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Molecular tests predicting the risk of distant recurrence (DR) can be used to assist therapy decision-making in oestrogen receptor-positive (ER+) and human epidermal growth factor receptor 2-negative (HER2-) breast cancer patients after considerations of standard clinical markers. The Oncotype DX Recurrence Score (RS) is a widespread tool used for this purpose. Here, we compared the RS with the StemPrintER Risk Score (SPRS), a novel genomic predictor with a unique biological basis in its ability to measure the expression of cancer stemness genes.We benchmarked the SPRS vs. RS, alone or in combination with clinicopathological variables expressed by the Clinical Treatment Score (CTS), for the prognostication of DR in a retrospective cohort of 776 postmenopausal patients with ER+/HER2-breast cancer enrolled in the translational arm of the randomised Arimidex, Tamoxifen, Alone or in Combination (ATAC) trial. Likelihood ratio (LR) with χIn all patients, the SPRS provided significantly more prognostic information than the RS for ten-year DR prognostication (C-index = 0.688, LR-χIn postmenopausal ER+/HER2- breast cancer patients, SPRS provided more prognostic information than RS for DR when used alone or in combination with the CTS. The SPRS could therefore potentially identify high-risk patients, who might benefit from aggressive treatments, from low-risk patients who might safely avoid adjuvant chemotherapy or prolongation of endocrine therapy.

Kaplan-Meier plots for 10 year distant recurrence for CTS and CTS+OMclin2 composite continuous score split by the score median in node-negative and node-positive patients, respectively. *Due to the distribution of CTS score, dividing the cohort by the score median resulted in slightly uneven groups. The numbers of patients at risk in each group at various time points are given below each graph. CTS, clinical treatment score; med, median; CI, confidence interval

PDF file - 134K, Supplementary Figure 3. Organotypic of MDA-MB-231 + beta6-1089 staining.

Supplementary Figure S1: Consort diagram to show the derivation of this cohort from the whole ATAC trial population.

Added prognostic information to NPI. Likelihood (χ²) for distant recurrence for all prognostic scores in all patients and subgroups. DR, distant recurrence; OMm, OncoMasTR Molecular Score; OMclin2, OncoMasTR Risk Score; NPI, Nottingham Prognostic Index.

Supplementary Table S1: Hazard Ratios (95% CI) and chi-square values for all recurrence and all individual genes and gene modules according to time periods in all patients, HER2-ve patients, and HER2-ve/Node-ve patients. Supplementary Table S2: Hazard Ratios (95% CI) and chi-square values for distant recurrence and all individual genes and gene modules according to time periods in all patients, HER2-ve patients, and HER2-ve/Node-ve patients. Supplementary Table S3: Comparison of GHI and IHC genes according to time period in all patients, HER2-ve, and HER2-ve/Node-ve patients.

PDF file - 106K, Supplementary Figure 1. Transient transfection of alphavbeta6 in primary myoepithelial cells.

Purpose: To identify the individual genes or gene modules that lead to the OncoptypeDx 21-gene recurrence score's reduced performance after 5 years and thereby identify indices of residual risk that may guide selection of patients for extended adjuvant therapy.Experimental Design: We conducted a retrospective assessment of the relationship between (i) the individual genes and gene modules of the Recurrence Score and (ii) early (0–5 years) and late (5–10 years) recurrence rates in 1,125 postmenopausal patients with primary estrogen receptor–positive breast cancer treated with anastrozole or tamoxifen in the Arimidex, Tamoxifen, Alone or Combined (ATAC) randomized clinical trial.Results: In the HER2-negative population (n = 1,009), estimates of recurrence risk were similar between years 0–5 and 5–10 for proliferation and invasion modules but markedly different for the estrogen module and genes within it (all split at the median): for low estrogen module, annual recurrence rates were similar across the two time windows (2.06% vs. 2.46%, respectively); for high estrogen module, annual rates were 1.14% versus 2.72%, respectively (Pinteraction = 0.004). Estrogen receptor transcript levels showed inverse prediction across the time windows: HR, 0.88 (0.73–1.07) and 1.19 (0.99–1.43), respectively (Pinteraction = 0.03). Similar time-, module-, and estrogen-dependent relationships were seen for distant recurrence.Conclusions: Patients with tumors with high estrogen receptor transcript levels benefit most from 5 years' endocrine therapy but show increased recurrence rates after 5 years and may benefit from extended therapy. Improved prognostic profiles may be created by considering period of treatment and follow-up time. Clin Cancer Res; 21(12); 2763–70. ©2015 AACR.

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PDF file - 108K, Supplementary Figure 2. alphavbeta6 functional assays in the beta6-1089 cell line using RNAi.

Purpose:To test the validity of OncoMasTR Molecular Score (OMm), OMclin1, and OncoMasTR Risk Score (OMclin2) prognostic scores for prediction of distant recurrence (DR) in estrogen receptor (ER)-positive/HER2-negative breast cancer treated with 5 years' endocrine therapy only and compare their performance with the Oncotype DX Recurrence Score (RS).Experimental Design:OMm incorporates three master transcription regulator genes. OMclin1 combines OMm, tumor size, grade, and nodal status; OMclin2 incorporates OMm, tumor size, and nodal status. OMclin1 and OMclin2 were evaluated for 646 postmenopausal patients with ER-positive/HER2-negative primary breast cancer with 0–3 involved lymph nodes in TransATAC. Patients were randomized to 5 years' anastrozole or tamoxifen without chemotherapy. RS was available in all cases. We used likelihood ratio-χ2, C-index, and Kaplan–Meier analyses to assess prognostic information.Results:OMm, OMclin1, and OMclin2 were highly prognostic for prediction of DR in years 0–10 among all patients [likelihood ratio (LR)-χ2 = 25.4, 48.7, and 45.0, respectively, all P < 0.001; C-index = 0.67, 0.71, and 0.71, respectively], compared with RS (LR-χ2 = 18.8; P < 0.001; C-index = 0.63). All three scores provided significant additional prognostic value beyond clinical treatment score, Nottingham Prognostic Index, and Ki67. OMclin1 and OMclin2 categorized 190 and 267 node-negative patients as low risk (DR rates: 2.9% and 4.9%, respectively). In comparison, RS categorized 296 node-negative patients as low-risk and 128 patients as intermediate-risk (DR rate: 6.6% and 17.3%, respectively).Conclusions:OMm, OMclin1, and OMclin2 were highly prognostic for early and late DR in women with early-stage ER-positive breast cancer receiving 5 years' endocrine therapy. In TransATAC, OMclin1 and the OncoMasTR Risk Score (OMclin2) were superior to RS in identifying patients at increased risk of DR.

Purpose: This study investigated the functional and clinical significance of integrin αvβ6 upregulation in myoepithelial cells of ductal carcinoma in situ (DCIS).Experimental Design: Archival samples of DCIS and DCIS with associated invasion (n = 532) were analyzed for expression of αvβ6 by immunohistochemistry and ability to predict recurrence and progression assessed in an independent, unique cohort of DCIS cases with long-term follow-up. Primary myoepithelial cells and myoepithelial cell lines, with and without αvβ6 expression, were used to measure the effect of αvβ6 on growth and invasion of tumor cell lines in vitro and in a xenograft mouse model. Involvement of TGFβ signaling was established using mink lung epithelial cell (MLEC) assay and antibody inhibition, and expression and activation of matrix metalloproteinase (MMP)-9 established by Real Time-PCR and zymography.Results: Expression of αvβ6 is significantly associated with progression to invasive cancer (P < 0.006) and with recurrence over a median follow-up of 114 months in a series of matched DCIS cases treated with local excision. We show that expression of αvβ6 drives myoepithelial cells to promote tumor cell invasion in vitro and enhances mammary tumor growth in vivo. The tumor-promoting effect of αvβ6-positive myoepithelial cells is dependent on TGFβ-driven upregulation of MMP9 and can be abrogated by inhibiting this pathway.Conclusion: These findings indicate that altered myoepithelial cells in DCIS predict disease progression and recurrence and show that upregulation of αvβ6 on myoepithelial cells generates a tumor promoter function through TGFβ upregulation of MMP-9. These data suggest that expression of αvβ6 may be used to stratify patients with DCIS. Clin Cancer Res; 20(2); 344–57. ©2013 AACR.

PDF file - 55K, Supplementary Table 1. Details relating to; treatment received, patient age, hormone replacement therapy, mean follow up and pathological characteristics for cases and controls.

Background We investigated the association between body mass index (BMI) and breast cancer risk in women at increased risk of breast cancer receiving tamoxifen or anastrozole compared with placebo using data from the International Breast Cancer Intervention Studies [IBIS-I (tamoxifen) and IBIS-II (anastrozole)]. Methods Baseline BMI was calculated from nurse assessed height and weight measurements for premenopausal (n = 3138) and postmenopausal (n = 3731) women in IBIS-I and postmenopausal women in IBIS-II (n = 3787). The primary endpoint was any breast cancer event (invasive and ductal carcinoma in situ). We used Cox proportional hazards regression to calculate hazard ratios (HRs) for risk after adjustment for covariates. Results There were 582 (IBIS-I) and 248 (IBIS-II) breast cancer events [median follow-up = 16.2 years (IQR 14.4–17.7) and 10.9 years (IQR 8.8–13.0), respectively]. In adjusted analysis, women with a higher BMI had an increased breast cancer risk in both IBIS-I [HR = 1.06 per 5 kg/m2 (0.99–1.15), p = 0.114] and in IBIS-II [HR per 5 kg/m2 = 1.21 (1.09–1.35), p 2 = 1.14 (1.03–1.26), p = 0.01] but not premenopausal women [adjusted HR per 5 kg/m2 = 0.97 (0.86–1.09), p = 0.628]. There was no interaction between BMI and treatment group for breast cancer risk in either IBIS-I (p = 0.62) or IBIS-II (p = 0.55). Conclusions Higher BMI is associated with greater breast cancer risk in postmenopausal women at increased risk of the disease, but no effect was observed in premenopausal women. The lack of interaction between BMI and treatment group on breast cancer risk suggests women are likely to experience benefit from preventive therapy regardless of their BMI. Trial registration Both trials were registered [IBIS-I: ISRCTN91879928 on 24/02/2006, retrospectively registered (http://www.isrctn.com/ISRCTN91879928); IBIS-II: ISRCTN31488319 on 07/01/2005, retrospectively registered (http://www.isrctn.com/ISRCTN31488319)]

PURPOSE The Onco type DX Recurrence Score (RS), Prosigna Prediction Analysis of Microarray 50 (PAM50) Risk of Recurrence (ROR), EndoPredict (EP), and Breast Cancer Index (BCI) are used clinically for estimating risk of distant recurrence for patients receiving endocrine therapy. Discordances in estimates occur between them. We aimed to identify the molecular features that drive the tests and lead to these differences. PATIENTS AND METHODS Analyses for RS, ROR, EP, and BCI were conducted by the manufacturers in the TransATAC sample collection that consisted of the tamoxifen or anastrozole arms of the ATAC trial. Estrogen receptor–positive/human epidermal growth factor receptor 2 (HER2)–negative cases without chemotherapy treatment were included in which all four tests were available (n = 785). Clinicopathologic features included in some tests were excluded from the comparisons. Estrogen, proliferation, invasion, and HER2 module scores from RS were used to characterize the respective molecular features. Spearman correlation and analysis of variance tests were applied. RESULTS There were moderate to strong correlations among the four molecular scores (ρ = 0.63-0.74) except for RS versus ROR (ρ = 0.32) and RS versus BCI (ρ = 0.35). RS had strong negative correlation with its estrogen module (ρ = −0.79) and moderate positive correlation with its proliferation module (ρ = 0.36). RS’s proliferation module explained 72.5% of ROR’s variance, while the estrogen module explained only 0.6%. Most of EP’s and BCI’s variation was accounted for by the proliferation module (50.0% and 54.3%, respectively) and much less by the estrogen module (20.2% and 2.7%, respectively). CONCLUSION In contrast to common understanding, RSs are determined more strongly by estrogen-related features and only weakly by proliferation markers. However, the EP, BCI, and particularly ROR scores are determined largely by proliferative features. These relationships help to explain the differences in the prognostic performance of the tests.

Background Anastrozole has been associated with substantial accelerated bone mineral density (BMD) loss during active treatment. Methods One thousand four hundred and ten women were included in a BMD substudy and stratified into three strata according to their baseline T-score at spine or femoral neck. The primary objective of this analysis was to investigate whether DXA BMD at the spine and hip changed two years after treatment cessation (between years 5 and 7) in those who did not receive risedronate. Results Five- and seven-year BMD data were available for a total of 528 women who did not receive risedronate. In women with normal BMD at baseline, an increase in BMD at the lumbar spine after anastrozole withdrawal was observed 1.25% (95% CI 0.73 to 1.77) (P = 0.0004), which was larger than in those on placebo (0.14% (−0.29 to 0.56))). At the hip, BMD remained unchanged between years 5 and 7 for those previously on anastrozole but continued to a decrease in those who had been randomised to placebo (−1.35% (−1.70 to −0.98)). Conclusions These are the first results reporting BMD changes after stopping anastrozole in a breast cancer prevention setting. Our results show that the negative effects of anastrozole on BMD in the preventive setting are partially reversible.

Background Studies in the adjuvant setting have shown that endocrine therapy related side effects predict breast cancer recurrence risk. Here, we assess the relationship between early reported side effects and incidence of breast cancer in women randomised to tamoxifen for cancer prevention in the International Breast Intervention Study (IBIS)–I trial. Methods Women randomised to tamoxifen in the IBIS-I trial and for whom side effect status was known at the 6-month follow-up visit were included in this analysis. Side effects included in this analysis were hot flushes, vaginal discharge, and vaginal dryness. The primary endpoint was all breast cancer and secondary endpoint was oestrogen receptor (ER) positive breast cancer. Cox proportional hazard models were used to investigate breast cancer incidence in the tamoxifen group with and without side effects reported within 6 months of randomisation. Results Women randomised to tamoxifen and reporting hot flushes at the 6-month follow-up visit had a non-statistically significant increase in breast cancer compared to those without hot flushes (HR = 1.26 (0.98–1.62), P = 0.08). A significant higher breast cancer risk was observed for postmenopausal women who reported hot flushes at the 6-month follow-up visit compared to those without hot flushes (HR = 1.59 (1.12–2.26), P = 0.01). A higher risk was observed for ER-positive breast cancer in postmenopausal women (HR = 1.81 (1.19–2.74), P = 0.01). No significant associations between gynaecological side effects and breast cancer occurrence was observed. Conclusions Overall, no association between side effects reported at 6 months and subsequent breast cancer occurrence was observed. Some side effects might be useful markers for breast cancer occurrence in postmenopausal women.

Background: Oncotype DX Recurrence Score (RS), Prosigna PAM50 ROR score, the EndoPredict score (EP, EPclin) and the Breast Cancer Index (BCI) all provide substantial molecular prognostic information and are used in clinical practice for guiding the treatment for large numbers of patients with primary ER+/HER2- breast cancer. Despite this, there is incomplete information on the concordance between these scores and very little understanding of the molecular features that drive the individual scores. Here we report from a unique database (i) the correlation between all 4 scores when measured by the respective manufacturers in the TransATAC set of samples and (ii) the degree to which each of the scores is driven by proliferation, oestrogen signalling, HER2 signalling and invasive properties insomuch as these are reflected by each component module of the RS test (PM, EM, HM and IM, respectively). Methods: The TransATAC tumour sample set was collected from patients in the tamoxifen or anastrozole arms from the ATAC clinical trial of 5 years of endocrine therapy in postmenopausal patients with primary hormone receptor positive breast cancer. Cases were included if HER2- and all 4 scores were available. None of the patients received adjuvant chemotherapy. EP and ROR both include some clinical features in their final score but components were excluded for the purposes of this study. The RS module scores were provided by the manufacturer. The genes that contribute to the module scores are as follows: PM: Ki67, STK15, Survivin, CCNB1, MYBL2; EM: ER, PGR, BCL2, SCUBE2; HM: GRB7, HER2; IM: MMP11, CTSL2). Spearman rank correlation was used to study the association between molecular components. Results: Data was available for all four molecular scores for 785 TransATAC patients. Correlations were moderate to strong for RS vs EP (r=0.63), ROR vs EP (r=0.68), ROR vs BCI (r=0.74) and EP vs BCI (r=0.67) but were weak for RS vs ROR (r=0.32) and RS vs BCI (r=0.35). The correlation between each score with the RS module scores are shown in the Table. Molecular scoreRS moduleRSROREPBCIPM0.360.860.710.74EM-0.790.00-0.38-0.10HM-0.04-0.14-0.15-0.18IM0.260.380.340.32 The RS had a strong negative correlation with its EM (r= -0.79) and a weak positive correlation with its PM (r= 0.36) although the latter was stronger when the PM was thresholded; 78% of the cases had a PM score below the threshold of 6.5 and were assigned a value of 6.5 in the RS with no correlation between RS and PM in these samples (r=-0.03). ROR showed a very strong correlation with the PM (r=0.86), but none with the EM (r: 0.00). EP and BCI both showed strong correlation with the PM (r= 0.71 and 0.74, respectively) and with low or near absent correlation with EM (r= -0.38 and -0.10, respectively). There was little correlation between the HM with each of the scores while the IM correlation was similar for each score varying from 0.26 to 0.38. Of the two module components of BCI, MGI was strongly associated with the PM (r= 0.84) and not at all with the EM while H/I moderately correlated with PM (r=0.51) and weakly correlated with EM (r= -0.31). Conclusion: EP scores are moderately correlated with each of the other 3 scores while RS scores are dissimilar to those from the ROR and BCI. In contrast to common thinking the RS is driven only weakly by proliferative features and more by those related to ER/PR pathways. The EP, BCI and particularly ROR are dominated by proliferative features. These relationships provide and explanation for the differences that we and others have reported in the prognostic performance of the tests for both early and late recurrence. Citation Format: Richard Buus, Ivana Sestak, Mitch Dowsett, Ralf Kronenwett, Sean Ferree, Catherine Schnabel, Frederick L Baehner, Jack Cuzick. Correlation between and molecular drivers of oncotype DX, prosigna, EndoPredict and breast cancer index: A TransATAC 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 P3-07-05.

The agricultural sector is a source of greenhouse gas emissions that directly affect the global problem of climate change and contribute approximately 11% in total greenhouse gas emissions in the world and in Croatia too. Irregular and irresponsible agricultural practices, such as excessive tillage and improper fertilization often lead to soil carbon loss and increased carbon dioxide emissions to the atmosphere. This field study provides results how agricultural practices affect carbon dioxide emissions from soil, carbon sequestration and soil quality during the cultivation of winter wheat. The field experiment was conducted in a temperate continental climate on distric Stagnosol. Four investigated treatments were: organic fertilization, mineral fertilization, control treatment and black fallow. The lowest carbon dioxide emission was recorded on bare soil and the highest on organic fertilization treatment. The application of manure, mineral fertilizers and calcification rendered significant effect on some soil chemical characteristics and daily carbon dioxide flux.

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.

Anastrozole has been shown to prevent breast cancer in postmenopausal women at high risk of the disease, but has been associated with substantial accelerated loss of bone mineral density (BMD) and increased fractures. Here, we investigate the effect of risedronate on BMD after 5 years of follow-up in the IBIS-II prevention trial. 1410 women were enrolled in the bone sub-study and stratified into three strata according to the lowest baseline T-score at spine or femoral neck. The objective was to compare the effect of oral risedronate (35 mg weekly) versus placebo in osteopenic women in stratum II who were randomised to anastrozole in the main study. 258 osteopenic, postmenopausal women at high risk of developing breast cancer for whom baseline and follow-up bone mineral density measurements were available. 5-year mean BMD change at the lumbar spine for osteopenic women randomised to anastrozole and risedronate was −0.4% compared to −4.2% for those not on risedronate (P, Highlights • Results confirm anastrozole induced bone mineral density decrease in women at increased risk of developing breast cancer • Risedronate counterbalances the negative effect of anastrozole induced bone mineral density loss at the lumbar spine • Long-term intake of risedronate did not prevent bone loss at the total hip in osteopenic women • N-Terminal Propeptide of Type I Collagen (PINP) can predict bone mineral density changes

Multi-gene prognostic signatures including the Oncotype® DX Recurrence Score (RS), EndoPredict® (EP) and Prosigna® (Risk Of Recurrence, ROR) are widely used to predict the likelihood of distant recurrence in patients with oestrogen-receptor-positive (ER+), HER2-negative breast cancer. Here, we describe the development and validation of methods to recapitulate RS, EP and ROR scores from NanoString expression data. RNA was available from 107 tumours from postmenopausal women with early-stage, ER+, HER2− breast cancer from the translational Arimidex, Tamoxifen, Alone or in Combination study (TransATAC) where previously these signatures had been assessed with commercial methodology. Gene expression was measured using NanoString nCounter. For RS and EP, conversion factors to adjust for cross-platform variation were estimated using linear regression. For ROR, the steps to perform subgroup-specific normalisation of the gene expression data and calibration factors to calculate the 46-gene ROR score were assessed and verified. Training with bootstrapping (n = 59) was followed by validation (n = 48) using adjusted, research use only (RUO) NanoString-based algorithms. In the validation set, there was excellent concordance between the RUO scores and their commercial counterparts (rc(RS) = 0.96, 95% CI 0.93–0.97 with level of agreement (LoA) of −7.69 to 8.12; rc(EP) = 0.97, 95% CI 0.96–0.98 with LoA of −0.64 to 1.26 and rc(ROR) = 0.97 (95% CI 0.94–0.98) with LoA of −8.65 to 10.54). There was also a strong agreement in risk stratification: (RS: κ = 0.86, p κ = 0.87, p κ = 0.92, p

Spectral data obtained from optical spaceborne sensors are being recognized as a valuable source of data that show promising results in assessing soil properties on medium and macro scale. Combining this technique with laboratory Visible-Near Infrared (VIS-NIR) spectroscopy methods can be an effective approach to perform robust research on plot scale to determine wildfire impact on soil organic matter (SOM) immediately after the fire. Therefore, the objective of this study was to assess the ability of Sentinel-2 superspectral data in estimating post-fire SOM content and comparison with the results acquired with laboratory VIS-NIR spectroscopy.The study is performed in Mediterranean Croatia (44° 05’ N; 15° 22’ E; 72 m a.s.l.), on approximately 15 ha of fire affected mixed Quercus ssp. and Juniperus ssp. forest on Cambisols. A total of 80 soil samples (0-5 cm depth) were collected and geolocated on August 22nd 2019, two days after a medium to high severity wildfire. The samples were taken to the laboratory where soil organic carbon (SOC) content was determined via dry combustion method with a CHNS analyzer. SOM was subsequently calculated by using a conversion factor of 1.724. Laboratory soil spectral measurements were carried out using a portable spectroradiometer (350-1050 nm) on all collected soil samples. Two Sentinel-2 images were downloaded from ESAs Scientific Open Access Hub according to the closest dates of field sampling, namely August 31st and September 5th 2019, each containing eight VIS-NIR and two SWIR (Short-Wave Infrared) bands which were extracted from bare soil pixels using SNAP software. Partial least squares regression (PLSR) model based on the pre-processed spectral data was used for SOM estimation on both datasets. Spectral reflectance data were used as predictors and SOM content was used as a response variable. The accuracy of the models was determined via Root Mean Squared Error of Prediction (RMSEp) and Ratio of Performance to Deviation (RPD) after full cross-validation of the calibration datasets.The average post-fire SOM content was 9.63%, ranging from 5.46% minimum to 23.89% maximum. Models obtained from both datasets showed low RMSEp (Spectroscopy dataset RMSEp = 1.91; Sentinel-2 dataset RMSEp = 0.99). RPD values indicated very good predictions for both datasets (Spectrospcopy dataset RPD = 2.72; Sentinel-2 dataset RPD = 2.22). Laboratory spectroscopy method with higher spectral resolution provided more accurate results. Nonetheless, spaceborne method also showed promising results in the analysis and monitoring of SOM in post-burn period.Keywords: remote sensing, soil spectroscopy, wildfires, soil organic matterAcknowledgment: This work was supported by the Croatian Science Foundation through the project "Soil erosion and degradation in Croatia" (UIP-2017-05-7834) (SEDCRO). Aleksandra Perčin is acknowledged for her cooperation during the laboratory work.

The OncoAnalyses for RS, ROR, EP, and BCI were conducted by the manufacturers in the TransATAC sample collection that consisted of the tamoxifen or anastrozole arms of the ATAC trial. Estrogen receptor-positive/human epidermal growth factor receptor 2 (HER2)-negative cases without chemotherapy treatment were included in which all four tests were available (n = 785). Clinicopathologic features included in some tests were excluded from the comparisons. Estrogen, proliferation, invasion, and HER2 module scores from RS were used to characterize the respective molecular features. Spearman correlation and analysis of variance tests were applied.There were moderate to strong correlations among the four molecular scores (ρ = 0.63-0.74) except for RS versus ROR (ρ = 0.32) and RS versus BCI (ρ = 0.35). RS had strong negative correlation with its estrogen module (ρ = -0.79) and moderate positive correlation with its proliferation module (ρ = 0.36). RS's proliferation module explained 72.5% of ROR's variance, while the estrogen module explained only 0.6%. Most of EP's and BCI's variation was accounted for by the proliferation module (50.0% and 54.3%, respectively) and much less by the estrogen module (20.2% and 2.7%, respectively).In contrast to common understanding, RSs are determined more strongly by estrogen-related features and only weakly by proliferation markers. However, the EP, BCI, and particularly ROR scores are determined largely by proliferative features. These relationships help to explain the differences in the prognostic performance of the tests.

PurposeClinical Treatment Score at 5 years (CTS5) is a prognostic tool to estimate distant recurrence (DR) risk after 5 years of endocrine therapy for postmenopausal women with oestrogen receptor-positive (ER-positive) breast cancer.MethodsThe validity of CTS5 was tested in a retrospective cohort of patients diagnosed with early ER-positive breast cancer. The primary endpoint was DR in years 5–10. The primary analysis cohort consisted of postmenopausal women, with premenopausal women as a secondary analysis cohort. Cox regression models were used to determine the prognostic value of CTS5 and Kaplan–Meier curves were used with associated 10-year DR risks (%).Results2428 women were included with a median follow-up of 13.4 years. The CTS5 was significantly prognostic in both postmenopausal (N = 1662, HR = 2.18 95% CI (1.78–2.67)) and premenopausal women (N = 766, HR = 1.84 95% CI (1.32–2.56)). The 10-year DR risks were 2.9% (1.9–4.5), 7.2% (5.3–9.9), and 12.9% (10.0–16.7) for low, intermediate and high risk in postmenopausal women and 3.8% (2.2–6.7), 6.9% (4.4–10.8), and 11.1% (7.4–16.5) in premenopausal women, respectively. The number of observed DRs was significantly greater than expected in those predicted to be at high risk by CTS5 but this discordance was lost when those receiving more than 60 months of endocrine therapy were excluded.ConclusionsThe CTS5 demonstrated clinical validity for predicting late DR within a large cohort of unselected postmenopausal patients but less so in premenopausal patients. Calibration of the CTS5 was good in patients who did not receive extended endocrine therapy. The CTS5 low-risk cohort has risk of DR so low as to not warrant extended endocrine therapy.

Purpose: Invasive lobular carcinoma (ILC) accounts for approximately 5%–15% of all invasive breast cancer cases. Most of the correlations between multigene assays and patient outcome were derived from studies based on patients with invasive ductal carcinoma (IDC) or without distinction between the subtypes. Here, we investigate the prognostic value of EndoPredict (EPclin) in a large cohort of ILCs pooled from three phase III randomized trials (ABCSG-6, ABCSG-8, TransATAC). Experimental Design: The primary objective of this analysis was to determine the prognostic value of EPclin for distant recurrence (DR) in years 0–10 in postmenopausal women with ILC. The primary outcome was DR. Results: 470 women (17.9%) presented with ILC, 1,944 (73.9%) with IDC, and 216 (8.2%) with other histologic types. EPclin was highly prognostic in women with ILC [HR = 3.32 (2.54–4.34)] and provided more prognostic value than the Clinical Treatment Score [CTS; HR = 2.17 (1.73–2.72)]. 63.4% of women were categorized into the low EPclin risk group and they had a 10-year DR of 4.8% (2.7–8.4) compared with 36.6% of women in the high-risk group with a 10-year DR risk of 26.6% (20.0–35.0). EPclin also provided highly prognostic information in women with node-negative disease [HR = 2.56 (1.63–4.02)] and node-positive disease [HR = 3.70 (2.49–5.50)]. Conclusions: EPclin provided highly significant prognostic value and significant risk stratification for women with ILC. Ten-year DR risk in the EPclin low-risk groups were similar between ILC and IDC. Our results show that EPclin is informative in women with ILC and suggest that it is equally valid in both histologic subtypes.

Fire is an important element of the ecosystems, nevertheless, high severity fires can have negative impacts on the ecosystems as consequence of the high temperatures reaches. High temperatures normally have detrimental impacts on soil properties. The objective of this work was to determine the relationship of spectral reflectance and soil pH, electrical conductivity (EC), carbonates (CaCO3) and total carbon (TC) content after a medium to high severity wildfire occurred in Croatia using linear and nonlinear calibration models.Soils were sampled 2 days after a medium to high severity wildfire in Zadar County, Croatia. A total of 120 soil samples (0-5 cm) were collected from three different treatments (n= 40 per treatment): control (C), mean severity (MS), high severity (HS). Soil pH, EC, CaCO3 and TC content were determined using standard laboratory methods. Soil spectral measurements were carried out using a portable spectroradiometer (20 per treatment, 60 in total). Linear statistical model - partial least squares regression (PLSR) and non-linear - artificial neural network (ANN) were generated to estimate changes in soil pH, EC, CaCO3 and TC content based on the original spectral reflectance and its first derivative in form of principal components (PC). One-way ANOVA revealed pH values were significantly different in all three treatments. EC, CaCO3 and TC were significantly higher in HS plots compared with the other treatments.Different wildfire severity indicated very collinear soil spectral response, but with certain variations of reflectance intensity. Control samples showed a higher reflectance than MS and HS samples. This is attributed to the low pH and TC content. Low reflectance of MS and HS samples could be explained by their increased pH and TC values. Soil pH was the only parameter that showed a high R2 and low root mean squared error (RMSE) after Savitzky Golay smoothing and the first derivation. In PLSR model, strong to very strong correlation and low RMSE were obtained. ANN model also showed a high R2 and lower RMSE for all properties except pH. Both models showed satisfactory results for prediction of the studied soil properties. ANN model predicted EC, CaCO3, and TC better, while PLSR proved to be a better model for pH prediction.Key words: soil reflectance, fire severity, principal components, partial least squares regression, neural networksAcknowledgementsThis work was supported by Croatian Science Foundation through the project "Soil erosion and degradation in Croatia" (UIP-2017-05-7834) (SEDCRO).

Background: Aromatase inhibitors (AIs) reduce the risk of breast cancer in women at increased risk and reduce recurrence in those with ductal carcinoma in situ (DCIS) (IBIS-2, MAP.3, NSABP B-33). The effectiveness of AIs depends on full adherence. We have previously reported adherence figures for the International Breast Cancer Intervention Studies 2 (IBIS-2) when 5 years of active treatment was not completed. Here, we assess reports of early symptoms on 5-year adherence with anastrozole in the prevention (versus placebo) and DCIS (versus tamoxifen) IBIS-2 after active treatment has been completed by all women. Methods: In IBIS-2, 3864 postmenopausal women in the prevention study were randomised to placebo vs. anastrozole (1mg/day) and 2980 postmenopausal women with DCIS were randomised to tamoxifen (20mg/day) vs. anastrozole (1mg/day). Women were excluded from the analyses (n=491 [262 prevention; 229 DCIS]) due to breast cancer, death, major adverse events, or failure to initiate preventive therapy. Adherence ( Results: In the IBIS-2 prevention study (N=3615), overall adherence to treatment was 67.7% and was statistically not significantly different between anastrozole (66.5%) and placebo (69.0%) (OR=0.89 (0.78-1.03), P=0.11). Adherence was significantly lower regardless of treatment allocation for those who developed arthralgia (68.3% vs. 72.8%, P=0.008) or gynaecological symptoms (vaginal changes, irregular bleeding) (65.1% vs. 72.2%, P=0.007), but not for those who reported hot flushes (71.1% vs. 71.8%, P=0.92), compared with those who did not report these symptoms at 6 months. In the IBIS-2 DCIS study (N=2759), adherence to treatment was 70.1% overall (anastrozole (70.2%) or tamoxifen (70.0%) (OR=1.01 (0.86-1.19), P=0.92)). Women treated with anastrozole reported significantly more arthralgia (30.6% vs. 20.5%, P Conclusions: In the IBIS-2 trials, we observed no significant differences in adherence between either anastrozole vs. placebo (prevention), or anastrozole vs. tamoxifen (DCIS). Significant associations between early symptoms and adherence were observed only in the prevention study, regardless of treatment allocation. Reporting symptoms in the first 6 months of preventive and adjuvant therapy is unlikely to explain non-adherence to medication. Further research is required to identify modifiable factors which may be altered by behavioural interventions to improve adherence. Citation Format: Sestak I, Smith S, Sleeth M, Howell A, Cuzick J. Participant-reported symptoms as predictors of long-term adherence of endocrine therapy in the International breast cancer intervention studies 2 (IBIS-2) [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 P5-15-01.

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.

The goal of this study is to shed more light on the influence of fertilisation on radionuclide transfer from soil to crops, which is a significant ecological issue in present-day agriculture due to the intensive use of soil amendments. Research on this subject has not been extensive, hence there are still numerous open questions that require an interdisciplinary approach involving agroecology and radioecology. Maize was chosen for our investigation because it has been used, in considerable quantities and worldwide, as both food and feed, which makes it part of numerous food chains with humans as final consumers. The agricultural part of the experiment was carried out on an acidic soil in northwestern Croatia, and it was based on seven treatments with different levels of NPK fertilisation. Radionuclide activity concentrations in sampled soil, stem, and grain were measured by means of high-resolution γ-ray spectrometry. We found that the radiological properties of the soil were not affected by the addition of the fertiliser. Soil-to-stem uptake for radionuclides of the whole 232Th decay chain was first suppressed by mild fertilisation, and then, at higher fertiliser concentrations, it stayed low and became independent of fertilisation level. The same effect was observed for the 238U decay chain before gaseous 222Rn. We present arguments in favour of the cause of the observed suppression being radionuclide complexation with constituents of the fertiliser. However, the concentration of 210Pb in stem did not depend on fertilisation, which was most probably a consequence of the decay of airborne 222Rn and the deposition of its progenies onto stem. Radionuclide translocation from stem to grain did not depend on fertilisation either, and it was appreciable only for 40K and 137Cs. In grain, radionuclide concentrations were lower than in stem. Overall, in our case, fertilisation had positive effects, reducing the presence of radionuclides in the maize while having no detectable impact on the radiological properties of the soil. This finding may have implications for future work, especially regarding concerns about the impact of soil amendments on food production.

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: 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.

Purpose: Extended adjuvant hormone therapy improves estrogen receptor (ER)-positive breast cancer outcome. The total duration of endocrine therapy is still a subject for debate. The CTS5 score, developed by Dowsett et al1., predicts late distant recurrence (LDR) between years 5 and 10 after diagnosis in ER-positive invasive breast cancers in postmenopausal women. This LDR risk might be used to select patients for extended endocrine therapy. The CTS5 score is calculated using age at the start of endocrine therapy, tumor size, grade, and lymph node status. The aim of this study is the validation of this tool in a series of women from the UZ Leuven. Methods: This retrospective cohort study included 1125 postmenopausal women consecutively diagnosed with invasive estrogen receptor (ER) positive breast cancer who stopped endocrine therapy after 4.5 to 5.5 years. Tissue was tested for ER positivity and considered positive if >1% of tumor nuclei were stained. HER2 status was defined according to ASCO guidelines. Statistical analysis was performed by Cox proportional hazards models which determined the prognostic performance of the CTS5 score for LDR. Results: A total of 1125 patients were included in this analysis, of which 1097 had a known HER2 status and 1023 were negative. 62 of 1125 (5.5%) developed an LDR between years 5 and 10. The continuous CTS5 was a significant predictor for LDR (HR =2.69 (1.99-3.60), p Conclusion: In our series of postmenopausal women, CTS5 accurately predicts late distant recurrence in ER-positive, HER2-negative early invasive breast cancers. The CTS5-score, identifying a patient group with LDR risk of ≥5%, might be used to discuss the benefits of extended endocrine therapy for individual patients. However, the exact predictive value for the benefit of prolonged therapy can only be based on a randomized controlled trial (RCT) using the LDR risk as a stratification factor. It would be of great value to expand the study population, especially HER2-positive tumors, and define the prognostic performance of the CTS5 score in premenopausal women. Further research is needed. 1. Dowsett, M. et al. Integration of Clinical Variables for the Prediction of Late Distant Recurrence in Patients With Estrogen Receptor-Positive Breast Cancer Treated With 5 Years of Endocrine Therapy: CTS5. J. Clin. Oncol. 36, 1941-1948 (2018). Citation Format: Josephine Van Cauwenberge, Ivana Sestak, Kevin Punie, Hans Wildiers, Giuseppe Floris, Ignace Vergote, Patrick Berteloot, Toon Van Gorp, Ann Smeets, Els Van Nieuwenhuysen, Sileny Han, Ines Nevelsteen, Caroline Weltens, Hilde Janssen, Patrick Neven. Predicting distant recurrence of ER+ HER2- breast cancer after 5 years of endocrine therapy: The CTS5-tool validation in real life [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 P5-06-08.

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: Two large clinical trials have shown a reduced rate of breast cancer development in high-risk women in the initial 5 years of follow-up after use of aromatase inhibitors (MAP.3 and International Breast Cancer Intervention Study II [IBIS-II]). Here, we report blinded long-term follow-up results for the IBIS-II trial, which compared anastrozole with placebo, with the objective of determining the efficacy of anastrozole for preventing breast cancer (both invasive and ductal carcinoma in situ) in the post-treatment period. Methods: IBIS-II is an international, randomised, double-blind, placebo-controlled trial. Postmenopausal women at increased risk of developing breast cancer were recruited and were randomly assigned (1:1) to either anastrozole (1 mg per day, oral) or matching placebo daily for 5 years. After treatment completion, women were followed on a yearly basis to collect data on breast cancer incidence, death, other cancers, and major adverse events (cardiovascular events and fractures). The primary outcome was all breast cancer. Findings: 3864 women were recruited between Feb 2, 2003, and Jan 31, 2012. 1920 women were randomly assigned to 5 years anastrozole and 1944 to placebo. After a median follow-up of 131 months (IQR 105–156), a 49% reduction in breast cancer was observed for anastrozole (85 vs 165 cases, hazard ratio [HR] 0·51, 95% CI 0·39–0·66, p

Purpose: To test the validity of OncoMasTR Molecular Score (OMm), OMclin1, and OncoMasTR Risk Score (OMclin2) prognostic scores for prediction of distant recurrence (DR) in estrogen receptor (ER)-positive/HER2-negative breast cancer treated with 5 years' endocrine therapy only and compare their performance with the Oncotype DX Recurrence Score (RS). Experimental Design: OMm incorporates three master transcription regulator genes. OMclin1 combines OMm, tumor size, grade, and nodal status; OMclin2 incorporates OMm, tumor size, and nodal status. OMclin1 and OMclin2 were evaluated for 646 postmenopausal patients with ER-positive/HER2-negative primary breast cancer with 0–3 involved lymph nodes in TransATAC. Patients were randomized to 5 years' anastrozole or tamoxifen without chemotherapy. RS was available in all cases. We used likelihood ratio-χ2, C-index, and Kaplan–Meier analyses to assess prognostic information. Results: OMm, OMclin1, and OMclin2 were highly prognostic for prediction of DR in years 0–10 among all patients [likelihood ratio (LR)-χ2 = 25.4, 48.7, and 45.0, respectively, all P < 0.001; C-index = 0.67, 0.71, and 0.71, respectively], compared with RS (LR-χ2 = 18.8; P < 0.001; C-index = 0.63). All three scores provided significant additional prognostic value beyond clinical treatment score, Nottingham Prognostic Index, and Ki67. OMclin1 and OMclin2 categorized 190 and 267 node-negative patients as low risk (DR rates: 2.9% and 4.9%, respectively). In comparison, RS categorized 296 node-negative patients as low-risk and 128 patients as intermediate-risk (DR rate: 6.6% and 17.3%, respectively). Conclusions: OMm, OMclin1, and OMclin2 were highly prognostic for early and late DR in women with early-stage ER-positive breast cancer receiving 5 years' endocrine therapy. In TransATAC, OMclin1 and the OncoMasTR Risk Score (OMclin2) were superior to RS in identifying patients at increased risk of DR.

Purpose EndoPredict (EPclin) is a prognostic test validated to inform decisions on adjuvant chemotherapy to endocrine therapy alone for patients with oestrogen receptor-positive, HER2-negative breast cancer. Here, we determine the performance of EPclin for estimating 10-year distant recurrence-free interval (DRFI) rates for those who received adjuvant endocrine therapy (ET) alone compared to those with chemotherapy plus endocrine therapy (ET + C). Methods A total of 3746 women were included in this joint analysis. 2630 patients received 5 years of ET alone (ABCSG-6/8, TransATAC) and 1116 patients received ET + C (GEICAM 2003-02/9906). The primary objective was to evaluate the ability of EPclin to provide an estimate of the 10-year DR rate as a continuous function of EPclin separately for ET alone and ET + C. Cox proportional hazard models were used for these analyses. Results EPclin was highly prognostic for DR in women who received ET alone (HR 2.79 (2.49–3.13), P