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.

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.

CYP3A5 and CYP3A4 are the predominant enzymes responsible for tacrolimus metabolism; however only a proportion of the population expresses CYP3A5 secondary to genetic variation. CYP3A5 is expressed in both the intestine and the liver and has been shown to impact both the bioavailability and metabolism of orally administered tacrolimus. Increasing the initial tacrolimus dose by 50% to 100% is recommended in patients who are known CYP3A5 expressers; however, whether this dose adjustment is appropriate for i.v. tacrolimus administration is unclear. The objective of this study was to evaluate the impact of CYP3A5 genotype as well as other pharmacogenes on i.v. tacrolimus exposure to determine whether the current genotype-guided dosing recommendations are appropriate for this formulation. In addition, this study aimed to investigate dose conversion requirements among CYP3A5 genotypes when converting from i.v. to p.o. tacrolimus. This study is a retrospective chart review of all patients who underwent allogeneic stem cell transplantation at Michigan Medicine between June 1, 2014, and March 1, 2018, who received i.v. tacrolimus at the time of their transplantation. Secondary use samples were obtained for genotyping CYP3A5, CYP3A4, and ABCB1. Patient demographic information, tacrolimus dosing and trough levels, and concomitant medications received at the time of tacrolimus trough were collected retrospectively from the patients’ medical records. The i.v. dose-controlled concentration (C/D) and the i.v.:p.o. exposure ratio was calculated for all tacrolimus doses and patients, respectively. The impact of CYP3A5, CYP3A4, and ABCB1 genotypes on the i.v. C/D were evaluated with linear mixed modeling. The impact of CYP3A5 genotype on the i.v.:p.o. ratio was evaluated while controlling for age and concomitant use of an azole inhibitor. CYP3A5 and CYP3A4 genotypes were significantly associated with the i.v. C/D, with CYP3A5 expressers and CYP3A4 rapid metabolizers having 20% lower tacrolimus exposure. Neither genotype remained significant in the multivariable model, although age, hematocrit, and concomitant use of strong azole inhibitors were associated with increased i.v. C/D. When controlling for patient age and sex, CYP3A5 expressers had significantly higher i.v.:p.o. ratios than CYP3A5 nonexpressers (3.42 versus 2.78; P = .04). Post hoc analysis showed that the i.v.:p.o. ratio may differ among different CYP3A5 genotypes and azole inhibitor combinations. This study demonstrates that the current genotype-guided tacrolimus dose adjustment recommendations are inappropriate for CYP3A5 expressers receiving i.v. tacrolimus. Although CYP3A5 genotype is likely a minor contributor to i.v. tacrolimus exposure, genotype, in addition to capturing concomitant CYP3A inhibitors, would likely improve i.v.:p.o. dose conversion selection. © 2021 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc.

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.

Aim: This study tested for associations between SLCO1B1 polymorphisms and circulating estrogen levels in women with breast cancer treated with letrozole or exemestane. Patients & methods: Postmenopausal women with hormone-receptor positive breast cancer were genotyped for SLCO1B1*5 (rs4149056) and rs10841753. Pretreatment and on-treatment plasma estrogens and aromatase inhibitor (AI) concentrations were measured. Regression analyses were performed to test for pharmacogenetic associations with estrogens and drug concentrations. Results: SLCO1B1*5 was associated with elevated pretreatment estrone sulfate and an increased risk of detectable estrone concentrations after 3 months of AI treatment. Conclusion: These findings suggest SLCO1B1 polymorphisms may have an effect on estrogenic response to AI treatment, and therefore may adversely impact the anticancer effectiveness of these agents.

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.

Aim: This study evaluated the impact of CYP3A5 genotype and other patient characteristics on sublingual (SL) tacrolimus exposure and compared the relationship with oral administration. Patients & methods: Tacrolimus concentrations were retrospectively collected for adult lung transplant recipients, who were genotyped for CYP3A5*3, CYP3A4*22, CYP3A7*1C, and POR*28. Regression analyses were performed to determine covariates that impacted the SL and oral tacrolimus concentration/dose ratios. Results: An interaction of CYP3A5 genotype and CYP3A inhibitor increased the SL concentration/dose, while cystic fibrosis decreased the SL concentration/dose. The oral concentration/dose was independently associated with these covariates and was increased by serum creatinine and number of tacrolimus doses. Conclusion: This study suggests personalized dosing strategies for tacrolimus likely need to consider characteristics beyond CYP3A5 genotype.

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/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.

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.

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.

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.

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.

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.

Background: The prognostic significance of increased levels of CD8+ tumor infiltrating lymphocytes(TILs) in ER- breast cancer has been described. We sought to identify possible immune-related biomarkers for predicting benefit from letrozol(LET) or tamoxifen(TAM) for recurrence in ER+ breast cancer. Patient and Methods: We used Illumina DASL Assay to measure gene expression in FFPE primary breast cancers from a subset of postmenopausal patients enrolled in the BIG 1-98 randomized phase 3 trial comparing 5 years LET (n=344) vs TAM (n=381) as adjuvant endocrine therapy. Gene sets (n=1910) that represent cell states and perturbations within the immune system from the Human Immunology Project Consortium were used in an exploratory analysis to identify possible predictive signatures. Results: We identified five distinct gene signatures from previously reported laboratory experiments associated with immune cell differentiation that are highly predictive of benefit (reduced breast cancer recurrence risk) of LET over TAM, each with gene signature p-values Conclusion: The role of selective estrogen receptor modulators on immune response has been well described, where TAM has been shown to prevent differentiation and activation of dendritic cells (Naibandian 2005). Similarly, it has been shown that MET inhibitors negatively regulate neutrophils suggesting that anti-MET drugs in cancer could impact immune response (Finisquerra 2015). These findings suggest that if TAM is a negative regulator of immune response why in the ATAC clinical trial, the combination therapy of anastrozole plus TAM were not significantly different from TAM alone were anastrozole was superior. With the increasing importance of understanding the role of immune response on outcome and the use of combination therapies the assessment of TILs in the neoadjuvant setting will be critical for guiding therapy. Citation Format: Willis S, Gray KP, Regan MM, Rae JM, Kammler R, Young B, Ditzel HJ, Lyng MB, Colleoni M, Viale G, Leyland-Jones B. Immune related gene expression signatures predict benefit of letrozole over tamoxifen in BIG 1-98. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-07-36.

Background: Anastrozole (A) blocks estrogen production by inhibiting the activity of CYP19 aromatase. Fulvestrant (F) blocks estrogen receptor (ER) signaling by competitive binding, leading to ER degradation by ubiquitination. SWOG S0226 ("Phase III Randomized Trial of Anastrozole versus Anastrozole and Fulvestrant (250mg LD) as First Line Therapy for Post Menopausal Women with Metastatic Breast Cancer," ClinicalTrials.gov Identifier:NCT00075764) demonstrated that combination of A+F is superior to A alone as first-line therapy for patients with ER positive metastatic breast cancer (Mehta et al, NEJM, 2012). Our functional preclinical studies have shown that single nucleotide polymorphisms (SNPs) in SULT1A1 and UGT1A4, drug conjugation enzymes that inactivate A and F, result in decreased enzyme activity toward these drugs (Edavana et al, DMD, 2013; Edavana et al Pharmgenomics Pers Med 2013). We therefore hypothesized that these SNPs will be associated with disease outcomes in S0226 patients due to altered drug levels. Methods: Germline DNA was available for 295 (43.5%) patients enrolled in S0226 overall (157 on A and 138 on A+F). SNPs in SULT1A1 and UGT1A4 were determined either by direct sequencing or allele-specific PCR (TaqMan) assays. Results: There was no difference in progression-free survival (PFS) or overall survival (OS) comparing patients with or without available germline DNA (p = 0.86 and 0.36, respectively). The SULT1A1 G902A allele (rs6839), which confers decreased mRNA and enzymatic activity, was associated with improved PFS (GG/GA vs. AA; HR 0.74, 95% CI 0.56-0.98, p=0.033) and OS (HR 0.70, 95% 0.50-0.98, p=0.039). In exploratory subset analyses of PFS, the SULT1A1 G902A association was similar across both treatment arms (A HR=0.75; 95% CI 0.51-1.10; A+F HR=0.73; 95% CI 0.48-1.11). For OS there was some evidence of a difference by treatment (A HR=0.60; 95% CI 0.38-0.96; A+F HR=0.82; 95% CI 0.50-1.32), though no significant interaction was evident (p=0.30). The UGT1A4 G-163A promoter variant, which leads to decreased protein expression, was not associated with PFS (AA/AG vs. GG HR 0.88, 95% CI 0.68-1.14, p=0.33); however, this variant was associated with OS (HR 0.71, 95% CI 0.52-0.96, p=0.027). In subset analyses with OS, the difference was marginally stronger in the A arm (HR 0.63, 95% CI 0.42-0.97, p=0.035) compared to the A+F arm (HR 0.77, 95% CI 0.49-1.21, p=0.25), though the interaction was not significant (p=0.40). Conclusion: SULT1A1 and UGT1A4 gene variants resulting in decreased enzyme activity were associated with better PFS, OS or both in patients enrolled in SWOG S0226. Planned validation studies correlating these SNPs with drug levels and disease outcomes in additional patient cohorts will establish their clinical utility in identifying patients who benefit from A and F alone or in combination. Funding: Supported by NIH/NCI CA118981; NIH/NCI/NCTN grants CA180888, CA180819, and CA180863; and in part by AstraZeneca. Citation Format: Kadlubar SA, Barlow WE, Mehta RS, Daniels JR, Albain KS, Vandengerg TA, Dakhil SR, Tirumali NR, Lew DL, Gralow JR, Livingston RB, Hortobagiyi GN, Hayes DF, Rae JM. Association between gene variants in SULT1A1 and UGT1A4 and disease outcomes in patients enrolled in SWOG S0226 and treated with anastrozole alone or in combination with fulvestrant for metastatic breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-07-64.

Background: Tamoxifen therapy for estrogen receptor positive (ER+) breast cancer reduces recurrence risk by about half. Steady-state concentrations of endoxifen, a potent anti-estrogenic tamoxifen metabolite, are reduced in women whose CYP2D6 genotypes confer poor enzyme function. Many studies have measured associations between genetically impaired CYP2D6 function and tamoxifen resistance. It has been suggested that the subset of studies using DNA extracted from tumor-infiltrated tissue may have been susceptible to genotyping error induced by loss of heterozygosity (LOH); the putative non-differential genotype misclassification may have biased these studies' estimates toward the null. We reviewed the clinical epidemiology studies conducted to date to assess the importance of loss-of-heterozygosity (LOH) at the CYP2D6 locus and its implications for assessing tamoxifen effectiveness. Methods: We searched for the terms "tamoxifen" and "CYP2D6" in PubMed, including all papers and abstracts through 31 May 2015 on the association of CYP2D6 gene variants and the risk of breast cancer recurrence or mortality. We used a quantitative bias analysis (QBA) to evaluate the importance of genotype misclassification in studies that extracted DNA from tumor-infiltrated tissue. We conducted a random effects meta-analysis to evaluate all studies simultaneously, and within groups according to whether DNA was derived from tumor-infiltrated tissue or non-neoplastic tissue. Results: Thirty-one studies investigated CYP2D6 genotype and breast cancer recurrence, yielding relative effect estimates ranging from 0.08 to 14. DNA was extracted from blood or non-neoplastic tissue in 21 of these 31 studies (68%), and from tumor-infiltrated tissue in the remaining 10 (32%). Our analysis of the association between variant/variant genotype compared with wildtype/wildtype genotype included 21 of the 31 studies. Sixteen (76%) of these 21 studies extracted DNA from blood or non-neoplastic tissue and five (24%) extracted DNA from tumor-infiltrated tissue. Genotype misclassification parameters for the QBA were estimated from six concordance studies. There was little difference between the effect estimates (EE) and 95% confidence/simulation intervals (95% CI/SI) before and after QBA (EE=1.71, 95%CI=1.24, 2.36, and 1.80 95%SI=1.28, 2.54, respectively). Studies using non-neoplastic DNA had higher variance than those based on tumor-infiltrated tissue DNA, half reported implausibly high EE, and many were susceptible to design and analysis errors that would bias estimates of association away from the null. Conclusions: We found little relative bias in the summary estimates of association, either overall or when limited to the tumor-infiltrated tissue DNA studies. Three guideline panels, based on robust evidence, recommend against CYP2D6 genotype-guided tamoxifen therapy. Alternatives for optimizing the effectiveness of tamoxifen therapy, such as assuring adherence and persistence, are more likely to achieve clinically important benefits. Citation Format: Ahern TP, Hertz DL, Damkier P, Ejlertsen B, Hamilton-Dutoit SJ, Rae JM, Regan MM, Thompson AM, Lash TL, Cronin-Fenton DP. CYP2D6 genotype and breast cancer recurrence in tamoxifen treated patients: An evaluation of the importance of loss-of-heterozygosity. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-07-23.

Background: Increased rates of locoregional recurrence have been observed in TNBC despite chemotherapy and radiation (RT). Thus, approaches that result in radiosensitizaton in TNBC are critically needed. We characterized the RT response of 21 breast cancer cell (BCC) lines using clonogenic survival assays. We paired this with high-throughput drug screen data to identify AR as a top target for radiosensitization and assess AR inhibition as a radiosensitization strategy for TNBC. Methods: Clonogenic survival assays were used to determine the intrinsic RT sensitivity of 21 BCC lines. IC50 values were determined for 130 clinically available compounds and correlation coefficients were calculated using IC50 values and SF-2Gy. Gene expression was measured using RNA Seq and protein expression was measured using RPPA arrays in human tumor samples (n=2,061) and BCC lines (n=51). AR function was assessed using siRNA knockdown or functional inhibition with MDV3100 (enzalutamide). We measured in vivo tumor growth with varying control and treatment groups (16-20 tumors/group). Kaplan-Meier analysis was performed to estimate local control and survival. A Cox proportional hazards model was used to identify factors of survival, and MVA was used to determine variables associated with LRF survival. Results: Our radiosensitizer screen nominated bicalutamide as a most effective drug in treating RT-resistant BCC lines (R2= 0.46, p-value 2000 human breast tumor samples and found significant heterogeneity in AR expression with enrichment of expression at the protein and RNA level in TNBC. This same heterogeneity was also identified in human BCC lines. There was a strong correlation between AR RNA expression and protein expression (R2= 0.72, p Conclusion: Our results implicate AR as a mediator of radioresistance in breast cancer and support the rationale for developing clinical strategies, including clinical trials, to inhibit AR as a novel radiosensitizing target in TNBC. Citation Format: Speers C, Zhao SG, Liu M, Rae JM, Hayes DF, Feng FY, Pierce LJ. Androgen receptor (AR): A novel target and mechanism for radiosensitization and treatment in triple-negative breast cancers (TNBC). [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-13-05.

Background: Exemestane is a second generation steroidal aromatase inhibitor (AI) used for the treatment of estrogen receptor (ER) positive breast cancer in postmenopausal women. Variability in AI treatment efficacy and side effects seen across patients may be due, in part, to inter-patient differences in drug exposure. This exposure variability is likely caused by patient genetics factors, such as single nucleotide polymorphisms (SNPs) in drug metabolizing enzymes, or clinical factors such as patient body size, organ function, and comorbidities. The objective of this secondary correlative analysis was to identify genetic and clinical characteristics that affect steady state exemestane concentration, with a specific focus on the influence of inherited genetic variants and baseline hepatic function. Methods: 500 patients were enrolled on the Exemestane and Letrozole Pharmacogenetics (ELPh) Study and randomized to either drug. Clinical data and DNA were collected at baseline and blood samples were collected after 1 or 3 months of treatment to measure steady-state exemestane concentration via HPLC/MS. Genotyping was performed on a custom Sequenom MassARRAY iPLEX that included the recently discovered low activity CYP3A4*22 (rs35599367) SNP and several other SNPs with putative functional consequence in enzymes thought to be involved in exemestane metabolism (CYP1A1/2, CYP1B1, CYP3A4, CYP4A11, AKR1C3/4, AKR7A2). Our primary hypothesis was that patients carrying CYP3A4*22 variants would have higher serum exemestane concentrations. Other SNPs and clinical characteristics (hepatic and renal function, age, body mass index (BMI), time of sample collection, prior chemotherapy) were assessed for independent association, and then adjusted for in a multivariable tobit regression model for CYP3A4*22 on log-transformed censored exemestane concentration. Results: 246 (225 randomized to exemestane arm, 21 crossed-over from letrozole arm) patients had exemestane steady state levels and were evaluable in this analysis. As hypothesized, the CYP3A4*22 polymorphism (minor allele frequency=0.06) was associated with a 54% increase in exemestane concentration (95% CI: 14% - 109%, p40) at baseline (95% CI: 2% - 104%, p=0.02), 1% lower per unit increase in BMI (95% CI: 0% - 3%, p=0.05), and 20% lower in patients who received prior chemotherapy (95% CI: 4% - 34%, p=0.03). Age, renal impairment, and other SNPs were not associated with exemestane concentration. After adjustment for significant clinical covariates the CYP3A4*22 SNP remained significant (p Conclusions: Genetic and clinical predictors of exemestane concentration were discovered in a large cohort of prospectively enrolled estrogen responsive breast cancer patients. Ongoing analyses will determine whether the variability in exemestane concentration was associated with downstream effects on estrogen depletion or treatment-related toxicity. If so, these genetic and clinical characteristics could be useful for individualizing dosing of exemestane to ensure that all patients are receiving maximal benefit with minimal toxicity. Citation Format: Hertz DL, Kidwell KM, Seewald NJ, Gersch CL, Desta Z, Flockhart DA, Storniolo AM, Stearns V, Skaar TC, Hayes DF, Henry NL, Rae JM. CYP3A4*22 polymorphism is associated with increased exemestane concentrations in postmenopausal breast cancer patients. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-12-05.

Background: The anti-estrogen tamoxifen is metabolized into the more potent anti-estrogen, endoxifen, primarily by polymorphic CYP2D6. An association between CYP2D6 genotype and tamoxifen efficacy was assessed in a meta-analysis of 4,973 breast cancer patients by the ITPC. A subgroup analysis in 1,996 estrogen receptor (ER)-positive postmenopausal patients receiving 20[thinsp]mg/day tamoxifen for 5 years (criterion 1) found CYP2D6 poor metabolizer genotype was associated with worse invasive disease free survival (IDFS; hazard ratio (HR) 1.25, 95% confidence interval (CI) 1.06-1.47, P=0.009). This meta-analysis did not include data from two prospective trials (Anastrozole, Tamoxifen, Alone or In Combination (ATAC) and Breast Intergroup (BIG) 1-98) both of which meet the patient criteria but failed to replicate the ITPC findings. Some ITPC investigators criticized ATAC and BIG 1-98 for genotyping CYP2D6 from tumor-derived DNA, positing this leads to genotyping errors detected by Hardy-Weinberg Equilibrium (HWE). However, ITPC analyses did not exclude tumor-derived CYP2D6 genotypes or report HWE. Therefore, we re-analyzed the ITPC data to investigate claims that tumor-derived genotyping causes HWE departure and masks the association between CYP2D6 genotype and tamoxifen efficacy. Methods: The ITPC dataset was filtered to patients fulfilling criterion 1. HWE for CYP2D6*4 was analyzed in Caucasian patients (n=1,619) by study, by DNA source (tumor or blood), and in the entire subgroup. The ITPC meta-analysis was rerun stratified by DNA source using the same specifications (patients, phenotype, genotype, statistical model, etc.) as in ITPC. Results: Significant HWE deviation for CYP2D6*4, the most common variant (MAF =0.2), was not observed in any study genotyped from tumor but was observed in one study genotyped from blood. Combining studies led to significant HWE deviations in studies genotyped from both blood and tumor, and for the entire subcohort (Table 1). Associations between CYP2D6 genotype and IDFS stratified by DNA source yielded similar, non-statistically significant, results (blood: n=933, HR=1.19, 95% CI 0.91-1.57, P=0.21; tumor: n=997, HR=1.19, 95% CI 0.99-1.44, P=0.07). Conclusions: HWE deviations for CYP2D6*4 are not uniformly or exclusively found in studies using tumor DNA, but can occur as a statistical consequence of combining genotypes from heterogeneous populations like in the multi-institution BIG 1-98 and ATAC studies. Re-analysis of the ITPC dataset stratified by DNA source refutes the hypothesis that genotyping tumor DNA masks a pharmacogenetic association. These findings reaffirm the validity of the BIG 1-98 and ATAC analyses and support inclusion of these studies in the ITPC meta-analysis to rigorously assess the association between CYP2D6 genotype and tamoxifen efficacy. CYP2D6*4 HWE Test in Caucasian Patients from Each ITPC Study, by DNA Source, and CombinedDNA SourceStudy NumberNHWEBlood2700.68 4530.03 6130.28 84640.12 930.56 102220.0002 Total8250.0004Tumor51970.41 62170.04 83800.04 Total7940.0037CombinedTotal16190.000006 Citation Format: Kidwell KM, Hertz DL, Leyland-Jones B, Regan MM, Dowsett M, Rae JM. Analysis of the International tamoxifen pharmacogenomics consortium (ITPC) dataset shows that genotyping DNA derived from tumor does not introduce CYP2D6 genotyping error or mask an association with tamoxifen efficacy. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-09-02.

Purpose: Mutations in the estrogen receptor (ER)α gene, ESR1, have been identified in breast cancer metastases after progression on endocrine therapies. Because of limitations of metastatic biopsies, the reported frequency of ESR1 mutations may be underestimated. Here, we show a high frequency of ESR1 mutations using circulating plasma tumor DNA (ptDNA) from patients with metastatic breast cancer. Experimental Design: We retrospectively obtained plasma samples from eight patients with known ESR1 mutations and three patients with wild-type ESR1 identified by next-generation sequencing (NGS) of biopsied metastatic tissues. Three common ESR1 mutations were queried for using droplet digital PCR (ddPCR). In a prospective cohort, metastatic tissue and plasma were collected contemporaneously from eight ER-positive and four ER-negative patients. Tissue biopsies were sequenced by NGS, and ptDNA ESR1 mutations were analyzed by ddPCR. Results: In the retrospective cohort, all corresponding mutations were detected in ptDNA, with two patients harboring additional ESR1 mutations not present in their metastatic tissues. In the prospective cohort, three ER-positive patients did not have adequate tissue for NGS, and no ESR1 mutations were identified in tissue biopsies from the other nine patients. In contrast, ddPCR detected seven ptDNA ESR1 mutations in 6 of 12 patients (50%). Conclusions: We show that ESR1 mutations can occur at a high frequency and suggest that blood can be used to identify additional mutations not found by sequencing of a single metastatic lesion. Clin Cancer Res; 22(4); 993–9. ©2015 AACR.

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

538 Background: 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 CYP2A6. The objective of this genome-wide association study (GWAS) was to identify genetic variants that affect steady state letrozole concentrations. Methods: The Exemestane and Letrozole Pharmacogenetics (ELPh) Study randomized 503 post-menopausal patients with HR+ non-metastatic breast cancer to exemestane or letrozole treatment. Germline DNA was collected pre-treatment and blood samples were collected after 1 or 3 months of treatment to measure steady-state letrozole concentration via HPLC/MS. Genome-wide genotyping was conducted on the Infinium Global Screening Array to the Haplotype Reference Panel ( > 2 million variants). The association of each polymorphism with square-root transformed letrozole concentration was tested in self-reported white patients via linear regression using the standard alpha for genome-wide significance (α = 5x10−8) assuming an additive genetic model and correcting for age and body mass index. Results: 228 patients met inclusion criteria and had all necessary data. Each variant allele of rs7937 a patient carried increased their letrozole concentration ~22.9 ng/mL (standard error = 4.01, p = 3.51x10−8, Table) and this variant explained 13% of the variability in letrozole concentrations. rs7937 is located ~50 kB upstream of CYP2A6, and has previously been identified in GWAS of CYP2A6-related phenotypes, including nicotine metabolism and lung cancer. Conclusions: This GWAS confirmed that steady-state letrozole concentrations are partially determined by germline polymorphisms affecting CYP2A6 activity. If letrozole concentrations affect treatment efficacy or toxicity, CYP2A6 genetics may be useful to individualize letrozole dosing to improve clinical outcomes in patients with HR+ breast cancer. [Table: see text]

Background Pazopanib is approved for metastatic renal cell carcinoma (RCC). We assessed the safety and efficacy of pazopanib with a low fat meal (LFM)

Personalized oncology, or more aptly precision oncogenomics, refers to the identification and implementation of clinically actionable targets tailored to an individual patient’s cancer genomic information. Banking of human tissue and other biospecimens establishes a framework to extract and collect the data essential to our understanding of disease pathogenesis and treatment. Cancer cooperative groups in the United States have led the way in establishing robust biospecimen collection mechanisms to facilitate translational research, and combined with technological advances in molecular testing, tissue banking has expanded from its traditional base in academic research and is assuming an increasingly pivotal role in directing the clinical care of cancer patients. Comprehensive screening of tumors by DNA sequencing and the ability to mine and interpret these large data sets from well-organized tissue banks have defined molecular subtypes of cancer. Such stratification by genomic criteria has revolutionized our perspectives on cancer diagnosis and treatment, offering insight into prognosis, progression, and susceptibility or resistance to known therapeutic agents. In turn, this has enabled clinicians to offer treatments tailored to patients that can greatly improve their chances of survival. Unique challenges and opportunities accompany the rapidly evolving interplay between tissue banking and genomic sequencing, and are the driving forces underlying the revolution in precision medicine. Molecular testing and precision medicine clinical trials are now becoming the major thrust behind the cooperative groups’ clinical research efforts.

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

Introduction: Fifty percent of ER positive MBC patients do not benefit from ET. Potential mechanisms of resistance to ET in this patient population include absence of ER expression by deletion or suppression, alteration in ER signaling pathway genes, or upregulation of multiple growth factor receptor pathways. We hypothesized that genotyping and phenotyping of CTC combined with genomic analysis of ptDNA will provide important insights into the multiple mechanisms of ET resistance and that a set of blood tests might serve as a "liquid biopsy" abrogating the need for tissue specimens. Methods: Twenty-four patients providing informed consent were enrolled into the Mi CTC-ONCOSEQ study, a companion trial to Mi-ONCOSEQ (the Michigan Oncology Sequencing Program). Seven of these patients (5 with ER immunohistochemistry (IHC) positive and 2 with ER negative cancers) who had available archived primary and metastatic cancer tissue, a research metastatic biopsy for genomic analysis, and who had ≥5CTC/7.5 ml whole blood (WB) characterized for ER protein (CTC-ER) are the focus of this report. All the patients were ET refractory. None of them was progressing on fulvestrant at the time of study entry. CTC enumeration and phenotyping was performed with CellSearch©. Circulating ptDNA was analyzed by droplet digital polymerase chain reaction (ddPCR). ER status from archived tissue was obtained from chart review. ER mRNA expression was determined in the research biopsy of metastatic tissue by using quantitative RNA sequencing. Mutational status of ER gene, ESR1, was determined by Next-gen Sequencing using the Illumina HiSeq 2500 platform. Results: The 2 control patients with triple negative breast cancer had negative CTC-ER. Discordance between CTC-ER and tissue ER by IHC was observed (Table). Two of the 5 ER positive patients retained CTC-ER positivity (39% and 19% of the CTC). One of them (#7) harbored an ESR1 mutation in the research biopsy tissue and in ptDNA, whereas the other (#14) had wild type (WT) ESR1. CTC-ER protein levels in patients #12, 17 and 24 were negative. All had WT ESR1 in the research biopsy tissue. Of note, patient #12 had WT ESR1 in the research biopsy, but an ESR1 mutation was detected in her ptDNA. Pt#CTC-ER Tissue-ER ESR1 status in research biopsyESR1 status in ptDNA N[deg]CTC/7.5ml WB% CTC-ER +Primary by IHCMet by IHCMet research biopsy by mRNA 71839%+++Y537SY537S141619%+NA+WTWT12130%+++WTD538G17160%++weakly+WTWT242750%+weakly+weakly+WTWT Conclusions: These exploratory data suggest heterogeneous mechanisms of resistance to ET in patients with previously determined ER-positive MBC, including ESR1 mutations in ER positive cases (seen in 2 patients) and loss of ER expression (seen in CTC of 3 patients). In contrast, other cancers continue to express WT ESR1, and therefore must have developed alternative mechanisms of resistance. At least 2 of these mechanisms can be detected and monitored with complementary circulating assays: CTC and ptDNA. Further investigations are needed to understand the heterogeneous mechanisms of resistance to ET. Citation Format: Paoletti C, Aung K, Cannell EM, Darga EP, Chu D, Kidwell KM, Thomas DG, Tokudome N, Brown ME, McNutt LM, Gersch C, Schott AF, Park BH, Robinson DR, Chinnaiyan AM, Rae JM, Hayes DF. Molecular analysis of cancer tissue, circulating tumor cells (CTC) and cell-free plasma tumor DNA (ptDNA) suggests variable mechanisms of resistance to endocrine therapy (ET) in estrogen receptor (ER) positive metastatic breast cancer (MBC). [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-05-01.

The University of Michigan College of Pharmacy has made substantial investment in the area of pharmacogenomics to further bolster its activity in pharmacogenomics research, implementation and education. Four tenure-track faculty members have active research programs that focus primarily on the discovery of functional polymorphisms (HJ Zhu), and genetic associations with treatment outcomes in patients with cancer (DL Hertz), cardiovascular disease (JA Luzum) and psychiatric conditions (VL Ellingrod). Recent investments from the University and the College have accelerated the implementation of pharmacogenetics broadly across the institution and in targeted therapeutic areas. Students within the PharmD and other health science professions receive substantial instruction in pharmacogenomics, in preparation for careers in biomedical health in which they can contribute to the generation, dissemination and utilization of pharmacogenomics knowledge to improve patient care.

Elevated S100A8 expression has been observed in cancers of the bladder, esophagus, colon, ovary, and breast. S100A8 is expressed by breast cancer cells as well as by infiltrating immune and myeloid cells. Here we investigate the association of elevated S100A8 protein expression in breast cancer cells and in breast tumor stroma with survival outcomes in a cohort of breast cancer patients. Tissue microarrays (TMA) were constructed from breast cancer specimens from 417 patients with stage I–III breast cancer treated at the University of Michigan Comprehensive Cancer Center between 2004 and 2006. Representative regions of non-necrotic tumor and distant normal tissue from each patient were used to construct the TMA. Automated quantitative immunofluorescence (AQUA) was used to measure S100A8 protein expression, and samples were scored for breast cancer cell and stromal S100A8 expression. S100A8 staining intensity was assessed as a continuous value and by exploratory dichotomous cutoffs. Associations between breast cancer cell and stromal S100A8 expression with disease-free survival and overall survival were determined using the Kaplan–Meier method and Cox proportional hazard models. High breast cancer cell S100A8 protein expression (as indicated by AQUA scores), as a continuous measure, was a significant prognostic factor for OS [univariable hazard ratio (HR) 1.24, 95% confidence interval (CI) 1.00–1.55, p = 0.05] in this patient cohort. Exploratory analyses identified optimal S100A8 AQUA score cutoffs within the breast cancer cell and stromal compartments that significantly separated survival curves for the complete cohort. Elevated breast cancer cell and stromal S100A8 expression, indicated by higher S100A8 AQUA scores, significantly associates with poorer breast cancer outcomes, regardless of estrogen receptor status. Elevated breast cancer cell and stromal S1008 protein expression are significant indicators of poorer outcomes in early stage breast cancer patients. Evaluation of S100A8 protein expression may provide additional prognostic information beyond traditional breast cancer prognostic biomarkers.

Inter-individual differences in estrogen concentrations during treatment with aromatase inhibitors (AIs) may contribute to therapeutic response and toxicity. The aim of this study was to determine plasma concentrations of estradiol (E2), estrone (E1), and estrone sulfate (E1S) in a large cohort of AI-treated breast cancer patients.In a randomized, multicenter trial of postmenopausal women with early-stage breast cancer starting treatment with letrozole (n = 241) or exemestane (n = 228), plasma estrogen concentrations at baseline and after 3 months were quantitated using a sensitive mass spectrometry-based assay. Concentrations and suppression below the lower limit of quantification (LLOQ) were compared between estrogens and between drugs.The ranges of baseline estrogen concentrations wereLLOQ-361 pg/mL for E2,LLOQ-190 pg/mL for E1, and 8.3-4060 pg/mL for E1S. For E2, the frequency of suppression below the LLOQ was not statistically significantly different between AIs (exemestane: 89.0%, letrozole: 86.9%, p = 0.51). However, patients on letrozole were more likely to achieve suppression below the LLOQ of both E1 (exemestane: 80.1%, letrozole: 90.1%, p = 0.005) and E1S (exemestane: 17.4%, letrozole: 54.9%, p = 4.34e-15). After 3 months of AI therapy, the ranges of estrogen concentrations wereLLOQ-63.8 pg/mL,LLOQ-36.7 pg/mL, andLLOQ-1090 pg/mL for E2, E1, and E1S, respectively. During treatment, 16 patients had an increased concentration compared to the baseline concentration of at least one estrogen.Letrozole had greater suppression of plasma E1 and E1S than exemestane, though the response was highly variable among patients. Additional research is required to examine the clinical relevance of differential estrogen suppression.