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PDF file - 109K, Regression estimates for associations between sex-hormone measures and risk factors in the SIBS cohort.

Background: Ovarian suppression in premenopausal women is known to reduce breast cancer risk. This study aimed to assess uptake and compliance with ovarian suppression using the luteinizing hormone releasing hormone (LHRH) analogue, goserelin, with add-back raloxifene, as a potential regimen for breast cancer prevention.Methods: Women at ≥30% lifetime risk breast cancer were approached and randomized to mammographic screening alone (C-Control) or screening in addition to monthly subcutaneous injections of 3.6 mg goserelin and continuous 60 mg raloxifene daily orally (T-Treated) for 2 years. The primary endpoint was therapy adherence. Secondary endpoints were toxicity/quality of life, change in bone density, and mammographic density.Results: A total of 75/950 (7.9%) women approached agreed to randomization. In the T-arm, 20 of 38 (52%) of women completed the 2-year period of study compared with the C-arm (27/37, 73.0%). Dropouts were related to toxicity but also the wish to have established risk-reducing procedures and proven chemoprevention. As relatively few women completed the study, data are limited, but those in the T-arm reported significant increases in toxicity and sexual problems, no change in anxiety, and less cancer worry. Lumbar spine bone density declined by 7.0% and visually assessed mammographic density by 4.7% over the 2-year treatment period.Conclusions: Uptake is somewhat lower than comparable studies with tamoxifen for prevention with higher dropout rates. Raloxifene may preserve bone density, but reduction in mammographic density reversed after treatment was completed.Impact: This study indicates that breast cancer risk reduction may be possible using LHRH agonists, but reducing toxicity and preventing bone changes would make this a more attractive option. Cancer Epidemiol Biomarkers Prev; 27(1); 58–66. ©2017 AACR.

PDF file - 108K, Regression estimates for associations between density measures and risk factors in the SIBS cohort.

Purpose: The majority of breast cancer patients who have estrogen receptor positive (ER+) tumors whose proliferation is reduced after estrogen deprivation by aromatase inhibitors (AI). This study investigates any link between proliferation and hypoxia, a major determinant of tumor biology, and defines the effect of estrogen deprivation on hypoxia-associated genes.Methods: Genome-wide expression profiles were obtained from tumor biopsies from 81 ER+ postmenopausal patients, before and after 2 weeks' anastrozole treatment. A hypoxia metagene was developed by identifying genes clustered with classical hypoxia-regulated genes, excluding those associated with proliferation. Proliferation was measured by Ki67 and a proliferation metagene derived from two published breast cancer data sets.Results: Hypoxia and proliferation metagenes were associated at baseline (Pearson correlation coefficient, r = 0.67, P < 10−4) and after 2 weeks (r = 0.71, P < 10−4). Hypoxia metagene at baseline was associated with 2-week Ki67 (r = 0.43, P = 0.0002) and more weakly with poor 2-week Ki67 change consistent with a weak association with AI resistance. Hypoxia metagene was significantly downregulated with AI. This downregulation was significantly associated with change in the proliferation metagene and with Ki67 but, importantly, not with the substantial change in expression of classical estrogen-dependent genes.Conclusions: Hypoxia metagene is closely associated with proliferation before and after AI treatment. The downregulation of hypoxia metagene after AI therapy is most likely the result of changes in proliferation. There may be a weak effect of hypoxia metagene on de novo resistance to AIs. These findings are important to consider in coapplication of antiproliferative agents with antiangiogenic or antihypoxia agents. Clin Cancer Res; 17(9); 3005–12. ©2011 AACR.

Supplementary Figure S1.

Supplementary Table 1. DXA scan measurements (Baseline, 1-yr and 2-yr.)

Background: Mammographic breast density and endogenous sex-hormone levels are both strong risk factors for breast cancer. This study investigated whether there is evidence for a shared genetic basis between these risk factors.Methods: Using data on 1,286 women from 617 families, we estimated the heritabilities of serum estradiol, testosterone, and sex-hormone binding globulin (SHBG) levels and of three measures of breast density (dense area, nondense area, and percentage density). We tested for associations between hormone levels and density measures and estimated the genetic and environmental correlations between pairs of traits using variance and covariance components models and pedigree-based maximum likelihood methods.Results: We found no significant associations between estradiol, testosterone, or SHBG levels and any of the three density measures, after adjusting for body mass index (BMI).The estimated heritabilities were 63%, 66%, and 65% for square root–transformed adjusted percentage density, dense area, and nondense area, respectively, and 40%, 25%, and 58% for log-transformed–adjusted estradiol, testosterone, and SHBG. We found no evidence of a shared genetic basis between any hormone levels and any measure of density, after adjusting for BMI. The negative genetic correlation between dense and nondense areas remained significant even after adjustment for BMI and other covariates (ρ = −0.34; SE = 0.08; P = 0.0005).Conclusions: Breast density and sex hormones can be considered as independent sets of traits.Impact: Breast density and sex hormones can be used as intermediate phenotypes in the search for breast cancer susceptibility loci. Cancer Epidemiol Biomarkers Prev; 21(12); 2167–75. ©2012 AACR.

Supplementary Figure S1; Supplementary Tables S1-S6.

Purpose: Targeting vascular endothelial growth factor (VEGF) and estrogen receptor signaling pathways concomitantly may enhance benefit in estrogen receptor–positive breast cancer. We had shown previously that the VEGF receptor tyrosine kinase inhibitor PTK787/ZK222584 (PTK/ZK) is a competitive aromatase inhibitor in vitro. Here we investigated (a) whether PTK/ZK shows both antiangiogenic and antiaromatase inhibitory properties in vivo, and (b) whether the combination of PTK/ZK and letrozole is superior to letrozole alone.Experimental Design: Estrogen-dependent human breast cancer cells engineered to express aromatase (MCF7 AROM 1 and BT474 AROM) were used. Mice were treated with vehicle, PTK/ZK (25, 50, or 100 mg/kg), letrozole, or PTK/ZK in combination with letrozole.Results: In MCF7 AROM 1 tumors, all treatments induced growth suppression and were associated with a reduction in cell turnover index, a composite measurement of both proliferation and apoptosis. PTK/ZK significantly reduced vessel density. Whereas letrozole caused tumor regression, PTK/ZK stabilized tumor volumes. The growth suppressive and antiangiogenic effects of PTK/ZK were confirmed in BT474 AROM xenografts. The addition of PTK/ZK did not enhance the growth-suppressive effects of letrozole. However, PTK/ZK decreased progesterone receptor (PgR) and TFF1 expression and uterine weight, indicating that PTK/ZK decreases 17β-estradiol (E2) signaling in vivo.Conclusion: The VEGF receptor inhibitor PTK/ZK showed effects on E2-dependent gene expression consistent with aromatase inhibition as well as antiangiogenesis in xenograft models of breast cancer. The combination with letrozole was not superior to letrozole alone. Overall, these results provide further support for a potential therapeutic approach of dual inhibition of VEGF and E2 signaling using a single agent. Clin Cancer Res; 16(16); 4178–87. ©2010 AACR.

Supplementary Figure and Table Legends from Premenopausal Mammographic Density in Relation to Cyclic Variations in Endogenous Sex Hormone Levels, Prolactin, and Insulin-like Growth Factors

Supplementary Tables 1-4 from Premenopausal Mammographic Density in Relation to Cyclic Variations in Endogenous Sex Hormone Levels, Prolactin, and Insulin-like Growth Factors

Supplementary Information from Premenopausal Mammographic Density in Relation to Cyclic Variations in Endogenous Sex Hormone Levels, Prolactin, and Insulin-like Growth Factors

Background: NET is offered to postmenopausal patients (pts) with clinical stage 2/3 ER+/HER2- BC to promote breast-conserving surgery. Also limited surgical accessibility during the COVID19 pandemic has increased NET utility. Inability to identify ET-resistant disease at diagnosis risks disease progression (PD) and delays more effective treatments. Dowsett et al. recently demonstrated that baseline levels of ER, progesterone receptor (PR), Ki67 (>15% vs ≤15%), and Ki67 (>10% vs ≤10%) 2-4 weeks (wks) after starting NET may improve appropriate patient (pt) selection for NET (PMC7280290). The ER, PR and Ki67-based prediction model divides pts with primary ER+/HER2- BC into 3 groups for appropriateness for NET: (Group 1) NET is likely to be inappropriate (Allred ER 10% has been associated with worse outcome (PMC5455353), or (Group 3) NET is appropriate (ER 8 and PgR ≥6). The ALTERNATE trial (NCT01953588) randomized postmenopausal women with clinical stage II or III, ER+ (Allred score 6-8)/HER2- BC to receive anastrozole (ANA), fulvestrant (FUL), or ANA + FUL for 6 months, unless Ki67 was >10% on wk 4 or 12 biopsy, in which case pts were triaged to receive neoadjuvant chemotherapy (NCT) or surgery. As previously reported, the ET-sensitive disease (mPEPI 0 plus pCR) rates were similar across the treatment arms and overall 22% (286 of 1,299) pts had Ki67 >10% at wk 4 or 12. The ALTERNATE trial therefore provides a large independent data set to evaluate the NET appropriateness model. Results: Among 1,299 eligible pts randomized to receive 6 months of NET, 214 were excluded due to absent HR Allred score (n=41) or absence of pre-treatment and wk 4 Ki67 determinations (n=173). The proportions of the remaining 1,085 pts in Group 1, 2 and 3 were 1% (n=10), 43% (n= 468), and 56% (n=607), respectively. On-study Ki67 >10% prompting conversion from NET to NCT/Surgery occurred in: Group 1 90% (9 of 10), Group 2 30% (141 of 468), and Group 3 17% (104 of 607) (Table 1). Among the 1,075 pts in Groups 2 and 3, 260 (24%) pts had Ki67 ≤15% at baseline (BL), among whom only 14 (5.4%) had Ki67 >10% at wk 4, compared to 231 of the 815 (28.3%) who had BL Ki67 >15% and subsequent Ki67 >10% at wk 4. 2% of pts who remained on NET due to on-treatment Ki67 Conclusion: ALTERNATE trial data support a model whereby levels of ER, PR and Ki67 at diagnosis can be used for the identification of postmenopausal pts with primary ER+/HER2- BC who are appropriate for NET. When baseline ER Allred scores are >6 and Ki67 ≤15%, there is a low likelihood of ET-resistant disease. When BL Ki67 is >15%, ET sensitivity is variable, and on-treatment biopsy for Ki67 may assist in triaging regarding NET appropriateness, particularly given the extremely low local PD rates seen in ALTERNATE when on-treatment Ki67 was Table 1 Baseline levels of ER, PR, and Ki67 in Relation to Wk 4 Ki67 (N=1,085)BaselineWeek 4GroupNERAllred ScorePRAllred ScoreKi67Ki67 ≤10%N (%)Ki67 >10%N (%)1N=2615%1 (12.5%)7 (87.5%)2AN=647 or 815%148 (63%)87 (37%)2BN=466 or 7≥6≤15%42 (91.3%)4 (8.7%)51 (30.2)N=1236 or 7≥6>15%76 (61.8%)47 (38.2%)3N=1508≥6≤15%143 (95.3%)7 (4.7%)104 (17.1)N=4578≥6>15%360 (78.8%)97 (21.2%) Citation Format: Matthew J Ellis, Vera Suman, A. Marilyn Leitch, Souzan Sanati, Kiran Vij, Gary W Unzeitig, Jeremy Hoog, Mark Watson, Olwen Hahn, Joseph Guenther, Abigail Caudle, Erika Crouch, Horacio Maluf, Mitch Dowsett, Amy Tiersten, Monica Mita, Wajeeha Razaq, Tina J Hieken, Yang Wang, Travis Dockter, Jo Anne Zujewski, Anna Weiss, Clifford Hudis, Eric P Winer, Kelly Hunt, Ann H Partridge, Cynthia X Ma, Lisa A Carey. Validation of a predictive model for potential response to neoadjuvant endocrine therapy (NET) in postmenopausal women with clinical stage II or III estrogen receptor positive (ER+) and HER2 negative (HER2-) breast cancer (BC): An ALTERNATE trial analysis (Alliance A011106) [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 PD2-10.

PURPOSETo update key recommendations of the American Society of Clinical Oncology/College of American Pathologists estrogen (ER) and progesterone receptor (PgR) testing in breast cancer guideline.METHODSA multidisciplinary international Expert Panel was convened to update the clinical practice guideline recommendations informed by a systematic review of the medical literature.RECOMMENDATIONSThe Expert Panel continues to recommend ER testing of invasive breast cancers by validated immunohistochemistry as the standard for predicting which patients may benefit from endocrine therapy, and no other assays are recommended for this purpose. Breast cancer samples with 1% to 100% of tumor nuclei positive should be interpreted as ER positive. However, the Expert Panel acknowledges that there are limited data on endocrine therapy benefit for cancers with 1% to 10% of cells staining ER positive. Samples with these results should be reported using a new reporting category, ER Low Positive, with a recommended comment. A sample is considered ER negative if < 1% or 0% of tumor cell nuclei are immunoreactive. Additional strategies recommended to promote optimal performance, interpretation, and reporting of cases with an initial low to no ER staining result include establishing a laboratory-specific standard operating procedure describing additional steps used by the laboratory to confirm/adjudicate results. The status of controls should be reported for cases with 0% to 10% staining. Similar principles apply to PgR testing, which is used primarily for prognostic purposes in the setting of an ER-positive cancer. Testing of ductal carcinoma in situ (DCIS) for ER is recommended to determine potential benefit of endocrine therapies to reduce risk of future breast cancer, while testing DCIS for PgR is considered optional. Additional information can be found at www.asco.org/breast-cancer-guidelines .

Purpose.—To update key recommendations of the American Society of Clinical Oncology/College of American Pathologists estrogen receptor (ER) and progesterone receptor (PgR) testing in breast cancer guideline.Methods.—A multidisciplinary international Expert Panel was convened to update the clinical practice guideline recommendations informed by a systematic review of the medical literature.Recommendations.—The Expert Panel continues to recommend ER testing of invasive breast cancers by validated immunohistochemistry as the standard for predicting which patients may benefit from endocrine therapy, and no other assays are recommended for this purpose. Breast cancer samples with 1% to 100% of tumor nuclei positive should be interpreted as ER positive. However, the Expert Panel acknowledges that there are limited data on endocrine therapy benefit for cancers with 1% to 10% of cells staining ER positive. Samples with these results should be reported using a new reporting category, ER Low Positive, with a recommended comment. A sample is considered ER negative if < 1% or 0% of tumor cell nuclei are immunoreactive. Additional strategies recommended to promote optimal performance, interpretation, and reporting of cases with an initial low to no ER staining result include establishing a laboratory-specific standard operating procedure describing additional steps used by the laboratory to confirm/adjudicate results. The status of controls should be reported for cases with 0% to 10% staining. Similar principles apply to PgR testing, which is used primarily for prognostic purposes in the setting of an ER-positive cancer. Testing of ductal carcinoma in situ (DCIS) for ER is recommended to determine potential benefit of endocrine therapies to reduce risk of future breast cancer, while testing DCIS for PgR is considered optional. Additional information can be found at www.asco.org/breast-cancer-guidelines.

Background: For women with ER+ early stage breast cancer who are disease-free after 5 years of scheduled endocrine therapy, recurrences occur at a steady rate to at least year 20 from diagnosis, and are strongly correlated with tumor and nodal status (TN). For women diagnosed in 1976-2011, 20-year distant recurrence (DR) risk was 13%, 20%, and 34% for T1N0, T1N1-3, and T1N4-9 disease, respectively (Pan et al NEJM 2017). Using updated data, we investigated whether DR risk is lower for women diagnosed more recently. Methods: Kaplan-Meier and Cox regression analyses, stratified by trial, TN status and treatment, included 82,598 women with ER+ breast cancer from 108 trials who were alive and disease free after 5 years of scheduled endocrine therapy, 19,675 more than in the previously published dataset, of whom 11,391 were diagnosed since 2005. Results: Estimates of DR during the period from 5 to 20 years were 1% to 2% lower in the updated dataset than reported in 2017. Compared to earlier years (before 1995), the hazard ratio (HR) for DR in years 5-9 was 0.83 (95%CI 0.77 - 0.90) in women diagnosed in 1995-99, decreasing to 0.64 (0.59 - 0.70) in 2000-04, and to 0.58 (0.52 - 0.65) in 2005-12. Those diagnosed after 2000 (median follow-up after year 5 = 2.7, IQR 1.1-4.3 years) had a 30% lower risk of DR (HR = 0.70 (95% CI 0.66 - 0.75) compared with women diagnosed before 2000 (median follow-up after year 5 = 6.1, IQR 4.4-9.9 years). The recurrence risk in years 5-10 after diagnosis in women diagnosed after 2000 was 3% for T1N0 and 5% for T2N0, with few events recorded after year 10. If these recurrence rates continue at the same rate, 20-year risk of DR for women diagnosed after 2000 is projected to be 8% and 14% for T1N0 and T2N0 stages, respectively, compared with 13% and 19% for those diagnosed before 2000. More detailed analyses and investigation of factors influencing the improvements in outcome will be presented. Conclusion: The risk of DR at 20 years after diagnosis for women with node-negative ER+ early stage breast cancer, who discontinue endocrine therapy at 5 years is likely to be about a third lower now than in our previous report. However, long-term follow-up of patients diagnosed more recently is required to accurately characterize long-term recurrence risks. Reference: Pan H, Gray R, Braybrooke J, Davies C, Taylor C, McGale P, et al. 20-year risks of breast-Cancer recurrence after stopping endocrine therapy at 5 years. N Engl J Med. 2017;377:1836-46. Citation Format: Hongchao Pan, Jeremy Braybrooke, Richard Gray, Richard Peto, Rosie Bradley, Robert Hills, Jonas Bergh, Sandra M Swain, Mitchell Dowsett, Daniel F Hayes, Richard D Gelber, for the EBCTCG. Improvements in long-term outcome for women with estrogen receptor positive (ER+) early stage breast cancer treated with 5 years of endocrine therapy: Analyses of 82,598 women in the Early Breast Cancer Trialists’ Collaborative Group (EBCTCG) database [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr GS2-04.

Background: Aromatase inhibitors (AI), which block the conversion of androgen to estrogen, are the mainstay therapy for estrogen receptor–positive (ER+) breast cancer (BC). However, many patients relapse. Purpose: To identify molecular alterations associated with long-term neoadjuvant AI therapy of ER+ BC. Methods: We retrospectively identified 137 patients diagnosed with ER+ BC and treated with neoadjuvant AI for at least 1 month at the Royal Marsden Hospital (2003–2016). Paired pre- (diagnosis) and post-AI (surgery) biopsies with >40% invasive cell areas were available from 87 ER+ BC patients. In all samples, we evaluated ER, PR and Ki67 immunostaining, ESR1 hot-spot mutations by droplet digital PCR and expression of 801 genes associated with BC and response to endocrine therapy by NanoString. Results: Mean time on neoadjuvant treatment was 26 weeks (range 5.6–92.3). Cell proliferation remained suppressed in most tumours indicating little evidence for acquired resistance: 56/87 (64%) showed residual Ki67 (Ki67r) 10%. This was paralleled by reduced expression of proliferation genes, ER (immunostaining and gene expression) and estrogen-regulated genes (ERG) at surgery compared with the diagnostic samples (all p6 months, giving a prevalence in this cohort of 5/34 (15%). Tumours with ESR1 mutations showed less suppression of ERGs (p=0.002) and proliferation (p=0.039) and increased ESR1 (p=0.016) expression at surgery compared with tumours without mutation. Pathway analysis confirmed lack of inhibition of estrogen response (FDR>5%) and less inhibition of cell cycle [enrichment score: -0.49 vs -0.78) and E2F targets (-0.47 vs -0.8) in tumours with mutation. Tumours without ESR1 mutation but with Ki67r>10% also showed reduced ERGs response (p=0.006) compared to tumours with Ki67r Conclusion: Overall most tumours showed no evidence for the emergence of resistant disease after neoadjuvant AI therapy even after many months of treatment. However, we detected an enrichment of ESR1 mutations (15% of cases) after long-term treatment as a putative driver of ERG expression and proliferation and thus reduced AI response. Therefore, mutant ER appears to be associated with ligand-independent ERG activity supporting the clinical validity of dual blockade with a selective ER down-regulator combined with a CDK4/6 inhibitor targeting the RB/E2F axis in this scenario. Citation Format: Leal MF, Haynes BP, Schuster E, Yeo B, Afentakis M, Dodson A, Buus R, Cheang MC, Martin L-A, Dowsett M. Acquired ESR1 mutation and persistent expression of estrogen regulated genes in ER+ breast cancers on long-term neoadjuvant treatment with aromatase inhibitors [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-15-01.

Background: During endocrine treatment of breast cancer, the occurrence of symptoms related to oestrogen depletion are important predictor of treatment efficacy. We have previously shown that appearance of vasomotor and joint related symptoms are a useful biomarker for a greater response to endocrine treatment (Cuzick et al., Lancet, 2008). Furthermore a prediction model including clinicopathological parameters (CTS5) has shown to be a good prognostic tool for the prediction of late distant recurrences (DR) (Dowsett et al., JCO, 2018). Here, we assessed whether the occurrence of endocrine related adverse events were predictive of late DR in the ATAC trial. Methods: The ATAC trial (N=4735) of postmenopausal women with estrogen receptor positive breast cancer treated with 5 years' tamoxifen or anastrozole was used for this analysis. Women who reported symptoms (hot flashes, joint symptoms, gynaecological symptoms) at any time during the trial were compared to those not reporting these symptoms for late DR. Time to late DR, defined beginning at 5 years after randomization, was the primary endpoint. Hazard ratios (HR) and corresponding 95% CIs were estimated by Cox proportional hazards regression models. Results: 2937 women (62%) who were recurrence free after 5 years, reported either hot flashes, joint symptoms, or gynaecological symptoms during the active treatment period. Women who reported joint symptoms (adjusted for CTS5: HR=0.74 (0.59-0.94)), or gynaecological symptoms (adjusted for CTS5: HR=0.68 (0.47-0.97)) had significantly fewer late DR compared to those not reporting these events during the active 5 years' treatment period. Those who reported any symptom during the treatment period had an overall 34% lower risk of a late DR (univariate: HR=0.66 (0.53-0.83), P Conclusions: This retrospective analysis of the ATAC trial showed that occurrences of endocrine related symptoms during the treatment period are associated with the risk of developing a late DR, irrespective of treatment allocation. Larger effects were found for joint or gynaecological symptoms and remained significant after adjustment for clinical parameters. These findings might help clinicians and patients in their decision making process about extended endocrine therapy. Citation Format: Sestak I, Dowsett M, Cuzick J. Importance of adverse events during endocrine treatment for the prediction of late distant recurrences [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-08-02.

Background: EndoPredict (EPclin) is a validated prognostic test combining expression of 12 cancer-related genes for breast cancer patients with estrogen receptor (ER) positive, HER2-negative disease who received 5 years of endocrine therapy (Buus et al., 2017; Dubsky et al. 2012) and for women who received chemotherapy (Martin et al., 2014). Here, we determine the EPclin and 10-year distant recurrence free interval (DRFI) rates for patients who received adjuvant endocrine therapy plus chemotherapy (ET+C) or endocrine therapy alone (ET) using data from five large clinical trials. Methods: A total of 3746 women with ER-positive, HER2-negative disease were included in this analysis. 2630 patients received 5 years of ET alone (ABCSG-6/8, TransATAC) and 1116 patients received ET+C (GEICAM 2003-02/9906). EPclin incorporates tumor size and nodal status and accounts for different EPClin scores between ET+C and ET alone cohorts. The primary objective was to evaluate the 10-year DRFI rates as a continuous function of EPclin separately for patients in ET+C and ET. Secondary objectives included assessing the difference in the prognostic ability of EPclin between ET+C and ET overall (years 0-10) and for specific follow-up periods (years 0-5 and years 5-10). The primary endpoint was DRFI and the secondary endpoint was breast cancer free interval (BCFI). Cox proportional hazard models were used to estimate 10-year DRFI rates and to assess the prognostic information provided by EPclin. Results: All of the women on ET alone and 49% of those on ET+C were postmenopausal. Women who received ET+C had more node positive disease, more poorly differentiated tumours, and higher EPclin scores than those who received ET alone. Women who received ET+C had significantly smaller increases in 10-year DRFI rates with increasing EPclin score than those receiving ET alone (Table). EPclin was highly prognostic for DRFI in all women who received ET alone (HR=2.79 (2.49-3.13), P Conclusion: In our results from a non-randomized analysis, we observed significantly smaller increases in 10-year DRFI rates with increasing EPclin scores for women who received ET+C compared to those who received ET alone. Our indirect comparisons suggest that a high EPclin score can predict chemotherapy benefit in women with ER-positive, HER2-negative disease. 10-year DRFI risks (%) (95% CI) according to EPclin score for patients who received ET+C versus ET alone. ET+CETEPclin10-year DR risk (%)10-year DR risk (%)11.1% (0.5-1.7)1.0% (0.6-1.4)22.5% (1.5-3.5)2.8% (2.1-3.5)35.7% (4.1-7.2)7.6% (6.4-8.8)412.4% (10.1-14.6)19.8% (17.6-22.0)525.8% (22.0-29.5)46.1% (40.2-51.4) Citation Format: Sestak I, Martin M, Dubsky P, Rojo F, Cuzick J, Filipits M, Ruiz A, Buus R, Hlauschek D, Rodriguez-Lescure A, Dowsett M, Gnant M. Prediction of distant recurrence by EndoPredict in patients with estrogen receptor-positive, HER2-negative breast cancer who received adjuvant endocrine therapy plus chemotherapy (ET+C) or endocrine therapy alone (ET) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-08-04.

AIM To determine whether a modified Clinical Treatment Score (CTS) based on continuous tumor size and 5 lymph node categories provided more prognostic information in an independent test set than the original CTS with and without the Immunohistochemical-4 (IHC4) algorithm for prediction of residual distant recurrence risk over 10-years. BACKGROUND Risk of recurrence information in patients with estrogen receptor-positive (ER+), early breast cancer informs decision-making on chemotherapy use. The CTS and IHC4 algorithms provide such information, particularly when used in combination (IHC4+C). Their derivation in the translational cohort of the Arimidex Tamoxifen Alone or in Combination trial (TransATAC) was described by Cuzick et al in 2011. In the original model tumor size and nodal status were each classified into three categories, causing prognostic information to be lost. METHODS We modeled a novel CTSn algorithm on outcome data from patients in the anastrozole and tamoxifen arms in ATAC incorporating tumor size as a continuous variable and sub-dividing nodal status into five categories. IHC4n was re-derived in the TransATAC cohort independent of CTSn. Patients were chemotherapy-naïve. We compared ability to predict risk of residual distant recurrence of the new IHC4n+Cn model with that of the original one when tested in a training cohort and in a validation set of chemotherapy-naïve patients from the Tamoxifen vs. Exemestane Adjuvant Multicentre (TEAM) trial using Cox regression models and the C-index. RESULTS The ATAC training set for CTSn comprised 4056 patients, the TransATAC training set for IHC4n comprised 1125 patients; 2591 patients were in the TEAM validation set. Patients in the TEAM set were older (median age in TransATAC: 63.5, TEAM: 68.3 years), had a higher nodal-burden (node-positive in TransATAC: 29.4%, TEAM: 51.8%) and had more Grade 3 tumors (TransATAC: 18.3%, TEAM: 32.2%). The new IHC4n+Cn was significantly prognostic, and non-significantly more prognostic than the original IHC4+C in both the training and validation cohorts. When assessed using the C-index statistic, IHC4n+Cn had a higher discriminatory ability than the original algorithm (Table 1). Table 1 TransATAC (N=1125)TEAM (N=2591) HR* (95% CI)C-indexHR* (95% CI)C-indexOld Models CTS2.26 (2.01-2.53)0.6811.88 (1.73-2.03)0.650IHC41.67 (1.46-1.91)0.6301.49 (1.35-1.63)0.604IHC4+C2.76 (2.40-3.18)0.7242.03 (1.87-2.21)0.671New Models CTSn2.64 (2.26-3.09)0.7212.16 (1.96-2.39)0.687IHC4n1.74 (1.52-2.01)0.6421.51 (1.36-1.68)0.603IHC4n+Cn2.91 (2.47-3.42)0.7382.28 (2.06-2.51)0.695(*Hazard Ratio for change in one Standard Deviation). CONCLUSION By separately remodellng the part of the IHC4+C score based on clinicopathological characteristics using the whole ATAC chemo-naïve cohort, and the part that uses IHC-derived information in chemo-naïve TransATAC patients, we increased the precision of the individual risk estimates produced by both CTSn and IHC4n compared to those given by the original algorithms. The new IHC4n+Cn shows a trend for improved prognostic ability compared to the original IHC4+C. Like its predecessor, it relies on information that is readily available to clinicians and integrates it in an evidence-based way to improve prognostication in ER+ early breast cancer. Citation Format: Dodson A, Sestak I, Bayani J, Dowsett M, Bartlett J, Cuzick J. A newly derived combined clinical treatment score and immunohistochemical-4 prognostic tool [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-07-02.

Introduction: While endocrine therapy is an effective, well-tolerated treatment for estrogen receptor positive breast cancer (ER+BC), a large proportion of initial responders will develop hormone therapy resistance, and relapse. A major challenge in determining the mechanisms underlying endocrine therapy resistance is our limited ability to recapitulate inter- and intra-tumour heterogeneity in vitro. In this study we developed ER+BC cells resistant to estrogen deprivation, and to palbociclib treatment, and subjected these to 2D and 3D high-throughput drug and siRNA screens, in order to elucidate the pathways underpinning the development of endocrine resistance. Methods: ER+BC cell lines modelling relapse on aromatase inhibitor therapy were derived by long-term culture in the absence of exogenous estrogen, and were termed long-term estrogen deprived (LTED). Two of these lines were then used to generate additional resistance to palbociclib, thus modelling endocrine-resistant, palbociclib-resistant disease. These lines were then subjected to screens performed in 2D and 3D. Effects of the siRNAs and drug compounds were assessed by measuring cell viability, with a robust Z score of -2 taken as a threshold of significance. Table 1 outlines the key characteristics of the cell lines, the details of the drug screens performed, and the top 3 drugs with their corresponding targets. The siRNA screens were performed in 2D and 3D with a 709 kinome library. Results: The siRNA screens highlighted PI3K-AKT-mTOR signalling as being dysregulated in multiple 3D and 2D models, while the drug screens showed that compounds targeting phosphoinositide 3-kinase alpha (PI3Kα) were significant common hits. This is consistent with the recent success of the BYLieve trial in targeting PI3Kα. A second key area of dependency was in cell cycle regulation, with CDK7 and CDK9 found to be significant hits in multiple models. The 709 kinase screens in 2D and 3D demonstrated broadly similar results (43 vs 37 median number of significant hits 2D vs 3D; Z-score Table 1.Cell line characteristics and drug screen findingsCell line and key characteristics2D drug screen of 396 compounds, at 3 concentrations*3D drug screen of 70 compounds at 1 concentrationNumber of significant drug screen hits (Z score Citation Format: Arany Soosainathan, Joanna Nikitorowicz-Buniak, Sunil Pancholi, Marjan Iravani, John Alexander, Syed Haider, Stephen R Johnston, Mitchell Dowsett, Lesley A Martin, Clare M Isacke. Exploiting novel models of endocrine-resistant breast cancer to identify new therapeutic targets [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-02-05.

Cancer organoids to model therapy response Cancer organoids are miniature, three-dimensional cell culture models that can be made from primary patient tumors and studied in the laboratory. Vlachogiannis et al. asked whether such “tumor-in-a-dish” approaches can be used to predict drug responses in the clinic. They generated a live organoid biobank from patients with metastatic gastrointestinal cancer who had previously been enrolled in phase I or II clinical trials. This allowed the authors to compare organoid drug responses with how the patient actually responded in the clinic. Encouragingly, the organoids had similar molecular profiles to those of the patient tumor, reinforcing their value as a platform for drug screening and development. Science , this issue p. 920

Background Estrogen-receptor positive (ER+) breast cancer (BC) accounts for over 75% of diagnosed cases. Despite treatment, a large proportion relapse with de novo or acquired endocrine resistant disease, making it one of the greatest challenges for BC research. Using a kinome siRNA library screen, we identified MPS1 that is required for recruitment of the spindle assembly checkpoint complex, as strongly associated with resistance to endocrine therapy and palbociclib. Until now, the target population for MPS1 inhibitors has focused on triple negative BC. Our unexpected finding shows, potential efficacy of MPS1 inhibitors in ER+ BC resistant to endocrine therapies and palbociclib, a prominent contemporary first-line combination for advanced disease. Methods ER+ BC cell lines (MCF7, SUM44, ZR75.1, HCC1428 and T47D) adapted to estrogen independent growth (LTED) and sequential resistance to palbociclib (991R) were subjected to a siRNA screen targeting 709 kinases. Z-scores were used to identify the most robust candidates. Cell viability upon MPS1 inhibition with CCT289346 (MPS1i) was assessed 2D and 3D. The class effect was confirmed with other compounds targeting MPS1. Impact of MPS1i on ER co-localisation and ER-transactivation was assessed using confocal microscopy and reporter assays, respectively. Effect of MPS1i on chromosomal alignment and time spent in mitosis was established by time lapse and confocal microscopy. BrdU incorporation and cell cycle were assessed by FACS. PARP cleavage was used to measure apoptosis. Global gene expression analysis of MPS1 was carried out in two independent neoadjuvant studies of aromatase inhibitor (AI) treated patients. Results Kinome knockdown identified targets associated with the G2/M checkpoint as strongly implicated in the LTED phenotype. In particular, MPS1 was the top common hit in LTED and 991R cell lines. Increase in MPS1 was evident in MCF7-LTED at both the transcript and protein level. Notably, the MPS1 inhibitor CCT289346 caused a significant reduction in viability of the majority of LTED and 991R cell lines tested. Table. Sensitivity of ER+ BC cell lines to CCT289346Cell LineModelIC50 nMMCF7Wt90 LTEDESR1wt80 LTEDESR1Y537C50 TAMR55 ICIR50 LTEDICIR60 991R40 LTED991R45 LTED991R-ICIR25T47DWt110 LTED110 991R50 LTED991R60SUM44LTED40 Upon inhibition of MPS1, cells demonstrated shorter time in mitosis, aberration of cell cycle and amplified mitotic errors, resulting in increased apoptosis. To evaluate the clinical relevance of MPS1 in ER+ BC treated with endocrine therapy, we interrogated publicly available datasets from patients treated with neoadjuvant AI therapy. In the anastrozole cohort, on-treatment gene expression of MPS1 (p Conclusion This novel finding shows MPS1 inhibitors are capable of inducing mitotic aberrations and apoptosis in ER+ BC models resistant to endocrine therapy and palbociclib providing a new therapeutic strategy. Citation Format: Nikitorowicz-Buniak J, Pancholi S, Simigdala N, Ribas R, Linardopoulos S, Dowsett M, Johnston SR, Martin L-A. Global knockdown of cellular kinases identifies MPS1 as a novel modulator of endocrine and palbociclib resistance highlighting a new role for MPS1 inhibitors [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-09-03.

AIMS To describe and analyse >168,000 sets of results from clinical breast biomarker testing carried-out between 2009 and 2016 in the UK and Republic of Ireland, focussing on biological relationships. To present robust confirmatory evidence on known associations and provide new data on previously undescribed or unconfirmed ones. BACKGROUND Between Jan-2009 and Jan-2016 the UK National External Quality Assessment Scheme for Immunocytochemistry and In-situ Hybridisation (UK NEQAS ICC&ISH) collected results on clinical breast cancer testing, comprehensively for human epidermal growth factor receptor-2 (HER2) and optionally for estrogen receptor (ER) and progesterone receptor (PR), from most UK and Irish testing centres. Primary objectives were to assess and, where indicated achieve improvements in testing quality. The size and scope of the data-set is unparalleled in the literature and represent a significant reference resource for the breast cancer community. METHODS UK NEQAS ICC&ISH created and curated an on-line data-entry system allowing centres to systematically collect their own HER2, ER and PR results from clinical testing; for HER2, immunohistochemical (IHC) and in-situ hybridisation (ISH) data were collected; for ER and PR, IHC results could be entered according to local practise format. Tools and guidance were provided enabling centres to analyse their data for local audit and quality assurance. Clinicopathologic data was also collected, including: patient age at diagnosis, histological tumor type, tumor grade, site/stage (primary, recurrence, metastasis) and sample type (core, excision). RESULTS Data are present on 168,793 patients. 173 centres contributed ≥100 entries (96% of total). Median age was 62 years (IQR: 51-72). Tumor type was stated in 42%: 76% were invasive ductal, and 13% invasive lobular carcinoma. Grade was stated in 56%: 15% were Grade 1, 55% Grade 2 and 30% Grade 3. Site/stage was stated in 56%: 92% were primary, 2% recurrent and 6% metastatic. Sample type was stated in 70%: 75% were cores and 25%, excision. Receptor statuses were available as follows; HER2 (100%): 87% negative, 13% positive; ER (45%): 15% negative, 85% positive; PR (31%): 29% negative, 71% positive. HER2 data were available as follows; category by IHC: 91%; amplification status by ISH: 15%; HER2 gene/chromosome 17 (CEP17) ratio: 15% (86% of which were IHC 2+); HER2 gene copy number: 7%; CEP17 copy number: 7%. HER2-positive rate was 24% in ER-negative and 11% in ER-positive cases. HER2-positive status was negatively associated with increasing ER positivity (rho -0.22, P Comprehensive description of significant associations for all parameters will be presented. CONCLUSION The unique size and scope of this data-set has allowed confirmation of known associations for HER2, ER and PR with clinicopathologic and biological characteristics in breast cancer to very high confidence levels, and has uncovered previously undescribed relationships in both ER-positive and ER-negative disease. Citation Format: Dodson A, Parry S, Ibrahim M, Bartlett J, Dowsett M, Miller K. ER, PR and HER2 biomarkers in UK and Irish clinical breast cancer testing: analysis of results from >168,000 patients [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-08-16.

Background: Invasive lobular carcinomas (ILC) account for around 15% of all oestrogen receptor (ER) positive invasive breast cancers. The EndoPredict assay (EPclin) is a multigene classifier to predict the likelihood of distant recurrence in ER-positive, HER2-negative breast cancer patients treated with adjuvant endocrine therapy and has been validated in several retrospective translational studies. However, these validation studies did not include an analysis of the histological subtypes. Here, we investigate the role of EPclin for the prediction of distant recurrence in women with ILC and compare the prognostication to those with invasive ductal carcinoma (IDC) in TransATAC. Methods: Data on EPclin and histological type of tumour were available for 928 postmenopausal women with ER-positive, HER2-negative disease treated with 5 years of tamoxifen or anastrozole. The primary endpoint was distant recurrence and the primary objective was to assess the prognostic value of EPclin for the prediction of distant recurrence in women with invasive lobular carcinoma. Kaplan–Meier and Cox regression analyses were used to determine distant recurrence risk for 0-10 years of follow-up. Likelihood ratio tests were used to assess the prognostic information provided by EPclin. Hazard Ratios (HR) are for a change in 1 Standard Deviation. Results: 141 (15.2%) had ILC, 710 (76.5%) IDC, and the remaining 77 (8.3%) were of different histological type. EPclin provided highly significant prognostic value for distant recurrence in women with ILC (HR=3.33 (2.33-4.77), P Conclusions: This is the first analysis to focus on the role of EPclin for the prognostic assessment of women with ILC. Although numbers of women with ILC in TransATAC were small, EPclin provided highly significant prognostic information and risk stratification for this subgroup of women. 10-year distant recurrence risk in the EPclin low risk groups were similar between ILC and IDC, suggesting that chemotherapy is not indicated, irrespective of tumour type. Our results show that EPclin is informative in women with ILC. Citation Format: Sestak I, Kronenwett R, Denkert C, Cuzick J, Dowsett M. Prognostic performance of EndoPredict in invasive lobular carcinoma [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-08-01.

This abstract was withdrawn by the authors.

Despite increasing evidence supporting the clinical utility of immune infiltration in the estrogen receptor-negative (ER-) subtype, the prognostic value of immune infiltration for ER+ disease is not well defined.Quantitative immune scores of cell abundance and spatial heterogeneity were computed using a fully automated hematoxylin and eosin-stained image analysis algorithm and spatial statistics for 1178 postmenopausal patients with ER+ breast cancer treated with five years' tamoxifen or anastrozole. The prognostic significance of immune scores was compared with Oncotype DX 21-gene recurrence score (RS), PAM50 risk of recurrence (ROR) score, IHC4, and clinical treatment score, available for 963 patients. Statistical tests were two-sided.Scores of immune cell abundance were not associated with recurrence-free survival. In contrast, high immune spatial scores indicating increased cell spatial clustering were associated with poor 10-year, early (0-5 years), and late (5-10 years) recurrence-free survival (Immune Hotspot: LR-χ2 = 14.06, P.001, for 0-10 years; LR-χ2 = 6.24, P = .01, for 0-5 years; LR-χ2 = 7.89, P = .005, for 5-10 years). The prognostic value of spatial scores for late recurrence was similar to that of IHC4 and RS in both populations, but was not as strong as other tests in comparison for recurrence across 10 years.These results provide a missing link between tumor immunity and disease outcome in ER+ disease by examining tumor spatial architecture. The association between spatial scores and late recurrence suggests a lasting memory of protumor immunity that may impact disease progression and evolution of endocrine treatment resistance, which may be exploited for therapeutic advances.

Background The efficacy and safety of palbociclib, a cyclin-dependent kinase 4/6 inhibitor, combined with fulvestrant and goserelin was assessed in premenopausal women with advanced breast cancer (ABC) who had progressed on prior endocrine therapy (ET). Patients and Methods One hundred eight premenopausal endocrine-refractory women ≥18 years with hormone receptor–positive (HR+)/human epidermal growth factor receptor 2–negative (HER2−) ABC were among 521 women randomized 2:1 (347:174) to fulvestrant (500 mg) ± goserelin with either palbociclib (125 mg/day orally, 3 weeks on, 1 week off) or placebo. This analysis assessed whether the overall tolerable safety profile and significant progression-free survival (PFS) improvement extended to premenopausal women. Potential drug-drug interactions (DDIs) and ovarian suppression with goserelin were assessed via plasma pharmacokinetics and biochemical analyses, respectively. (ClinicalTrials.gov identifier: NCT01942135) Results Median PFS for premenopausal women in the palbociclib (n = 72) versus placebo arm (n = 36) was 9.5 versus 5.6 months, respectively (hazard ratio, 0.50, 95% confidence interval: 0.29–0.87), and consistent with the significant PFS improvement in the same arms for postmenopausal women. Any-grade and grade ≤3 neutropenia, leukopenia, and infections were among the most frequent adverse events reported in the palbociclib arm with concurrent goserelin administration. Hormone concentrations were similar between treatment arms and confirmed sustained ovarian suppression. Clinically relevant DDIs were not observed. Conclusion Palbociclib combined with fulvestrant and goserelin was an effective and well-tolerated treatment for premenopausal women with prior endocrine-resistant HR+/HER2− ABC. Inclusion of both premenopausal and postmenopausal women in pivotal combination ET trials facilitates access to novel drugs for young women and should be considered as a new standard for clinical trial design.

Background Neoadjuvant endocrine therapy (NAE) is often a good option for postmenopausal (PM) women with estrogen receptor positive (ER+) breast cancers(BC). Fall in Ki67 is widely accepted as valid for predicting favorable tumor response to NAE and improved outcome. We report our planned correlative study to investigate if intrinsic subtype impacts on Ki67 changes (ΔKi67) as measured by immunohistochemistry. We also explored the correlation of several ER+ BC relevant molecular features at baseline(B) with ΔKi67. Patients and methods POETIC is a phase III, randomized 2:1 study for 4486 PM patients with ER+ BC to determine whether peri-operative aromatase inhibitor (AI) followed by standard adjuvant therapy improves outcome compared with standard adjuvant therapy alone. The proliferation rate was estimated as percentage (%) of cancer cells staining for Ki67. Primary biological endpoint was defined as two-week (2wk) change in Ki67 (2wkΔKi67): ln[(2wk Ki67+0.1)/(B Ki67+0.1)]. Secondary endpoint: “responders”, was % change of Ki67 defined as (2wk Ki67 – B Ki67) *100/B Ki67. “Responder” was defined as follows: reduction 75% as good responder (GR). Human whole genome expression(GE) Illumina BeadChips were performed. Data was obtained from 137 paired samples from the treatment group(T) and 49 pairs from the control(C) group with GE data passing quality check and baseline Ki67≥5% to minimise the impact of extreme values based on proportional ΔKi67. Intrinsic subtype and risk of recurrence(ROR) groups were calculated using PAM50. GE scores from Oncotype Dx, MammaPrint, p53 mutation/wildtype(Troester 2006), ER+ early response (ERE)(Hatzis 2011), estrogen-regulated genes subtypes (Oh 2006) and markers for 23 different immune cell types(Bindea 2013) were calculated. Associations of GE scores to endpoints of response were determined by Spearman correlation and chi-square tests. Bonferroni correction was used to control error rate with p Results At B of the 137 paired T, 64% were Luminal A (LumA), 22% Luminal B (LumB), 9% as HER-2 enriched (HER2-E), 2% as Basal-like (BLBC) and 3% as Normal-like. Subtypes at B were associated with response, with LumA showing the biggest reduction of Ki67 (p=0.0001) and GR. All GE, except ERE, correlated significantly with 2wkΔKi67 and response: higher risk groups associated with lowest reduction rate. None of immune cell types correlated with 2wkΔKi67, except that tumors enriched with T-helper 1 cell type were associated with PR (p < 0.000001). Comparing subtypes between time-points, 85% of LumB and 42% of HER2-E were assigned instead as LumA at 2wk regardless of response. Of the 15 ROR defined high-risk group, only 33% were assigned instead as low-risk at 2wk. Conclusion Both LumA and LumB are endocrine sensitive. A fall of Ki67 was observed in majority of cases. Most tumors estimated as high-risk by molecular profiling showed less response and most remained moderate or high risk of recurrence on endocrine therapy. Whether molecular profiling at 2wk after starting AI predicts for long-term outcome in PM women with ER+ better than at diagnosis will need to be determined. Citation Format: Cheang MCU, Morden J, Gao Q, Parker J, López-Knowles E, Detre S, Hills M, Zabaglo L, Tomiczek M, Mallon E, Robertson J, Smith I, Bliss J, Dowsett M, On Behalf of the POETIC Trialists. The impact of intrinsic subtypes and molecular features on aromatase inhibitor induced reduction of proliferation marker of Ki67 in primary ER+ breast cancer: A POETIC study (CRUK/07/015) [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 P2-10-02.

BACKGROUND Gene expression (expr) analyses are increasingly used for characterising the pharmacodynamic response of primary BC. This includes assessing ER+ BC's dependence on estrogen (E) by measuring gene expr changes after AI-treatment. However, differences in tissue sampling and other preanalytic procedures between samples taken at diagnosis (D) and surgery (S), may lead to systematic artifactual changes that are falsely ascribed to the intervention. To identify genes whose expr is truly affected by AI, we measured global gene expr changes from paired core-cut biopsies at D and S from patients in the POETIC presurgical window trial. METHODS In POETIC, 4486 postmenopausal women with primary ER+ BC were randomised 2:1 to receive perioperative AI (2 weeks pre + 2 weeks post surgery, termed Tr) or no perioperative treatment (termed Con), allowing gene expr changes to be compared between Tr and Con. RNA was extracted from paired RNA-later stored core-cuts of 56 Con and 157 Tr patients and arrayed on Illumina whole genome expr BeadChips. Raw data was extracted, transformed, normalised and batch-corrected. Probes not detected (p>0.01) in >=25% of samples were discarded. Impact of AI on genes was evaluated based on difference of the expr mean changes (log2(S/D)) of the Tr and Con samples. RESULTS In the Con group, expr of 73 genes significantly changed (FDR 615 genes were down-regulated and 472 up-regulated in Tr but not Con. The majority of down-regulated genes were cell cycle or proliferation-associated or E-regulated, including ESR1, PDZK1, GREB1, HSPB1. Functional mapping showed changes in the regulation of cyclins and cyclin dependent kinases impacting on G1/S and G2/M. Of note, up-regulated genes included CDK6 (target for CDK4/6 inhibitors) and CCND2, involved in G1/S checkpoint regulation; SNAI2, TGFB3, TGFBR2, associated with tumour invasion and metastasis; and other genes involved in aryl hydrocarbon receptor, Glioblastoma Multiforme, HIPPO and p53 signalling. CONCLUSION Expr of certain genes is altered by processes involved in presurgical window studies. In the absence of a Con group, these may be wrongly ascribed to an experimental intervention or wrongly considered as unaffected by the intervention (eg MYC in this study). Down-regulation of E-responsive and proliferation genes was an expected response to AI but increased expr of genes such as SNAI2, CCND2 and CDK6 indicates immediate tumour re-wiring and provides mechanistic support for benefit from combination therapy with a CDK4/6 inhibitor. Citation Format: Gao Q, López-Knowles E, Cheang MCU, Morden J, Martin L-A, Sidhu K, Evans D, Martins V, Dodson A, Skene A, Holcombe C, Mallon E, Abigail E, Bliss J, Robertson J, Smith I, Dowsett M. True effect of aromatase inhibitor (AI) treatment on global gene expression (expr) changes in postmenopausal ER+ breast cancer (BC) patients: A POETIC study (CRUK/07/015) [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 P2-09-02.

Background Higher levels of the proliferation marker Ki67 at breast cancer (BC) diagnosis are increasingly recognised to indicate poorer prognosis. Change in ΔKi67 in response to endocrine treatment reflects response. The precise relationship between Ki67 and other clinico-path factors and how associations are affected by short exposure to aromatase inhibitor (AI) has been unclear. Methods POETIC was a UK-wide, phase III, randomised trial which tested perioperative use of AI (anastrozole (A), letrozole (L)) in postmenopausal women with early BC (Dowsett JNCI Monogr 2011). Ki67 was measured in a single central lab at diagnosis (B=baseline) and 2 weeks later at surgery (S) allowing in vivo assessment of AI sensitivity. POETIC recruited 4483 women from 130 UK centres. Paired biopsies were available for 96%. Relationship between Ki67 and clinico-path factors is described by summary statistics (median) and independent associations explored in multivariable linear regression models (MVM). Analyses of Ki67 at S and ΔKi67 (reduction) were adjusted for B Ki67 and surgical sample type. Results Absolute Ki67 (B) was associated with each classic prognostic factor. Factors affecting ΔKi67 in control pts were Ki67 (B), grade (B) and surgical sample type (core cut 4.1%; excision 17.7% p Baseline Ki67 %Ki67 change (%) with 2 weeks AIVariableNMedianln(Ki67) P value (MVM)NMedianln (Ki67(S)/Ki67 (B)) P Value (MVM)All patients391315.2 252877.6 PgR (local) 0.01 5cm17416.9 11474.1 Histological type (B) Summary POETIC provides the largest multi-centre series of women in whom centrally assessed Ki67 has been correlated with classic clinico-path factors and impact of short term AI exposure explored. Choice of AI and surgical sample type are both dictated by participating site and their inter-relationship requires further review. Whether the greater suppression of Ki67 following L, a drug associated with better E2 suppression and aromatisation than A has clinical consequences is beyond the scope of this work. Relationships found (e.g. sample type, grade) are critical for interpretation of studies using Ki67 for prognosis and ΔKi67 as a pharmacodynamic response marker. Citation Format: Bliss JM, Morden J, Evans A, Holcombe C, Horgan K, Mallon E, Raghavan V, Skene A, Dodson A, Hills M, Detre S, Zabaglo L, Graf M, Banerji J, Gillman A, Robertson J, Dowsett M, Smith I, On Behalf of the POETIC Trialists. Clinico-pathological relationships with Ki67 in POETIC (CRUK/07/015) – Critical lessons for assessing Ki67 for prognosis and as a pharmacodynamic marker [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 P2-05-01.

Background: Previous reports from the Anastrozole Tamoxifen Alone or in Combination (ATAC) trial have shown significantly prolonged disease-free survival, lower rates of recurrence and distant recurrence, and reduced contralateral breast cancer in patients treated with anastrozole compared to tamoxifen (Cuzick et al., Lancet, 2010). Here, we compare the long-term effects of anastrozole versus tamoxifen in patients randomised to either monotherapy arm in the ATAC trial. Methods: Postmenopausal women with hormone receptor positive breast cancer randomised to anastrozole or tamoxifen in the main ATAC trial were eligible for the LATTE observational study. The primary objective was to compare the long-term effects of tamoxifen and anastrozole on time to recurrence and death beyond 10 years after randomisation. Secondary objectives included time to distant recurrence, cancer-specific survival, new breast primaries, other cancers, fractures, and cardiac/cerebrovascular events. Cox proportional hazard methods were used to compute hazard ratios (95% CI) for recurrence from the time of last publication (10 years median follow-up). Results: 2452 women from 11 countries were entered into the LATTE study. 40 women withdrew consent and 759 women died or had a recurrence within 10 years, which left 1653 women for analysis (838 anastrozole vs. 815 tamoxifen). A total of 118 breast events (69 anastrozole (8.2%) vs. 49 tamoxifen (6.0%)) were reported. No significant difference between the two treatment arms were observed (HR=1.36 (0.94-1.97), P=0.098). 57 women had a distant recurrence (33 (3.9%) vs. 24 (2.9%)), 41 reported a loco-regional recurrence (23 (2.7%) vs. 18 (2.2%)), and 26 contra-lateral breast cancer were recorded (17 (2.0%) vs. 9 (1.1%)). None of the treatment comparisons were statistically significant. 305 deaths were recorded (147 (17.5%) vs. 158 (19.4%)), of which 31 were due to breast cancer. Significantly fewer gynaecological cancers were recorded with anastrozole (7 vs. 16; OR=0.42 (0.15-1.09), P=0.05), but overall the effect on other cancers was not significant (54 (6.4%) vs. 64 (7.9%). Fractures, cardiovascular, and cerebrovascular events were evenly distributed between the treatment arms. Conclusions: Although anastrozole was associated with significant fewer recurrences compared to tamoxifen in the first 10 years of follow-up, in this analysis, with limited number of patients, we could not find a significant difference between the two treatment arms. Citation Format: Cuzick J, Sestak I, Bianco A, Strobbe L, Bergh J, Hanusch C, Neven P, Dowsett M, Forbes JF, Buzdar A, Smith R, Howell A. Long-term comparison of anastrozole versus tamoxifen: Results from LATTE/ATAC [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 P2-09-03.

Background: EndoPredict (EPclin) and Oncotype Recurrence Score (RS) have both shown their prognostic ability for women with estrogen receptor positive (ER), HER2-negative breast cancer, with EPclin outperforming RS specifically in node-positive population and for the prediction of late distant recurrence (DR). Age is an important risk factor for breast cancer but no data is available on the performance of genomic assays according to patient’s age. Here, we investigate whether EPclin and RS provide differential prognostic value in different age groups. Methods: 928 postmenopausal women with ER-positive, HER2-negative breast cancer were included in this analysis for whom information on EPclin and RS were available. None of the patients included in this analysis received chemotherapy. Age at diagnosis was treated as a categorical variable (≤60 years, 60-70 years, >70 years). Primary endpoint was DR and we used Cox regression models to assess the prediction of DR by EPclin and RS according to age. Results: 300 (32.3%) women were aged 60 years or younger, 377 (40.6%) between 60 and 70 years, and 251 (27.1%) were older than 70 years. Women aged 60 years or older had significantly more node-positive disease, higher grade tumors, and more DR than those aged 60 or younger. RS scores were similar between age groups whereas EPclin scores were significantly higher in older women compared to younger women, reflecting the combined molecular plus clinical nature of the EPclin. Overall, both RS and EPclin were prognostic but with decreasing prognostic value with increasing age (Table). We observed a significant interaction between continuous age and both tests (RS: P=0.004; EPclin: P=0.026). In women aged 70 years and older, RS did not provide any prognostic information (HR=1.22 (0.96-1.55); LR-χ2=2.5) whereas EPclin showed significant prognostic ability in this age group (HR=2.02 (1.59-2.57); LR-χ2=30.4) (Table). Kaplan-Meier estimates were significantly different between low and high risk groups for both tests in women aged less than 70 years. No significant risk stratification was achieved by RS in women aged 70 years or older whereas EPclin distinctively stratified older patients into low and high risk groups with significant different 10-year DR risks. Conclusion: Genomic assays are increasingly used in the clinic to determine prognosis for women with ER-positive, HER2-negative breast cancer. Here, we show that the effect sizes and prognostic value of EPclin and RS are different across age groups, and only the EPclin was prognostic in elderly patients. Our results indicate that tests that incorporate clinical parameters in their score are likely to be more prognostic in elderly patients and should be used in the decision-making process when using genomic assays. Hazard Ratios (HRs (95% CI)) for continuous EPclin/RS scores and risk groups according to age groups≤60 years60-70 years>70 years(N=300)(N=377)(N=251)RS2.23 (1.57-3.15)1.59 (1.32-1.91)1.22 (0.96-1.55)LowReferenceReferenceReferenceIntermediate3.79 (1.40-10.24)2.74 (1.44-5.23)1.75 (0.97-3.15)High8.86 (3.07-25.57)6.55 (3.25-13.19)1.63 (0.67-3.96)EPclin3.47 (2.28-5.27)2.74 (2.17-3.45)2.02 (1.59-2.57)LowReferenceReferenceReferenceHigh9.60 (3.60-25.57)5.55 (2.91-10.61)3.68 (1.89-7.17) Citation Format: Ivana Sestak, Ralf Kronenwett, Richard Buus, Jack Cuzick, Mitchell Dowsett. Prognostic value of EndoPredict and Oncotype Recurrence Score according to patient's age [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-06-04.

Background: Several multigene assays are available for managing ER-positive, HER2-negative early stage breast cancer but with little direct comparative information within this patient population. Existing evidence suggests that current multigene assays provide broadly equivalent risk information for the population of women with ER-positive, HER2-negative breast cancers. However, for the individual patient tests may provide differing risk categorization (Bartlett et al, JNCI 2016). We have previously demonstrated the prognostic value of different commercially available tests including Prosigna (ROR) and Oncotype Dx (RS) (Dowsett et al, JCO 2013; Sestak et al, JAMA Onc 2018). Here, we investigate risk classification and 10-year DR rates in patients that had discordant risk categorization between ROR and RS. Methods: A total of 663 postmenopausal women with ER-positive, HER2-negative, node-negative early stage breast cancer from the TransATAC study for whom both signatures were available were included in this analysis. The primary endpoint was distant recurrence (DR). Predefined ROR cut-off points for low/intermediate and intermediate/high of 40/60 were used. Comparisons were made using two sets of RS cut-off points 1) TailoRX cut-off points of 10/25 and 2) original cut-off points of 18/31. Kaplan-Meier curves were used to estimate the mean risk of DR after 10 years of follow-up in predefined risk groups. Results: Using original RS cut-off points, 25 patients (3.8%) had a high-low discordance compared to ROR, with a total of 295 (44.5%) discordant cases. Using the TailoRx cut-off points, 40 patients (6.0%) had a high-low discordance compared to ROR, with a total of 396 (59.7%) discordant cases. Applying original RS cut-off points, patients categorized into ROR low but into intermediate or high by RS (86/365 (23.6%)) had a 10-year DR rate of only 6.3% (2.7-14.6) (Table). In contrast, patients categorized into RS low but into intermediate or high by ROR (144/423 (48.6%)) had a 10-year DR rate of 13.4% (8.3-21.2). Using the TailoRx cut-off points, patients categorized into ROR low but into intermediate or high by RS (261/365 (71.5%)) had a 10-year DR rate of 2.9% (1.4-5.9), while patients with an RS low but intermediate or high by ROR (62/166 (37.3%)) had a 10-year DR rate of 18.2% (10.2-31.2) (Table). Conclusion: Discordance in risk categorization at the individual patient-level between commercially available multigene assays for early stage breast cancer is not uncommon. Our results show that node-negative patients that were classified by ROR as low risk, regardless of RS risk stratification, had an excellent 10-year DR risk with endocrine therapy alone. In contrast, patients that were classified as RS low risk by either cut-off points but were classified as intermediate or high risk by ROR had a significantly worse 10-year outcome. Our results indicate that ROR better categorized women with node negative disease into respective risk groups. Risk stratification and 10-year DR risk (%) according to ROR and RS cut-off points.ROR cut-offsLow (N=365)Intermediate/High(N=298)Original RS cut-offsNumber10-year risk (%)Number10-year risk (%)Low (N=423)2791.5%14413.4%Intermediate/High (N=240)866.3%15424.8%TailoRx cut-offsLow (N=166)1042.1%6218.2%Intermediate/High (N=497)2612.9%23619.5% Citation Format: Ivana Sestak, Sean Ferree, Itay Shemesh, Wesley Buckingham, Jack Cuzick, Mitchell Dowsett. Discordant classification and outcomes between Prosigna and Oncotype Dx Recurrence Score for ER-positive, HER2-negative, node-negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-06-05.

Background Experimental evidence (Fisher et al, 1989) & a small clinical trial (IMPACT) respectively suggested peri-operative endocrine therapy (ET) may improve long-term disease-related outcome in patients undergoing primary surgery for ER positive (ER+) breast cancer (BC) & that tumor Ki67 levels after 2 weeks of peri-operative aromatase inhibitor (POAI) therapy might offer an effective way of predicting outcome & the need for additional adjuvant treatment. POETIC (*Peri-Operative Endocrine Therapy - Individualising Care) is a phase III randomized controlled trial designed to test these hypotheses & provide data to determine whether 2 week Ki67 improves prediction beyond that by baseline Ki67 of the group who have a higher risk of relapse in the first years after diagnosis in spite of best current standard of care. Patients & methods Postmenopausal patients with ER+ BC were randomised 2:1 to either, POAI (centre choice: letrozole 2.5mg or anastrozole 1mg daily) for 14 days prior to & 14 days following surgery or no POAI (Control). Randomization was stratified by treating center; adjuvant treatment was per UK routine practice. Tissue samples were collected at baseline & surgery (FFPE) for blinded Ki67 testing. Primary endpoint was Time to Recurrence (TTR: time from randomization to loco-regional or distant recurrence or BC death). A secondary endpoint was Ki67 at baseline & after 2 weeks of AI. Results Between 2008 & 2014, 4480 patients (2976 AI, 1504 Control) were randomized from 130 UK centers. Median age was 67 (IQR 62-75), 18% had grade 3 tumors, 39% were node positive and 61% had tumor size>2cm. For adjuvant ET 314 patients (7.2%) received tamoxifen (Tam), 3695 (84.6%) an AI, 251 (5.7%) Tam changing to AI and 109 (2.5%) changing from AI to Tam. On 8 August 2017, median follow-up was 60.7 months (IQR 49.5 to 72.2). 408/4480 (9.1%) patients have had a TTR event; 263 (8.8%) allocated to POAI compared to 145 (9.6%) controls: HR=0.91 (95%CI: 0.74, 1.12) Log-rank p=0.37. Adjusted HR=0.91 (95%CI: 0.74, 1.11). The relationship of Ki67 (baseline & after 2 weeks) with TTR in both the POAI & control groups will be presented for the overall ER+ population & HER2 defined sub-groups. Discussion There was no significant evidence that four weeks of POAI improved TTR compared with no POAI. POETIC will provide definitive evidence on the role of 2 week POAI-treated Ki67 to inform future practice & trials in terms of the potential to identify a group of patients for whom current standard of care appears insufficient in the few years post diagnosis. Citation Format: Robertson JFR, Dowsett M, Bliss JM, Morden JP, Wilcox M, Evans A, Holcombe C, Horgan K, Kirwan C, Mallon E, Sibbering M, Skene A, Vidya R, Cheang M, Banerji J, Kilburn L, Dodson A, Smith I. Peri-operative aromatase inhibitor treatment in determining or predicting longterm outcome in early breast cancer – The POETIC* Trial (CRUK/07/015) [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr GS1-03.

Despite increasing evidence supporting the clinical relevance of tumour infiltrating lymphocytes (TILs) in invasive breast cancer, TIL spatial distribution pattern surrounding ductal carcinoma in situ (DCIS) and its association with progression is not well understood.To characterize the tissue microecology of DCIS, we designed and tested a new deep learning pipeline, UNMaSk (UNet-IM-Net-SCCNN), for the automated detection and simultaneous segmentation of DCIS ducts. This new method achieved the highest sensitivity and recall over cutting-edge deep learning networks in three patient cohorts, as well as the highest concordance with DCIS identification based on CK5 staining.Following automated DCIS detection, spatial tessellation centred at each DCIS duct created the boundary in which local ecology can be studied. Single cell identification and classification was performed with an existing deep learning method to map the distribution of TILs. In a dataset comprising grade 2-3 pure DCIS and DCIS adjacent to invasive cancer (adjacent DCIS), we found that pure DCIS cases had more TILs compared to adjacent DCIS. However, TILs co-localise significantly less with DCIS ducts in pure DCIS compared with adjacent DCIS, suggesting a more inflamed tissue ecology local to adjacent DCIS cases.Our experiments demonstrate that technological developments in deep convolutional neural networks and digital pathology can enable us to automate the identification of DCIS as well as to quantify the spatial relationship with TILs, providing a new way to study immune response and identify new markers of progression, thereby improving clinical management.

Background: Aromatase inhibitors (AIs) have an established role in the treatment of estrogen receptor alpha positive post-menopausal breast cancer. Recently we have developed and validated a microarray-derived 4-gene test (Edinburgh EndoResponse4) to predict response to AIs in the neoadjuvant setting. We have also demonstrated the translational potential of this test in predicting accurately clinical response when mRNA is measured for these genes by polymerase chain reaction (PCR) or the gene protein is measured by immunohistochemistry (IHC). There is a major clinical need for biomarkers to predict which patients are likely to recur on adjuvant endocrine therapy so alternative or additional treatments can be provided to reduce recurrence and improve outcome. The aim of this study was to determine if Endoresponse4 and IHC of these gene proteins could do this. Methods: The original microarray assay used pre- and on-treatment (14-days) biopsies from 73 post-menopausal women with ER-rich breast cancer receiving 3 months of neoadjuvant letrozole prior to surgery with 10 years follow-up after adjuvant letrozole. Matched formalin-fixed paraffin embedded (FFPE) tissue sections from 42 of these patients were used for IHC and antibodies were optimised against 3 of the 4 proteins (where validated antibodies were available) using Envision technology. The ability of our test to estimate recurrence-free (RFS) and breast cancer specific overall survival (OS) using both PCR and IHC was then tested in a unique validation cohort of 140 post-menopausal women with ER-rich breast cancer treated with 2 weeks of neoadjuvant letrozole or anastrozole prior to surgery followed by adjuvant endocrine therapy and 10 years of follow up.. Results: Within our training cohort (n=73) using Kaplan-Meier analysis our 4-gene test predicted neoadjuvant clinical response and demonstrated a significant association with both RFS (P=0.029) and OS (P=0.009). This approach predicts outcomes within 2-weeks rather than 4-months of treatment required in other studies such as P024. Using IHC in the training cohort (n=42), two gene markers in combination (IL6ST at diagnosis and MCM4 after 2-weeks treatment) predicted both RFS (P=0.017) and OS (P=0.009) with great accuracy. The 140 patient group is being analysed and the findings are so far are consistent with the initial training cohort and indicate a significant association with outcomes. Conclusion: • A 4 gene model with clinical potential has been developed and validated to predict response to neoadjuvant aromatase inhibitors. • This 4 gene model predicts for response and also predicts relapse free and overall survival. • Proteins encoded by 2 of these 4 genes measured by IHC in an initial test set of 42 patients predict accurately both EFS and OS • A validation cohort (n=140) with over 10-years of follow-up will be available at SABCS 2015 to determine if this 2 biomarker test can predict outcome on adjuvant endocrine therapy. Citation Format: Turnbull AK, Arthur LM, Webber V, Thomas J, Uddin S, Webb H, Dunbier A, Dowsett M, Renshaw L, Sims AH, Dixon JM. A validated test for neoadjuvant clinical response to endocrine therapy in breast cancer that estimates accurately recurrence-free and overall survival. [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 P3-07-20.

Background: Breast Cancer Index (BCI) is a gene expression-based assay that reports two distinct results: 1) BCI predictive based on HoxB13/IL17BR ratio (H/I), and 2) BCI prognostic based on an algorithm incorporating H/I with the Molecular Grade index (MGI). Both biomarkers have been validated independently in randomized trial cohorts. However, integrated results to better correlate recurrence risk with endocrine response have not been evaluated. The aim of this post-hoc analysis was to examine patient outcomes within BCI prognostic and predictive groups using the translational arm of the Arimidex, Tamoxifen, Alone or in Combination trial (TransATAC). Methods: Primary tumor samples (N=742) from hormonal receptor-positive, N0 breast cancer patients treated with 5 years of tamoxifen (TAM) or anastrozole (ANA) in the ATAC trial were examined. Kaplan-Meier analysis was used to examine the risk of distant recurrence (DR) in patient subgroups derived from BCI and H/I results. A separate series of clinical cases submitted for BCI testing (N=853) were analyzed to determine distribution of the combined BCI and H/I groups in clinical practice. Results: Summary of patient distribution across the 6 BCI clinical subgroups showed that a large number of patients (331/742, 45%) were BCI low risk with low likelihood of benefit, whereas 108/742 (15%) of patients with endocrine responsive disease (High H/I) were classified as BCI low risk (Table 1). Kaplan-Meier analysis demonstrated that patients classified as BCI low risk had a very similar 10-year risk of DR irrespective of H/I status (H/I low: 5.5% vs. H/I high: 4.0%), indicating that prognosis was largely determined by BCI vs H/I. Table 1: Distribution of BCI and H/I risk groups in TransATAC BCI: PrognosticH/I: PredictiveLow RiskIntermediate RiskHigh RiskTotalLow Likelihood3318717435 (59%)High Likelihood10895104307 (41%)Total439 (59%)182 (25%)121 (16%)742 In 853 node negative cases submitted for BCI clinical testing, the distribution of BCI and H/I risk groups were similar to that from the TransATAC cohort (Table 2). Table 2: Distribution of BCI and H/I risk groups in clinical cases submitted for BCI testing BCI: PrognosticH/I: PredictiveLow RiskIntermediate RiskHigh RiskTotalLow Likelihood36410523492 (58%)High Likelihood96107158361 (42%)Total460 (54%)212 (25%)181 (21%)853 Discussion: Both prognostic and predictive components reported from the BCI assay may be used to stratify patients into 6 clinical subgroups based on prognostic risk of distant recurrence and endocrine responsiveness. Findings from this analysis indicate that patients classified as BCI low risk, regardless of H/I status, had sufficiently low DR rates and identifies patients that may be adequately treated with 5 years of endocrine therapy. Citation Format: Zhang Y, Sestak I, Schroeder BE, Dowsett M, Cuzick J, Schnabel CA, Sgroi DC. Prognostic impact of the combined risk groups by breast cancer index and HOXB13/IL17BR ratio in hormonal receptor positive, node negative breast cancer: A TransATAC study. [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 P5-08-03.

Background: BCI is a genomic signature (Molecular Grade Index (MGI) and HOXB13/IL17BR (H/I)) that significantly predicts risk of distant recurrence (DR) in hormonal receptor-positive, lymph node negative (LN-) breast cancer. As previously shown in the TransATAC and MA.14 studies, BCI was also prognostic for DR in lymph node-positive (LN+) patients. Here, a distinct BCI model that integrates tumor size and grade was evaluated for prediction of DR in women with 1-3 lymph node positive disease. Methods: 219 primary tumor samples from hormonal receptor-positive patients with 1-3 positive lymph nodes treated with 5 years of tamoxifen or anastrozole were examined. Women with four or more positive lymph nodes were excluded. BCI was combined with tumour size and grade into a comprehensive risk score, BCIN+. Kaplan-Meier (KM) estimates of 10 year DR and hazard ratios (HR) and 95% confidence intervals (CI) were estimated. Change in likelihood ratio 2 (LR-2) values were used to measure prognostic information of each variable alone or combined in new score. New cutpoints for low versus high risk groups for the new model were determined to ensure the low risk patients had minimal 10-year residual disease. Results: In 219 LN+ patients, BCI alone provided substantial additional prognostic information to tumor size (LR-2=11.83, P=0.0006) and grade (LR-2=8.33, P=0.004). Both clinical variables provided additional significant prognostic information to BCI alone (BCI alone: LR-2=9.59, P=0.0004; T: LR-2=7.09, P=0.008; G: LR-2=27.59, P Discussion: Integration of tumor size and grade significantly enhanced the prognostic ability of BCI to predict 10 year DR risk in hormonal receptor-positive patients with 1-3 positive nodes. A significant number of patients have been identified to have a very low 10-year risk for DR, who may choose to safely forego unnecessary adjuvant chemotherapy or extended adjuvant endocrine therapy. Validation of BCIN+ in other datasets is ongoing. Citation Format: Sestak I, Zhang Y, Schroeder B, Dowsett M, Sgroi D, Cuzick J, Schnabel CA. Integration of tumor size and grade with the breast cancer index (BCI) for prediction of distant recurrence in hormone receptor-positive breast cancer with 1-3 positive lymph nodes. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-08-12.

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.

Background: Poor prognosis in TNBC can be predicted in the significant fraction of patients with large volume residual cancer burden (RCB-II/III) after neoadjuvant chemotherapy (NACT). Whilst residual disease has been characterised to identify “driver” mutations and copy number variations, the contribution of the immune response within its tumour microenvironment remains unclear. Here we aimed to: 1) assess the potential spatial heterogeneity of immune transcript related gene expression between areas of tumour approximately 1cm apart as might still occur with a radiologically guided biopsy through the residual disease; and 2) assess the immune stroma composition of the TNBC high metastatic risk RCB II/III disease. Method: 12 TNBC post NACT RCB II/III residual cases were identified from the KHP biobank. H&E sections were reviewed and areas of tumor 1cm apart within a residual resection specimen marked as area A and area B. HistoQuest analysis software was used to quantify the proportion of tumor infiltrating lymphocytes (per total cell count) within both areas. RNA was extracted from both areas and immune gene expression profiling performed using a Nanostring nCounter® on all 24 samples. The immune PanCancer panel consisted of 770 genes combining markers for different immune cell populations. Differential genes between paired samples were compared and unsupervised hierarchical clustering using 770 genes and immune cell types performed. Results:Quantitative comparison of the tumour infiltrating lymphocytes (TILS) between area A and B revealed that 73% (8/11) of the cases had a 2-fold (range 2.03-3.16) difference in the TILS. When comparing the 770 gene expression profiles between sampling areas in the same tumour, we found little spatial heterogeneity with areas A/B clustering together in 10 out of 12 cases. Interestingly, the two cases that revealed spatial heterogeneity within the paired samples displayed little immune cell heterogeneity histologically (i.e. Conclusion: The findings that high metastatic risk residual disease can be further characterized as either “immunologically inert” or “immunologically enriched” at the level of extensive immunological transcript gene expression and by histological assessment of TILS requires further investigation; and is being validated in a larger sample set. Citation Format: Irshad S, Cheang M, Gazinka P, Naidoo K, Buus R, Pinder S, Dowsett M, Tutt A. Immune profiling of post neoadjuvant high metastatic risk (RCB-II/III) residual disease in patients with early triple negative breast cancers [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 P2-04-07.

Background: The nuclear proliferation biomarker Ki67 has multiple potential roles in breast cancer, including prognosis-based decisions, but unacceptable between-laboratory variability has limited its clinical value. The International Ki67 Working Group (IKWG) has undertaken a systematic program to determine whether Ki67 immunohistochemistry can be analytically validated and standardized across laboratories. Technological advances and broader availability of devices for automated assessment of stained slides raise the possibility that these machines may improve on reproducibility of traditional pathologist-based visual Ki67 assessment. Aims: To characterize reproducibility of automated machine-measured Ki67 expression using slides previously analyzed in the IKWG phase 3 study that evaluated reproducibility of visual Ki67 assessment. Methods: Two sets of 30 previously stained slides containing core-cut biopsy sections of breast tumors were circulated to 14 laboratories for scanning and automated assessment of Ki67 expression. Sites were instructed to return average and maximum percentage of tumor cells positive for Ki67 for each slide, where maximum is designed to reflect “hot spot” analysis. Two laboratories returned scores from 2 operators; not all laboratories reported values for maximum Ki67 scores. Different operators were treated as distinct laboratories in analyses. Sixteen and 10 score sets were available for average and maximum Ki67 analyses, respectively, encompassing 7 unique scanner and 10 software platforms. Pre-specified analyses included evaluation of reproducibility across all laboratories as well as within a subgroup limited to those using Aperio scanners. The primary reproducibility metric was intraclass correlation coefficient between laboratories (ICC), regardless of device platform or software. Results: Geometric means across 30 cases for 16 operators ranged from 11.06% to 38.11% with overall mean 16.75% (95% CI:14.45-19.42) for average scores. Geometric means for 10 operators ranged from 16.44% to 68.73% with overall mean 25.16% (95% CI: 18.71-33.84) for maximum scores. ICC for automated average scores across 16 operators was 0.83 (95% CI: 0.73-0.91) and ICC for maximum scores across 10 operators was 0.63 (95% CI: 0.44-0.80) although one outlier lab dramatically affected results. For the laboratories using the Aperio platform (8 score sets), ICC for automated average scores was 0.89 (95% CI; 0.81-0.96). These results are similar to ICC of 0.87 (95%CI; 0.81-0.93) reported using these same slides in the Phase 3 visual assessment reproducibility study in which observers counted 500 cells per slide (Leung et al, NPJBrCancer, in press). Conclusions: Between-laboratory reproducibility for automated machine assessment of average Ki67 is similar to that for pathologist-based visual assessment of Ki67. However, the observed ICC was markedly numerically lower for the maximum score method compared to the average method, suggesting that the maximum score may not be useful as a reproducible measure of proliferation. Automated average scoring methods show promise for standardization of Ki67 scoring, supporting future studies to clinically validate Ki67. Citation Format: Rimm DL, McShane LM, Leung SCY, Bai Y, Bane AL, Bartlett JMS, Bayani J, Chang MC, Dean M, Denkert C, Enwere E, Galderisi C, Gholap A, Hugh JC, Jadhav A, Kornaga E, Laurinavicius A, Levenson R, Lima J, Miller K, Pantanowitz L, Piper T, Ruan J, Srinivasan M, Virk S, Wu Y, Yang H, Hayes DF, Nielsen TO, Dowsett M. An international multicenter study to evaluate reproducibility of automated scoring methods for assessment of Ki67 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 P1-03-01.

1020 Background: Accurate prediction of distant metastasis (DM) in early stage ER+/HER2- breast cancer (BC) patients is vital to avoid over/under-treatment with adjuvant chemotherapy (CT). The OncotypeDX Recurrence Score (RS) is a widely used tool to assist clinical decision-making for CT. The StemPrintER Risk Score (SPRS) is an alternative genomic predictor based on the biology of cancer stem cells that predicts recurrence risk in ER+/HER2- BCs (Pece S. et al., EBioMedicine 2019). Here, we analyze the prognostic value of SPRS in the TransATAC cohort of post-menopausal ER+/HER2- BC patients, and compare the prognostic information provided by SPRS and RS for 10-year risk of DM. Methods: The likelihood χ2 (LRχ2) and Kaplan-Meier survival analyses were used to assess prognostic information provided by SPRS, RS and the clinical treatment score (CTS) in 818 TransATAC patients treated with anastrozole or tamoxifen for 5 years. Comparative analyses were made for DM risk over the 10-year follow-up, as well as in the early (0-5 years) or late (5-10 years) interval, according to nodal status. Results: Used as a continuous variable, SPRS was an independent predictor of DM in years 0-10 among all patients when adjusted for clinical parameters as expressed by the CTS [HR=1.43 (1.18-1.73), PHIGH vs. LOW=2.96 (1.85-4.73); P2=33.4; RS: HR=1.52, P2=22.1), with even greater differences in late DM prediction in N0 patients. SPRS also provided more prognostic information than RS to CTS (ΔLRχ2: SPRS+CTS vs. CTS= 14.9; RS+CTS vs. CTS= 9.7). Conclusions: In ER+/HER2- TransATAC BC patients, SPRS was highly prognostic for DM and was superior to RS in providing additional prognostic information to conventional clinicopathological parameters.

Background: Two large clinical trials have shown the benefit of aromatase inhibitors in healthy women to reduce the risk of developing breast cancer (MAP.3 and IBIS-II). Here, we report blinded 10-year median follow-up efficacy data for the IBIS-II trial, which compared anastrozole to placebo in women at increased risk of developing breast cancer. Material and Methods: 3864 postmenopausal women at increased risk of developing breast cancer were recruited into a double-blind trial of anastrozole (N=1920) versus matching placebo (N=1944) for 5 years. The primary objective of this study was to determine the efficacy of anastrozole in preventing breast cancer (both invasive and ductal carcinoma in situ (DCIS)), overall and particularly for the post 5-year time period. Secondary endpoints included prevention of oestrogen receptor positive breast cancer, breast cancer mortality, non-breast cancer deaths, other cancers, cardiovascular disease, fractures, and musculoskeletal events. Results: After a median follow-up of 10.9 years (IQR 8.8-13.0), a total of 241 breast cancers have been reported (HR=0.50 (0.38-0.65), P Conclusion: This updated analysis of the IBIS-II trial confirms the significant reduction in breast cancer occurrence with anastrozole in the post-treatment follow-up period. These results indicate a long-term preventive benefit with anastrozole for ER-positive breast cancer in postmenopausal women. Table: Number of events and Hazard Ratios (95% CI) according to follow-up period.Number of eventsHR (95% CI)P-valueOverall241 (81 vs. 160)0.50 (0.38-0.65) Citation Format: Jack Cuzick, Ivana Sestak, John Forbes, Mitchell Dowsett, Simon Cawthorn, Robert Mansell, Sibylle Loibl, Bernardo Bonanni, Anthony Howell, IBIS-II investigators. Ten year results of the international breast cancer intervention study II [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr GS4-04.

Background: The Clinical Treatment Score at 5 years (CTS5) is a prognostic tool using clinicopathological parameters (nodal status, tumor size, grade, and age) to estimate distant recurrence (DR) risk after 5 years of endocrine treatment for postmenopausal women with estrogen receptor (ER) positive breast cancer. It was developed and validated in the ATAC and BIG 1-98 trials. Here, we evaluate whether the CTS5 is prognostic in women who were distant recurrence (DR) free 5 years after adjuvant treatment who participated in the TAILORx study. Methods: 10273 women with ER-positive, HER2-negative, and node-negative disease were entered into the TAILORx study and assigned or randomized to four treatment arms according to their Oncotype Recurrence Score (RS) (endocrine therapy alone (ET) or chemo-endocrine therapy (CET)). Primary endpoint of this analysis was DR and the primary objective of this analysis was to evaluate the CTS5 in the TAILORx study, stratified by chemotherapy use. Secondary objectives included validation of the CTS5 in two specific age groups (age≤50 years vs. age>50 years) and in the four separate treatment arms. Cox regression models were used to determine the prognostic value of CTS5. Kaplan-Meier curves were used to estimate conditional 9-year DR risks (%). Hazard ratios are reported for an increase of 1-standard deviation of CTS5 in the overall analysis population. Results: 7353 (71.6%) women were DR free 5 years after diagnosis with follow-up beyond 5 years, had information for all clinical parameters, and were included in this analysis. Median follow-up after 5 years was 2.9 years (1.8-3.9). Overall, mean age was 55.7 years (SD 9.0), mean tumor size 17.1mm (SD 7.8), and the vast majority had low or moderately differentiated disease (84.7%). CTS5 was significantly higher for women in arm D (RS 26-100, CET) compared to the other three arms (mean CTS5 3.09 vs. 2.82, P Conclusions: Low risks of late DR of 3.1% (2.4-4.0) for RS scores 0-25 (ET) and 3.8% (2.9-4.8) for RS scores 11-100 (CET) were observed in this ER-positive, HER2-negative and node negative TAILORx cohort. We confirm the prognostic ability of the CTS5 for late DR in the TAILORx cohort, specifically for patients older than 50 years and/or those who were deemed intermediate or high risk by Oncotype Dx RS (11-100). Our results show that the CTS5 is less prognostic for late DR in women aged 50 years or younger who received 5 years of endocrine therapy only. Further evaluation of the CTS5 in premenopausal cohorts is needed before it can be applied to younger patients. Table: Hazard Ratios (95% CI) for prognostic value of CTS5 overall and according to sub-groups.HR (95% CI )P-valueAll (N=7353)1.57 (1.36-1.82) 50 years (N=5094)1.78 (1.48-2.14) Citation Format: Ivana Sestak, Michael Crager, Jack Cuzick, Mitchell Dowsett, Steven Shak, Gong Tang, Robert Gray, Joseph Sparano. Validation of the clinical treatment score post 5 years (CTS5) in women with hormone receptor positive, HER2-negative, node-negative disease from the TAILORx study [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr GS4-03.

Purpose To update key recommendations of the American Society of Clinical Oncology/College of American Pathologists human epidermal growth factor receptor 2 (HER2) testing in breast cancer guideline. Methods Based on the signals approach, an Expert Panel reviewed published literature and research survey results on the observed frequency of less common in situ hybridization (ISH) patterns to update the recommendations. Recommendations Two recommendations addressed via correspondence in 2015 are included. First, immunohistochemistry (IHC) 2+ is defined as invasive breast cancer with weak to moderate complete membrane staining observed in > 10% of tumor cells. Second, if the initial HER2 test result in a core needle biopsy specimen of a primary breast cancer is negative, a new HER2 test may (not “must”) be ordered on the excision specimen based on specific clinical criteria. The HER2 testing algorithm for breast cancer is updated to address the recommended work-up for less common clinical scenarios (approximately 5% of cases) observed when using a dual-probe ISH assay. These scenarios are described as ISH group 2 ( HER2/chromosome enumeration probe 17 [CEP17] ratio ≥ 2.0; average HER2 copy number < 4.0 signals per cell), ISH group 3 ( HER2/CEP17 ratio < 2.0; average HER2 copy number ≥ 6.0 signals per cell), and ISH group 4 ( HER2/CEP17 ratio < 2.0; average HER2 copy number ≥ 4.0 and < 6.0 signals per cell). The diagnostic approach includes more rigorous interpretation criteria for ISH and requires concomitant IHC review for dual-probe ISH groups 2 to 4 to arrive at the most accurate HER2 status designation (positive or negative) based on combined interpretation of the ISH and IHC assays. The Expert Panel recommends that laboratories using single-probe ISH assays include concomitant IHC review as part of the interpretation of all single-probe ISH assay results. Find additional information at www.asco.org/breast-cancer-guidelines .