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To assess the long-term (20-year) endocrine therapy benefit in premenopausal patients with breast cancer.Secondary analysis of the Stockholm trial (STO-5, 1990-1997) randomly assigning 924 premenopausal patients to 2 years of goserelin (3.6 mg subcutaneously once every 28 days), tamoxifen (40 mg orally once daily), combined goserelin and tamoxifen, or no adjuvant endocrine therapy (control) is performed. Random assignment was stratified by lymph node status; lymph node-positive patients (n = 459) were allocated to standard chemotherapy (cyclophosphamide, methotrexate, and fluorouracil). Primary tumor immunohistochemistry (n = 731) and gene expression profiling (n = 586) were conducted in 2020. The 70-gene signature identified genomic low-risk and high-risk patients. Kaplan-Meier analysis, multivariable Cox proportional hazard regression, and multivariable time-varying flexible parametric modeling assessed the long-term distant recurrence-free interval (DRFI). Swedish high-quality registries allowed a complete follow-up of 20 years.In estrogen receptor-positive patients (n = 584, median age 47 years), goserelin, tamoxifen, and the combination significantly improved long-term distant recurrence-free interval compared with control (multivariable hazard ratio [HR], 0.49; 95% CI, 0.32 to 0.75, HR, 0.57; 95% CI, 0.38 to 0.87, and HR, 0.63; 95% CI, 0.42 to 0.94, respectively). Significant goserelin-tamoxifen interaction was observed (This study shows 20-year benefit from 2 years of adjuvant endocrine therapy in estrogen receptor-positive premenopausal patients and suggests differential treatment benefit on the basis of tumor genomic characteristics. Combined goserelin and tamoxifen therapy showed no benefit over single treatment. Long-term follow-up to assess treatment benefit is critical.

Background: Women with estrogen receptor (ER)-positive disease have a long-term risk of distant recurrence. The poor survival of premenopausal women diagnosed with breast cancer combined with the otherwise long life expectancy makes it especially important to identify tumor characteristics associated with long-term survival. Intra-tumor heterogeneity, having cancer cells of varying characteristics across the tumor, may promote therapeutic resistance and metastatic capacity. We have previously shown that high intra-tumor heterogeneity of ER increases the risk of fatal breast cancer. To our knowledge, the relation between intra-tumor heterogeneity of progesterone receptor (PR) expression and long-term survival is not known. We aimed to study the association between intra-tumor heterogeneity of PR and long-term survival of premenopausal women in the Stockholm tamoxifen trial (STO-5), with 20-years complete follow-up. Methods: This study was a secondary analysis of 504 ER-positive/PR-positive premenopausal women from the STO-5 trial (1990-1997). 451 women had human epidermal growth factor receptor 2 (HER2)-negative tumors. Patients were randomized to receive either 2 years of endocrine treatment (tamoxifen and/or goserelin) or no endocrine treatment. Lymph node-positive patients (n=251) also received standard chemotherapy (CMF). Immunohistochemical analysis, including estimating the proportion of tumor cells for each PR intensity level (0, 1+, 2+, or 3+), was completed in 2020. Intra-tumor heterogeneity of PR was calculated using Rao’s quadratic entropy and then categorized into low and high intra-tumor heterogeneity groups, using a predefined cutoff at the second tertile. Complete long-term (20 year) follow-up was obtained from high-quality Swedish registries. Long-term distant recurrence-free interval (DRFI) was assessed using univariate Kaplan-Meier analysis and multivariable Cox proportional hazard modeling, adjusting for patient and tumor characteristics. Results: A statistically significant difference in 20-year DRFI was seen between patients with high and low intra-tumor heterogeneity of PR in the univariate Kaplan-Meier analysis (log-rank P< 0.01). Survival proportions for DRFI at 20 years were 60.2% (95% CI, 53.2%-68.1%) and 73.3% (95% CI, 68.6%-78.3%) for patients with high and low PR intra-tumor heterogeneity, respectively. Analysis in patients with HER2-negative tumors yielded similar results. In the multivariable analysis, women with high intra-tumor heterogeneity of PR had a significantly increased long-term risk of distant recurrence, compared to women with low intra-tumor heterogeneity, hazard ratio (HR)=1.49 (95% CI, 1.08-2.06). The same pattern was seen in HER2-negative women, HR=1.50 (95% CI, 1.06-2.12), see Table. Conclusions: This study suggests an increased long-term risk of distant recurrences in ER-positive/PR-positive premenopausal women with high intra-tumor heterogeneity of PR as compared to women with low intra-tumor heterogeneity of PR, independent of HER2 status. Ongoing analyses include using deep learning for image analysis of breast cancer tumors to examine intra-tumor heterogeneity at higher resolution and for various tumor characteristics. Better understanding of tumor characteristics associated with long-term risk in premenopausal women is needed, given the poor prognosis and early onset of the disease. Long-term risk of distant recurrence by PR intra-tumor heterogeneity and HER2 status Multivariable Cox proportional hazard regression modeling of 20-year distant recurrence-free interval (DRFI) by high and low PR intra-tumor heterogeneity. ER-positive/PR-positive and ER-positive/PR-positive/HER2-negative premenopausal women were analyzed separately. The crude model was adjusted for age, randomization year, lymph node status, and endocrine treatment. The full model was adjusted for age, randomization year, lymph node status, endocrine treatment, tumor size, Ki-67 status, and HER2 status. Citation Format: Oscar Danielsson, Huma Dar, Gizeh Perez-Tenorio, Anna Nordenskjöld, Christina Yau, Christopher C. Benz, Laura J. Esserman, Bo Nordenskjöld, Olle Stål, Tommy Fornander, Annelie Johansson, Linda S. Lindström. Long-term survival and intra-tumor heterogeneity of progesterone receptor expression in estrogen receptor-positive/progesterone receptor-positive premenopausal women with breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-05-03.

Purpose: In vitro, p21-activated kinase 1 (Pak1) phosphorylates the serine 305 residue of the estrogen receptor α (ERα) and influences the response of breast cancer cells to tamoxifen. We investigated the influence of Pak1 and pERαser305 on breast cancer prognosis and results of tamoxifen therapy.Experimental Design: We examined Pak1 and pERαser305 protein by immunohistochemistry in a series of 912 tumors from node-negative breast cancer patients randomized to tamoxifen or no adjuvant endocrine treatment.Results: Cytoplasmic Pak1 correlated to large tumors and ER negativity, whereas nuclear Pak1 and pERαser305 correlated to small tumors and ER positivity. Nuclear expression of Pak1 and pERαser305 predicted reduced response to tamoxifen in patients with ERα-positive tumors (tamoxifen versus no tamoxifen: hazard ratio (HR), 1.33; 95% confidence interval (95% CI), 0.42-4.2; P = 0.63), whereas patients lacking this combination benefitted significantly from tamoxifen (HR, 0.43; 95% CI, 0.30-0.62; P < 0.0001). Similar nonsignificant trends were detected in analyses of the proteins separately. Pak1 in the cytoplasm was an independent prognostic marker, indicating increased recurrence rate (HR, 1.79; 95% CI, 1.17-2.74; P = 0.0068) and breast cancer mortality (HR, 1.98; 95% CI, 1.14-3.46; P = 0.016) for patients randomized to no adjuvant treatment.Conclusion: Our results suggest that patients with tumors expressing Pak1 and pERαser305 in combination are a group in which tamoxifen treatment is insufficient. In addition, the pathway may be of interest as a drug target in breast cancer. Furthermore, the findings support previous studies showing that Pak1 has differential roles in the cytoplasm and the nucleus. Clin Cancer Res; 16(5); 1624–33

Supplementary Data from Estrogen Receptor-α Phosphorylation at Serine 305, Nuclear p21-Activated Kinase 1 Expression, and Response to Tamoxifen in Postmenopausal Breast Cancer

ERRα protein expression and clinicopathological variables with respect to estrogen receptor (ER) and HER2 status.

Supplementary figure 1: Validation of the ERRα antibody used for immunohistochemical analysis; Supplementary Figure 2: The prognostic value of ERRα protein expression and subcellular location in adjuvant-untreated ER-negative patients; Supplementary Figure 3: ERRα protein expression is not prognostic in ER-positive breast cancer.

Purpose: Estrogen-related receptor alpha (ERRα) signaling has recently been implicated in breast cancer. We investigated the clinical value of ERRα in randomized cohorts of tamoxifen-treated and adjuvant-untreated patients.Experimental Design: Cox proportional hazards regression was used to evaluate the significance of associations between ERRα gene expression levels and patient DMFS in a previously published microarray dataset representing 2,000 breast tumor cases derived from multiple medical centers worldwide. The 912 tumors used for immunostaining were from a tamoxifen-randomized primary breast cancer trial conducted in Stockholm, Sweden, during 1976–1990. Mouse model was used to study the effect of tamoxifen treatment on lung colonization of MDA-MB-231 control cells and MDA-MB-231 cells with stable knockdown of ERRα. The phenotypic effects associated with ERRα modulation were studied using immunoblotting analyses and wound-healing assay.Results: We found that in ER-negative and triple-negative breast cancer (TNBC) adjuvant-untreated patients, ERRα expression indicated worse prognosis and correlated with poor outcome predictors. However, in tamoxifen-treated patients, an improved outcome was observed with high ERRα gene and protein expression. Reduced ERRα expression was oncogenic in the presence of tamoxifen, measured by in vitro proliferation and migration assays and in vivo metastasis studies.Conclusions: Taken together, these data show that ERRα expression predicts response to tamoxifen treatment, and ERRα could be a biomarker of tamoxifen sensitivity and a prognostic factor in TNBC. Clin Cancer Res; 22(6); 1421–31. ©2015 AACR.

Background: Tamoxifen is a standard endocrine therapy for both pre- and postmenopausal ER-positive breast cancer patients. Patients with ER-positive disease have a long-term risk of distant recurrence, thus, long-term follow-up studies are essential to understand true treatment benefit. Clinically used tumor characteristics are prognostic 5-10 years after primary diagnosis, however, whether these characteristics are predictive of long-term tamoxifen benefit is largely unexplored. Therefore, we aimed to determine the long-term tamoxifen therapy benefit by the clinically used tumor characteristics in pre- vs postmenopausal patients in the Stockholm tamoxifen (STO)-trials with 20-years complete follow-up. Methods: Secondary analysis of 1242 ER-positive/HER2-negative patients from the STO-trials, randomized to at least 2 years of 40 mg tamoxifen vs no endocrine therapy (control). Premenopausal lymph node-positive patients were allocated to chemotherapy as standard of care and postmenopausal high-risk patients were further randomized to chemotherapy vs radiotherapy. Tumor immunohistochemical analysis was recently conducted. Complete 20-year follow-up was obtained from Swedish high-quality registries. Long-term distant recurrence-free interval (DRFI) was assessed by multivariable Cox proportional hazard regression and time-varying analysis using flexible parametric modelling. Results: Premenopausal patients showed significantly improved long-term DRFI from tamoxifen vs control if they were lymph node-negative (Hazard Ratio [HR]=0.46; 95% CI, 0.24-0.87), PR-positive (HR=0.61; 95% CI, 0.41-0.91), or of genomic low risk (HR=0.47; 95% CI, 0.26-0.85), see Table. In postmenopausal patients, significantly improved long-term DRFI from tamoxifen vs control was seen for all good prognosis tumor characteristics, i.e. small tumor size (pT≤20mm: HR=0.55; 95% CI, 0.39-0.77), tumor grade 1-2 (HR=0.55; 95% CI, 0.41-0.73), lymph node-negative (HR=0.44; 95% CI, 0.30-0.64), PR-positive (HR=0.60; 95% CI, 0.44-0.80), Ki-67-low (< 15%: HR=0.51; 95% CI, 0.38-0.68), and genomic low risk (HR=0.53; 95% CI, 0.37-0.74), see Table. Also, postmenopausal patients with large tumor size (pT>20mm: HR=0.64; 95% CI, 0.44-0.94) and PR-negative tumors (HR=0.51; 95% CI, 0.32-0.81) showed significant long-term tamoxifen benefit. Time-varying analysis in premenopausal patients indicated that tamoxifen therapy benefit diminished over time. Significant tamoxifen benefit until year 5, 10, and 15 after primary diagnosis was observed for PR-positive, lymph node-negative, and genomic low-risk patients, respectively. Postmenopausal patients had a significant long-term tamoxifen benefit if they had tumors of small or large tumor size, tumor grade 1-2, lymph node-negative status, PR-positive status, low Ki-67 levels, or genomic low risk. Conclusions: This study suggests a differential long-term tamoxifen therapy benefit in pre- vs postmenopausal patients. Clinically defined low-risk postmenopausal patients have long-term tamoxifen benefit, whereas the benefit is absent or diminish over time for premenopausal patients. Improved long-term prognostic and endocrine therapy predictive markers in premenopausal breast cancer patients with poor prognosis and long life-expectancy is needed, which could involve molecular tools. Long-term tamoxifen benefit in premenopausal and postmenopausal breast cancer patients by the clinically used tumor characteristics. Table Multivariable Cox proportional hazard regression analysis of 20-year distant recurrence-free interval (DRFI) for patients with ER-positive/HER2-negative tumors, comparing patients randomized to tamoxifen vs patients randomized to no endocrine therapy (control). Adjusted for age, randomization year, tumor size, tumor grade, lymph node status, PR status, Ki-67 status, chemotherapy, radiotherapy, and type of surgery. Citation Format: Annelie Johansson, Huma Dar, Anna Nordenskjöld, Gizeh Perez-Tenorio, Christina Yau, Christopher C. Benz, Laura J. Esserman, Laura Van’t Veer, Bo Nordenskjöld, Olle Stål, Tommy Fornander, Linda S. Lindström. Differential Long-Term Benefit from Adjuvant Tamoxifen Therapy in Estrogen Receptor (ER)-Positive/Human Epidermal Growth Factor Receptor 2 (HER2)-Negative Premenopausal and Postmenopausal Breast Cancer Patients [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-03-11.

Background: The risk for patients with estrogen receptor (ER)-positive breast cancer remains stable decades after primary diagnosis, with a large proportion of distant metastatic events occurring late. Thus, long-term follow-up studies are essential to understand true treatment benefit. Tamoxifen (TAM) therapy is a fundamental endocrine treatment for ER-positive breast cancer. We have previously shown a long-term tamoxifen therapy benefit for patients with less aggressive tumor characteristics in ER-positive/HER2-negative patients. However, ER-positive breast cancer is highly heterogenous and can be separated into different molecular subpopulations that behave differently during extended follow-up. The long-term tamoxifen benefit by molecular subtype is largely unexplored. We therefore aimed to investigate the long-term benefit from tamoxifen by clinically used tumor characteristics in Luminal A vs Luminal B patients in the Stockholm tamoxifen (STO)-trials with complete 20-years follow-up. Methods: Secondary analysis of ER-positive/HER2-negative patients with Luminal A or B molecular subtype (n=952) from the STO-trials (1976-1997) randomized to TAM (40 mg) vs no endocrine therapy (control). Gene expression data was generated using custom designed Agilent arrays from FFPE breast cancer tumor tissue and was used to define PAM50 molecular subtypes. The clinically used markers were reannotated in 2014 and 2020. Complete 20-year follow-up was obtained from Swedish high-qualitative registries. The long-term distant recurrence-free interval (DRFI) was assessed by Kaplan-Meier, multivariable Cox proportional hazard regression and time-varying analysis, using flexible parametric modelling. Results: Multivariable analysis showed significantly improved long-term DRFI from TAM vs control for both Luminal A (HR=0.59; 95% CI, 0.44-0.81) and Luminal B (HR=0.67; 95% CI, 0.46-0.99) patients. Time-varying analysis showed that patients with Luminal A subtype significantly benefitted from TAM for 15 years (HR=0.60, 95% CI, 0.39-0.94 at year 15), whereas patients with Luminal B subtype had a significant TAM benefit for 5 years (HR=0.64, 95% CI, 0.42-0.98 at year 5), see Table. Furthermore, a significant long-term TAM benefit for patients with large tumor size (pT>20mm: HR=0.46; 95% CI, 0.25-0.84), tumor grade 1-2 (HR=0.55; 95% CI, 0.40-0.77), lymph node-negative (HR=0.51, 95% CI, 0.34-0.78), PR-positive (HR=0.57, 95% CI, 0.40-0.81) and Ki-67-low tumors (HR=0.55, 95% CI, 0.39-0.77) was seen for Luminal A patients. In Luminal B patients, significantly improved long-term DRFI from TAM vs control was seen for small tumor size (pT≤20mm: HR=0.44; 95% CI, 0.24-0.80), tumor grade 1-2 (HR=0.51; 95% CI, 0.29-0.90), lymph node-negative (HR=0.40, 95% CI, 0.19-0.83), PR-positive (HR=0.59, 95% CI, 0.38-0.93) and Ki-67-low tumors (HR=0.45, 95% CI, 0.25-0.84). Conclusions: This study suggests a 15 years tamoxifen benefit for patients with Luminal A subtype tumors, whereas the benefit for patients with Luminal B tumors is up to five years after diagnosis. Furthermore, our study suggests a long-term tamoxifen benefit for patients with less aggressive tumor characteristics regardless of tumor subtype, except for tumor size in Luminal A patients. ER-positive breast cancer is a heterogeneous disease and long-term follow-up studies in molecular subtypes are essential to understand differences in treatment benefit. Table. Time-varying analysis of long-term tamoxifen therapy benefit by PAM50 molecular subtype. Time-varying analysis of distant recurrence-free interval (DRFI) to assess how tamoxifen benefit varied over the 20 year of follow-up using flexible parametric survival modelling. Patients included in analyses with no missing values. Estimated hazard ratios (HR) at year 5, 10, 15 and 20 for patients with Luminal A or Luminal B molecular subtype tumors randomized to tamoxifen therapy, as compared with patients randomized to no endocrine therapy (control). Adjusted for age, period of primary breast cancer diagnosis, tumor size, tumor grade, lymph node status, PR status, Ki-67 status, chemotherapy, radiotherapy, type of surgery, and menopausal status. *indicates significant findings P Citation Format: Huma Dar, Annelie Johansson, Anna Nordenskjöld, Gizeh Perez-Tenorio, Christina Yau, Christopher C. Benz, Laura J. Esserman, Bo Nordenskjöld, Olle Stål, Tommy Fornander, Linda S. Lindström. Long-Term Benefit from Adjuvant Tamoxifen in Luminal A and Luminal B Breast Cancer Patients [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-03-04.

BACKGROUND The molecular breast cancer subgroups comprise diverse immune biology; for human epidermal growth factor receptor 2-positive (HER2+) breast cancer (BC) and triple-negative breast cancer (TNBC) abundance of tumour infiltrating lymphocytes (TILs) have been shown to indicate good prognosis and could predict pathological complete response after neoadjuvant chemotherapy. In contrast, the clinical relevance of TILs in estrogen receptor-positive/HER2-negative (ER+/HER2-) tumours is not settled. We primary aimed to analyse the prognostic effect of TILs on BC-free interval (BCFi) in premenopausal patients, stratified by molecular subgroups. The secondary aim was to investigate the predictive value of TILs on tamoxifen benefit. METHODS Archival tissue from primary tumours was collected from patients included in the SBII:2pre trial. In this study, 564 premenopausal women were randomised between two years of adjuvant tamoxifen or no systemic treatment regardless of hormone-receptor status. TILs were scored on whole tissue sections and the tumours were divided into ER+/HER2- (n = 255), HER2+ (n = 65) and TNBC (n = 95) molecular subgroups by immunohistochemistry and in situ hybridisation (ISH). The prognostic value of TILs was evaluated in patients allocated to no systemic therapy, whose tumours were successfully scored for TILs and had IHC/ISH data for generating molecular subgroups (n = 221). All patients with ER+ tumours and successfully annotated TILs were considered for prediction of tamoxifen benefit (n = 321). The median follow-up was 28 years and the prognostic and predictive analyses were performed by cumulative incidence curves and Cox regression analyses. RESULTS TILs were successfully scored in 477 tumours with available ER status and the proportion of low ( CONCLUSIONS We demonstrate a long-term favorable prognostic value of levels of TILs in a cohort of premenopausal BC patients. This effect seems to be extended to the ER+/HER2- subgroup. A beneficial effect of tamoxifen in ER+ patients was observed in patients with tumours of low TILs infiltration as compared to patients with tumours of high immune infiltration, but we could not confirm a treatment predictive effect. Table 1. Uni- and multivariable Cox regression analyses of BCFi and OS (control arm)Univariable*MultivariableEndpointBCFiOSBCFiOSHR (95% CI); P valueTILs, category***All molecular subgroups(n = 221)(n = 213)Low (Ref.)1.001.001.001.00Intermediate1.10 (0.78-1.54); 0.611.26 (0.88-1.80); 0.210.61 (0.40-0.93); 0.020.65 (0.41-1.02); 0.06High0.40 (0.22-0.71); 0.0020.52 (0.29-0.95); 0.030.22 (0.11-0.43); 20 mm vs. ≤20), NHG (1 vs. 2 vs. 3), ER (positive vs. negative), PR (positive vs. negative), HER2 (positive vs. negative), LVI (present vs. absent) and TILs (high vs. intermediate vs. low). Stratified by Region.*** TILs were categorised as low: Citation Format: Christine Lundgren, Pär-Ola Bendahl, Maria Ekholm, Mårten Fernö, Carina Forsare, Ute Krüger, Bo Nordenskjöld, Olle Stål, Lisa Rydén. Tumour infiltrating lymphocytes of prognostic value in different molecular breast cancer subgroups and as a suggestive predictive factor for adjuvant tamoxifen benefit in premenopausal patients after 30 years follow-up [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS5-09.

The metastatic potential of estrogen receptor (ER)-positive breast cancers is heterogeneous and distant recurrences occur months to decades after primary diagnosis. We have previously shown that patients with tumors classified as ultralow risk by the 70-gene signature have a minimal long-term risk of fatal breast cancer. Here, we evaluate the previously unexplored underlying clinical and molecular characteristics of ultralow risk tumors in 538 ER-positive patients from the Stockholm tamoxifen randomized trial (STO-3). Out of the 98 ultralow risk tumors, 89% were luminal A molecular subtype, whereas 26% of luminal A tumors were of ultralow risk. Compared to other ER-positive tumors, ultralow risk tumors were significantly (Fishers test, P < .05) more likely to be of smaller tumor size, lower grade, progesterone receptor (PR)-positive, human epidermal growth factor 2 (HER2)-negative and have low Ki-67 levels (proliferation-marker). Moreover, ultralow risk tumors showed significantly lower expression scores of multi-gene modules associated with the AKT/mTOR-pathway, proliferation (AURKA), HER2/ERBB2-signaling, IGF1-pathway, PTEN-loss and immune response (IMMUNE1 and IMMUNE2) and higher expression scores of the PIK3CA-mutation-associated module. Furthermore, 706 genes were significantly (FDR < 0.001) differentially expressed in ultralow risk tumors, including lower expression of genes involved in immune response, PI3K/Akt/mTOR-pathway, histones, cell cycle, DNA repair, apoptosis and higher expression of genes coding for epithelial-to-mesenchymal transition and homeobox proteins, among others. In conclusion, ultralow risk tumors, associated with minimal long-term risk of fatal disease, differ from other ER-positive tumors, including luminal A molecular subtype tumors. Identification of these characteristics is important to improve our prediction of nonfatal vs fatal breast cancer. Funding Agencies|Swedish Research Council (Vetenskapsradet)Swedish Research Council [2020-02466]; Swedish Research Council for Health, Working life and Welfare (FORTE) [2019-00477]; Gosta Milton Donation Fund (Stiftelsen Gosta Miltons donationsfond); Swedish Cancer SocietySwedish Cancer Society [180385, 190140]; Stockholm Cancer Society (Cancerforeningen i Stockholm); California Breast Cancer Research Program (CBCRP) [180B-0065]; National Institutes of Health (NIH)United States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [U01-CA196406]

PurposeThe protein tyrosine phosphatase PTPN2 dephosphorylates several tyrosine kinases in cancer-related signalling pathways and is thought to be a tumour suppressor. As PTPN2 is not frequently studied in breast cancer, we aimed to explore the role of PTPN2 and the effects of its loss in breast cancer.MethodsProtein expression and gene copy number of PTPN2 were analysed in a cohort of pre-menopausal breast cancer patients with immunohistochemistry and droplet digital PCR, respectively. PTPN2 was knocked down in three cell lines, representing different breast cancer subtypes, with siRNA transfection. Several proteins related to PTPN2 were analysed with Western blot.ResultsLow PTPN2 protein expression was found in 50.2% of the tumours (110/219), gene copy loss in 15.4% (33/214). Low protein expression was associated with a higher relapse rate in patients with Luminal A and HER2-positive tumours, but not triple-negative tumours. In vitro studies further suggested a subtype-specific role of PTPN2. Knockdown of PTPN2 had no effect on the triple-negative cell line, whilst knockdown in MCF7 inhibited phosphorylation of Met and promoted that of Akt. Knockdown in SKBR3 led to increased Met phosphorylation and decreased Erk phosphorylation as well as EGF-mediated STAT3 activation.ConclusionWe confirm previous studies showing that the PTPN2 protein is lost in half of the breast cancer cases and gene deletion occurs in 15-18% of the cases. Furthermore, the results suggest that the role of PTPN2 is subtype-related and should be further investigated to assess how this could affect breast cancer prognosis and treatment response. Funding Agencies|Swedish Cancer Society; ALF Grants, Region Ostergotland; LiU Cancer Foundation; Cancer Research Foundations of Radiumhemmet; Onkologiska Klinikernas i Linkoping Forskningsfond

This abstract was withdrawn by the authors. Citation Format: Ekholm M, Bendahl P-O, Fernö M, Nordenskjöld B, Stål O, Rydén L. Withdrawn [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 P4-14-10.

Clinically used breast cancer markers, such as tumor size, tumor grade, progesterone receptor (PR) status, and Ki-67 status, are known to be associated with short-term survival, but the association of these markers with long-term (25-year) survival is unclear.To assess the association of clinically used breast cancer markers with long-term survival and treatment benefit among postmenopausal women with lymph node-negative, estrogen receptor [ER]-positive and ERBB2-negative breast cancer who received tamoxifen therapy.This study was a secondary analysis of data from a subset of 565 women with ER-positive/ERBB2-negative breast cancer who participated in the Stockholm tamoxifen (STO-3) randomized clinical trial. The STO-3 clinical trial was conducted from 1976 to 1990 and comprised 1780 postmenopausal women with lymph node-negative breast cancer who were randomized to receive adjuvant tamoxifen therapy or no endocrine therapy. Complete 25-year follow-up data through December 31, 2016, were obtained from Swedish national registers. Immunohistochemical markers were reannotated in 2014. Data were analyzed from April to December 2020.Patients in the original STO-3 clinical trial were randomized to receive 2 years of tamoxifen therapy vs no endocrine therapy. In 1983, patients who received tamoxifen therapy without cancer recurrence during the 2-year treatment and who consented to continued participation in the STO-3 study were further randomized to receive 3 additional years of tamoxifen therapy or no endocrine therapy.Distant recurrence-free interval (DRFI) by clinically used breast cancer markers was assessed using Kaplan-Meier and multivariable Cox proportional hazards analyses adjusted for age, period of primary diagnosis, tumor size (T1a and T1b [T1a/b], T1c, and T2), tumor grade (1-3), PR status (positive vs negative), Ki-67 status (low vs medium to high), and STO-3 clinical trial arm (tamoxifen treatment vs no adjuvant treatment). A recursive partitioning analysis was performed to evaluate which markers were able to best estimate long-term DRFI.The study population comprised 565 postmenopausal women (mean [SD] age, 62.0 [5.3] years) with lymph node-negative, ER-positive/ERBB2-negative breast cancer. A statistically significant difference in long-term DRFI was observed by tumor size (88% for T1a/b vs 76% for T1c vs 63% for T2 tumors; log-rank P .001) and tumor grade (81% for grade 1 vs 77% for grade 2 vs 65% for grade 3 tumors; log-rank P = .02) but not by PR status or Ki-67 status. Patients with smaller tumors (hazard ratio [HR], 0.31 [95% CI, 0.17-0.55] for T1a/b tumors and 0.58 [95% CI, 0.38-0.88] for T1c tumors) and grade 1 tumors (HR, 0.48; 95% CI, 0.24-0.95) experienced a significant reduction in the long-term risk of distant recurrence compared with patients with larger (T2) tumors and grade 3 tumors, respectively. A significant tamoxifen treatment benefit was observed among patients with larger tumors (HR, 0.53 [95% CI, 0.32-0.89] for T1c tumors and 0.34 [95% CI, 0.16-0.73] for T2 tumors), lower tumor grades (HR, 0.24 [95% CI, 0.07-0.82] for grade 1 tumors and 0.50 [95% CI, 0.31-0.80] for grade 2 tumors), and PR-positive status (HR, 0.38; 95% CI, 0.24-0.62). The recursive partitioning analysis revealed that tumor size was the most important characteristic associated with long-term survival, followed by clinical trial arm among patients with larger tumors.This secondary analysis of data from the STO-3 clinical trial indicated that, among the selected subgroup of patients, tumor size followed by tumor grade were the markers most significantly associated with long-term survival. Furthermore, a significant long-term tamoxifen treatment benefit was observed among patients with larger tumors, lower tumor grades, and PR-positive tumors.

Resistance to endocrine therapy in estrogen receptor-positive (ER+) breast cancer is a major clinical problem with poorly understood mechanisms. There is an unmet need for prognostic and predictive biomarkers to allow appropriate therapeutic targeting. We evaluated the mechanism by which minichromosome maintenance protein 3 (MCM3) influences endocrine resistance and its predictive/prognostic potential in ER+ breast cancer. We discovered that ER+ breast cancer cells survive tamoxifen and letrozole treatments through upregulation of minichromosome maintenance proteins (MCMs), including MCM3, which are key molecules in the cell cycle and DNA replication. Lowering MCM3 expression in endocrine-resistant cells restored drug sensitivity and altered phosphorylation of cell cycle regulators, including p53(Ser(315,33)), CHK1(Ser(317)), and cdc25b(Ser(323)), suggesting that the interaction of MCM3 with cell cycle proteins is an important mechanism of overcoming replicative stress and anti-proliferative effects of endocrine treatments. Interestingly, the MCM3 levels did not affect the efficacy of growth inhibitory by CDK4/6 inhibitors. Evaluation of MCM3 levels in primary tumors from four independent cohorts of breast cancer patients receiving adjuvant tamoxifen mono-therapy or no adjuvant treatment, including the Stockholm tamoxifen (STO-3) trial, showed MCM3 to be an independent prognostic marker adding information beyond Ki67. In addition, MCM3 was shown to be a predictive marker of response to endocrine treatment. Our study reveals a coordinated signaling network centered around MCM3 that limits response to endocrine therapy in ER+ breast cancer and identifies MCM3 as a clinically useful prognostic and predictive biomarker that allows personalized treatment of ER+ breast cancer patients. Funding Agencies|Danish Cancer SocietyDanish Cancer Society; Danish Cancer Research Foundation; Danish Research CouncilDet Frie Forskningsrad (DFF); Danish Strategic Research CouncilDanske Strategiske Forskningsrad (DSF); A Race Against Breast Cancer; Region of Southern Denmark Research Foundation; Odense University Hospital Research Council

Background Invasive lobular carcinoma (ILC) comprises 8–15 % of all invasive breast cancers and large population-based studies with >10 years of follow-up are rare. Whether ILC has a long-time prognosis different from that of invasive ductal carcinoma, (IDC) remains controversial. Purpose To investigate the excess mortality rate ratio (EMRR) of patients with ILC and IDC and to correlate survival with clinical parameters in a large population-based cohort. Material and methods From 1989 through 2006, we identified 17,481 patients diagnosed with IDC (n = 14,583) or ILC (n = 2898), younger than 76 years from two Swedish Regional Cancer Registries. Relative survival (RS) during 20 years of follow up was analysed. Results ILC was significantly associated with older age, larger tumours, ER positivity and well differentiated tumours. We noticed an improved survival for patients with ILC during the first five years, excess mortality rate ratio (EMRR) 0.64 (CI 95 % 0.53–0.77). This was shifted to a significant decreased survival 10–15 years after diagnosis (EMRR 1.49, CI 95 % 1.16–1.93). After 20 years the relative survival rates were similar, 0.72 for ILC and 0.73 for IDC. Conclusions During the first five years after surgery, the EMRR was lower for patients with ILC as compared to patients with IDC, but during the years 10–15 after surgery, we observed an increased EMRR for patients with ILC as compared to IDC. These EMRR between ILC and IDC were statistically significant but the absolute difference in excess mortality between the two groups was small., Highlights • This is a large population-based study with more than 17,000 patients with a follow up exciding 20 years. • There is clinically important differences between invasive lobular and ductal carcinoma of the breast. • Lobular carcinoma shows better survival during the first period but significantly worse at the late period of observation.

BackgroundBreast cancer tumors are heterogenous, including their metastatic potential and time to distant metastasis. Patients with estrogen receptor (ER)-positive tumors have a continuous long-term risk of fatal disease decades after diagnosis. Recently, ER-positive breast cancer patients classified as having ultralow risk tumors by the 70-gene expression signature (MammaPrint) were associated with a minimal risk of fatal disease. However, the underlying tumor characteristics of ultralow risk tumors are unknown.MethodsSecondary analysis of the Stockholm tamoxifen randomized trial (STO-3, 1976-1990) enrolling postmenopausal lymph node-negative breast cancer patients. Immunohistochemistry of the clinically used breast cancer markers (n=727 patients) and gene expression profiling by Agilent microarrays (n=652 patients) were performed in 2014. Ultralow risk tumors, identified by the 70-gene signature, were compared to other ER-positive tumors (of low/high risk) and Luminal A and B PAM50 subtype tumors (ER-positive, low/high risk) by the clinical markers and multi-gene modules, representative of specific biological processes and pathways, using Fisher’s exact test. Furthermore, differential gene expression analysis was performed contrasting ultralow risk tumors to other ER-positive tumors (of low/high risk) using t-statistics and false discovery rate (FDR).ResultsUltralow risk tumors were significantly (P

Background Tumour-infiltrating lymphocytes (TILs) are of important prognostic and predictive value in human epidermal growth factor receptor 2-positive (HER2+) breast cancer (BC) and triple-negative breast cancer (TNBC), but their clinical relevance in oestrogen receptor-positive/HER2-negative (ER+/HER2−) remains unknown. The primary study aim was to analyse the prognostic effect of TILs on the BC-free interval (BCFi) in premenopausal patients stratified by BC subtypes. The secondary aim was to investigate if TILs are predictive of tamoxifen (TAM) benefit. Methods Archival tissues from primary breast tumours were collected from patients from the SBII:2pre trial, in which 564 premenopausal women were randomised to 2 years of adjuvant TAM or no systemic treatment, regardless of hormone receptor status. TILs were scored on whole tissue sections from 447 patients with available ER status. Tumours were divided into ER+/HER2−, HER2+ and TNBC subtypes by immunohistochemistry and in situ hybridisation. The prognostic value of TILs was analysed in systemically untreated patients (n = 221); the predictive information was investigated in the ER+ subgroup (n = 321) by cumulative incidence curves and Cox regression analyses. The median follow-up was 28 years. Results High (≥ 50%) infiltration of TILs was a favourable prognostic factor in terms of BCFi (univariable analysis: hazard ratioBCFi (HRBCFi) 0.40; 95% confidence interval (CI) 0.22–0.71; P = 0.002). Similar effects were observed across all BC subtypes. The effect of adjuvant TAM was stronger in patients with ER+ tumours and TILs BCFi 0.63; 95% CI 0.47–0.84; P = 0.002) than in patients with high immune infiltration (≥ 50%) (HRBCFi 0.84; 95% CI (0.24–2.86); P = 0.77). However, evidence for differential effects of TAM in categories of TILs, i.e. interaction, was weak. Conclusions We demonstrate a long-term favourable prognostic value of high infiltration of TILs in a cohort of premenopausal BC patients and the positive prognostic effect was extended to the ER+/HER2− subgroup. A beneficial effect of TAM in ER+ patients was observed in patients with tumours of low TIL infiltration, but evidence for a treatment predictive effect was weak. Trial registration This trial is registered in the ISRCTN database, trial ID: ISRCTN12474687.

PURPOSE The Luminal intrinsic subtypes and Luminal-like surrogate subtypes of breast cancer are based on gene expression and pathological markers, respectively. We aimed to study the prognostic value of PAM50 intrinsic subtypes and risk of recurrence (ROR) score in premenopausal women with early breast cancer. Prognostic differences of Luminal PAM50 vs. surrogate subtyping, and the predictive effect of Luminal PAM50 subtypes on tamoxifen benefit were further evaluated. METHODS PAM50 intrinsic subtypes and ROR score (n = 437) were determined by gene expression analyses (NanoString’s Breast Cancer 360 TM assay (BC360)) after RNA isolation of breast cancer tissues from patients included in the randomised SBII:2pre trial. Premenopausal women were randomised between two years of adjuvant tamoxifen vs. control. Surrogate subtyping was performed according to the St. Gallen 2013 classification. The endpoints breast cancer-free interval (BCFi) and overall survival (OS) (median follow-up: 28 and 33 years, respectively) were evaluated using the maximum follow-up and the two disjoint time intervals 0-10 years and >10 years. RESULTS After maximum follow-up, patients with Luminal B tumours by PAM50 subtyping had worse prognosis as compared to those with Luminal A tumours (Hazard ratio (HR)BCfi 1.60; 95% confidence interval (CI) 1.17-2.18; P = 0.004; Table 1). For this time interval, high vs. low ROR score was associated with higher incidence of breast cancer events (HRBCFi 1.70; 95% CI 1.01-2.85; P = 0.04), with a similar trend seen in node-negative patients. In total, 58% of tumours classified as Luminal B-like by surrogate subtyping were re-classified into Luminal A by PAM50 subtyping. At 10 years follow-up, patients whose tumours were classified as Luminal B-like/Luminal A, had a better prognosis as compared to those uniformly classified as Luminal B-like/Luminal B (HRBCFi 0.52; 95% CI 0.33-0.83; P = 0.006) but the effect weakened with longer follow-up (Table 1). According to 10 years data, the incidence of breast cancer events was reduced by two thirds by tamoxifen in patients with PAM50 Luminal A tumours, while no effect was seen in those with Luminal B tumours (HRBCFi 0.41 and HRBCFi 1.19, respectively, (Pinteraction = 0.02); Table 2). CONCLUSIONS PAM50 and ROR score provided prognostic information also in premenopausal women. More than 50% of patients with Luminal B-like tumours defined by surrogate subtyping were re-classified as Luminal A according to PAM50 subtyping. These patients had an improved prognosis at 10 years of follow-up, but the effect attenuated with time. At 10 years, PAM50 Luminal A, but not Luminal B, intrinsic subtype was associated with tamoxifen benefit. Table 1.Prognostic effect of PAM50 intrinsic subtypes and St. Gallen 2013 surrogate subtyping regarding BCFi and OS for different time intervals (uni- and multivariable analyses)UnivariableMultivariableaBCFiOSBCFiOSHR (95% CI); P valuePAM50 intrinsic subtype0–10 years(n = 437, n = 218 events)(n = 437, n = 176 events)(n = 411, n = 203 events)(n = 411, n = 166 events)LumA (Ref.)1.001.001.001.00LumB1.88 (1.31–2.71); 0.0012.33 (1.52–3.58); 10 yearsb(n = 210, n = 68 events)(n = 261, n = 124 events)(n = 199, n = 66 events)(n = 245, n = 119 events)LumA (Ref.)1.001.001.001.00LumB1.05 (0.55–2.00); 0.890.85 (0.52–1.39), 0.501.92 (0.92–4.01); 0.081.16 (0.66–2.04); 0.62HER2-E0.51 (0.20–1.29); 0.150.70 (0.38–1.29); 0.251.27 (0.33–4.90): 0.731.77 (0.76–4.14); 0.19Basal-like0.95 (0.50–1.82); 0.880.69 (0.40–1.17); 0.172.58 (0.94–7.08); 0.071.90 (0.86–4.18); 0.11Maximum follow-up timec(n = 437, n = 286 events)(n = 437, n = 300 events)(n = 411, n = 269 events)(n = 411, n = 285 events)LumA (Ref.)1.001.001.001.00LumB1.60 (1.17–2.18); 0.0041.41 (1.03–1.93); 0.031.66 (1.17–2.37), 0.0051.48 (1.04–2.10); 0.03HER2-E1.84 (1.32–2.56); 10 yearsb(n = 117, n = 42 events)(n = 147, n = 70 events)(n = 115, n = 41 events)(n = 145, n = 70 events)LumB-like/LumB (Ref.)1.001.001.001.00LumB-like/LumA1.30 (0.62–2.69); 0.491.11 (0.62–1.98); 0.720.64 (0.26–1.60); 0.340.59 (0.30–1.15); 0.12LumA-like/LumB----LumA-like/LumA0.45 (0.16–1.23); 0.120.81 (0.41–1.59); 0.540.26 (0.08–0.80); 0.020.39 (0.18–0.84); 0.02Maximum follow-up timec(n = 207, n = 129 events)(n = 207, n = 130 events)(n = 205, n = 128 events)(n = 205, n = 130 events)LumB-like/LumB (Ref.)1.001.001.001.00LumB-like/LumA0.70 (0.47–1.02); 0.060.65 (0.44–0.96); 0.030.58 (0.37–0.90); 0.020.50 (0.32–0.79); 0.003LumA-like/LumB0.34 (0.08–1.41); 0.140.37 (0.09–1.52); 0.170.53 (0.12–2.24); 0.380.48 (0.11–2.05): 0.32LumA-like/LumA0.39 (0.23–0.67); 0.0010.51 (0.32–0.83); 0.0070.39 (0.24–0.70); 0.0020.43 (0.25–0.74); 0.003Abbreviations: BCFi, breast cancer free-interval; CI, confidence interval; HR, hazard ratio; Lum, Luminal; NHG, Nottingham histological grade; OS, overall survival; TAM, tamoxifenaAll analyses were stratified by study region and adjusted for age (continuous), tumour size (>20 mm vs. ≤20), NHG (1 vs. 2 vs. 3), nodal status (N0 vs. N1 vs. N2) and treatment arm in addition to surrogate/molecular subtype b10 years–maximum follow-up time, starting at year 10c32 and 36 years regarding BCFi and OS, respectively Table 2.Predictive value (univariable analyses) of Lum PAM50 intrinsic subtypes for TAM response with respect to BCFi and OS (ER-positive/HER2-negative cohort)BCFiOSHR (95% CI); P value0-10 years(n = 217, n = 92 events)(n = 217, n = 64 events)TAM vs. control in Lum A0.41 (0.23-0.74); 0.0030.61 (0.30-1.26); 0.18TAM vs. control in Lum B1.19 (0.63-2.27); 0.591.76 (0.85-3.63); 0.13Interaction Lum PAM50 subtype x TAM (HR ratio)0.34 (0.14-0.83); 0.020.35 (0.13-0.97); 0.04>10 yearsa(n = 121, n = 43 events)(n = 153, n = 74 events)TAM vs. control in Lum A0.69 (0.35-1.37); 0.290.74 (0.44-1.25); 0.26TAM vs. control in Lum B0.17 (0.04-0.80); 0.030.25 (0.08-0.77); 0.02Interaction Lum PAM50 subtype x TAM (HR ratio)4.05 (0.74-22.1); 0.112.95 (0.85-10.2); 0.09Maximum follow-up timeb(n = 217, n = 135 events)(n = 217, n = 138 events)TAM vs. control in Lum A0.52 (0.34-0.81); 0.0040.71 (0.46-1.08); 0.11TAM vs. control in Lum B0.80 (0.45-1.41); 0.440.87 (0.49-1.54); 0.63Interaction Lum PAM50 subtype x TAM (HR ratio)0.65 (0.32-1.34); 0.240.82 (0.40-1.65); 0.57Abbreviations: BCFi, breast cancer-free interval; CI, confidence interval; ER, oestrogen receptor; HER2, human epidermal growth factor receptor 2; HR, hazard ratio; Lum, Luminal; TAM, tamoxifenAll analyses were stratified by study regiona10 years-maximum follow-up time, starting at year 10b32 and 36 years regarding BCFi and OS, respectively Citation Format: Christine Lundgren, Pär-Ola Bendahl, Maria Ekholm, Mårten Fernö, Carina Forsare, Ute Krüger, Bo Nordenskjöld, Olle Stål, Lisa Rydén. Prognostic and tamoxifen-predictive effect of PAM50 and ROR score in premenopausal women included in the randomised SBII:2 trial [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD9-05.

Purpose The protein tyrosine phosphatase, non-receptor type 2 (PTNP2) regulates receptor tyrosine kinase signalling, preventing downstream activation of intracellular pathways like the PI3K/Akt pathway. The gene encoding the protein is located on chromosome 18p11; the 18p region is commonly deleted in breast cancer. In this study, we aimed to evaluate PTPN2 protein expression in a large breast cancer cohort, its possible associations to PTPN2 gene copy loss, Akt activation, and the potential use as a clinical marker in breast cancer. Methods PTPN2 protein expression was analysed by immunohistochemistry in 664 node-negative breast tumours from patients enrolled in a randomised tamoxifen trial. DNA was available for 146 patients, PTPN2 gene copy number was determined by real-time PCR. Results PTPN2 gene loss was detected in 17.8% of the tumours. Low PTPN2 protein expression was associated with higher levels of nuclear-activated Akt (pAkt-n). Low PTPN2 as well as the combination variable low PTPN2/high pAkt-n could be used as predictive markers of poor tamoxifen response. Conclusion PTPN2 negatively regulates Akt signalling and loss of PTPN2 protein along with increased pAkt-n is a new potential clinical marker of endocrine treatment efficacy, which may allow for further tailored patient therapies. Funding Agencies|Swedish Cancer Society; Cancer Research Foundations of Radiumhemmet; Cancer Society in Stockholm; King Gustav V Jubilee Clinical Research Foundation; ALF grants Region Ostergotland; Onkologiska Klinikernas i Linkoping Forskningsfond

Background We and others have shown that the clinically used breast cancer markers alter their expression throughout tumor progression, influencing patient survival (Lindström et al, JCO 2012). What are the likely explanations to our findings? Here, we aimed to determine whether breast cancer intra-tumor heterogeneity of the estrogen receptor (ER) is a marker of tumor aggressiveness and benefit of tamoxifen therapy in a large randomized trial. Material and methods The Stockholm Tamoxifen (STO-3) trial enrolled postmenopausal lymph node negative breast cancer patients with a tumor size of less than 30 mm, between 1976 and 1990, to be randomized to receive adjuvant tamoxifen versus not. From the original randomized trial cohort approximately half of the patients (778 patients) had primary tumor formalin-fixed paraffin-embedded blocks available and were included in our study. No significant differences in age and period of diagnosis, type of surgery received, receptor status, tumor grade and size were observed between the treatment arms. All tumor slides were immunostained in a central laboratory using the SP1 antibody. ER slides were scored by two independent breast cancer pathologists assessing the fraction of cancer cells for each ER intensity level (0, +1, +2 or +3) compared to established standards. The resulting distribution of ER stained tumor cells defines intra-tumor heterogeneity of ER (Rao's quadratic entropy (QE),Potts et al, Lab Invest 2012). Intra-tumor heterogeneity was categorized using the third tertile as cut-off for high heterogeneity (726 patients). Analyses of long-term breast cancer specific survival (25 years) by intra-tumor heterogeneity of ER were performed using univariate Kaplan-Meier and multivariate Cox proportional hazard modeling adjusting for treatment arm, age and period of diagnoses, ER, progesterone receptor (PR), HER2, Ki-67, tumor grade, and tumor size. Further, a test of correlation was performed to investigate whether intra-tumor heterogeneity of ER was correlated to the percentage of ER positive cells, the H-Score or the Luminal A and B subtype (PAM50). Results In the univariate Kaplan-Meier analyses, a statistically significant difference in long-term survival by intra-tumor heterogeneity of ER was seen for all patients (log rank, P=0.018), tamoxifen treated arm (log rank, P=0.0033), but not untreated arm (log rank, P=0.19). However in the multivariate analysis, patients with high intra-tumor heterogeneity of ER in the treated arm as well as in the untreated arm had an almost two-fold increased long-term risk of fatal breast cancer disease as compared to patients with low or intermediate heterogeneity (Treated arm: HR, 2.06; 95% CI, 1.04-4.07 and Untreated arm: HR, 1.71; 95% CI, 1.01-2.87). No significant correlation of intra-tumor heterogeneity to the tested variables was seen. Conclusions Patients with high intra-tumor heterogeneity of ER had less benefit from tamoxifen therapy and an increased long-term risk of fatal breast cancer disease. Our findings should be clinically relevant since therapy benefit was evaluated in a randomized trial with long-term follow-up. Citation Format: Lindström LS, Yau C, Czene K, Thompson CK, van't Veer LJ, Nordenskjöld B, Stål O, Fornander T, Benz CC, Borowsky AD, Esserman LJ. Intra-tumor heterogeneity of the estrogen receptor predicts less benefit from tamoxifen therapy and poor long-term breast cancer patient survival – Retrospective analyses of the STO-3 randomized trial [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 P2-05-03.

Aims: The primary aim was to compare 2 years of adjuvant tamoxifen versus no systemic treatment in premenopausal patients with oestrogen receptor (ER) epositive tumours, regarding breast cancerefree interval (BCFi) and distant recurrenceefree interval (D-RFi), with 30 years of follow-up and for specified intervals. Moreover, we aimed to investigate the effects of adjuvant tamoxifen on the incidence of secondary malignancies and survival after distant recurrence. Methods: Premenopausal patients with primary breast cancer were randomised to 2 years of tamoxifen (n=277) or no systemic treatment (n=287), irrespective of ER status. Information regarding events was collected by a review of medical records and from national registers. Results: The median follow-up for all patients without events was 28 years, and only four of the patients alive had a follow-up of amp;lt;20 years. With 30 years of follow-up, tamoxifen prolonged BCFi in the intention-to-treat population (hazard ratio [HR] = 0.76, 95% confidence interval (CI) 0.61-0.94, p = 0.011) compared with no treatment. In patients with ER-positive tumours (n = 362), tamoxifen prolonged BCFi (HR = 0.62, 95% CI 0.47-0.82, p = 0.001) and D-RFi (HR = 0.73, 95% CI 0.54-0.99, p = 0.043). The positive effect on BCFi was significant also for the interval amp;gt; 15-30 years (HR = 0.53, 95% CI 0.28-0.98, p = 0.042). For patients with ER-positive tumours who were diagnosed with distant recurrence (n=165), survival after distant recurrence was shorter among tamoxifen-treated patients (median, 29 months versus 43 months). The incidence of contralateral breast cancer was 42% lower in the tamoxifen group (HR=0.58, 95% CI 0.35-0.96, p=0.035), whereas no differences were observed regarding other secondary malignancies. Conclusions: With three decades of follow-up, 2 years of adjuvant tamoxifen reduced the incidence of breast cancererelated events and distant recurrence, and the carryover effect seems to extend beyond 15 years. Moreover, adjuvant tamoxifen seems to be associated with shorter survival after diagnosis of distant recurrence. (C) 2019 The Authors. Published by Elsevier Ltd. Funding Agencies|Futurum-the Academy of Health and Care; Jonkoping County Council; Foundation for Clinical Cancer Research in Jonkoping; FORSS (Medical Research Council of Southeast Sweden); Gunnar Nilsson Cancer Foundation; Mrs. Berta Kamprad Foundation; Anna and Edwin Bergers Foundation; Swedish Cancer and Allergy Foundation; Governmental Funding of Clinical Research within the Swedish National Health Service; Swedish Cancer Society

Background Better tools are needed to identify breast cancer patients at very low risk of dying from their disease. We applied a previously specified 'Ultralow risk' threshold for the FDA-cleared MammaPrint 70-gene expression score to predict long-term absence of breast cancer-specific mortality in a Swedish randomized trial of breast cancer patients treated with tamoxifen (Tam) versus not and followed for more than 25 years. Methods Between 1976-1990 the Stockholm Tamoxifen (STO) trial enrolled and randomized node negative breast cancer patients with tumor size less than 30 mm to receive 2 years of Tam versus not, without regard to hormone receptor status. In the Tam-treated arm, patients without relapse at 2 years were further randomized to receive 3 additional years of Tam versus no additional endocrine therapy. From the original STO randomized trial cohort, about half (778 cases) had remaining formalin-fixed paraffin-embedded primary tumor blocks for additional tumor characterization and MammaPrint analysis. In this validation dataset, which now has >25 years follow-up, breast cancer-specific survival was assessed between MammaPrint scored 'Ultralow risk', 'Low risk' or 'High risk' categories by Kaplan-Meier analyses and multivariate Cox proportional hazard modeling, adjusting for treatment, age, period of diagnosis, tumor size, grade, receptor (ER, PR, HER2) and Ki-67 status. Results: In this unscreened patient population MammaPrint scored 15% of patients as 'Ultra-low risk', 43% as 'Low risk' and 42% as 'High risk'. At 20 years, a statistically significant difference in survival between risk categories was seen for all patients (log rank, P=0.0001), the Tam treated arm (log rank, P=0.0088) and the untreated arm (log rank, P=0.014). Ultra-low risk patients have significantly lower risk of disease-specific death relative to Low and High risk patients in our multivariate Cox analysis. Among Ultra-low risk patients there were no deaths out to 15 years in the Tam-treated arm; by 20 years, disease-specific survival in Tam-treated and untreated arms were 94% and 90%. The majority of Tam-treated patients received only 2 years of treatment, with ∼35% going on to receive 5 years of treatment. All Ultra-low risk cases were HR+HER2-. 78% were luminal A by PAM50; but only 31% of luminal A were Ultra-low risk. Invasive ductal (no-special-type) carcinomas were the most frequent, but lobular, tubular, invasive papillary and invasive cribriform subtypes were enriched and mucinous types were absent. Conclusions: Node-negative, T15 y) metastatic recurrence risk exists if left untreated, 'Ultralow risk' status could identify women for whom lumpectomy alone and a short course of adjuvant endocrine therapy is sufficient. Given the high frequency (∼50%) of such 'Ultralow risk' tumors detected in modern screening cohorts (e.g. MINDACT), a substantial fraction of newly diagnosed women could benefit from more targeted and less aggressive local and adjuvant therapy. Citation Format: Esserman LJ, Thompson CK, Yau C, van 't Veer LJ, Borowsky AD, Tobin NP, Nordenskjöld B, Fornander T, Stål O, Benz CC, Lindström LS. Identification of tumors with an indolent disease course: MammaPrint ultralow signature validation in a retrospective analysis of a Swedish randomized tamoxifen trial. [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 P6-09-01.

Deregulated PI3K/mTOR signals can promote the growth of breast cancer and contribute to endocrine treatment resistance. This report aims to investigate raptor and its intracellular localization to further understand its role in ER-positive breast cancer. Raptor protein expression was evaluated by immunohistochemistry in 756 primary breast tumors from postmenopausal patients randomized to tamoxifen or no tamoxifen. In vitro, the MCF7 breast cancer cell line and tamoxifen-resistant MCF7 cells were studied to track the raptor signaling changes upon resistance, and raptor localization in ER alpha-positive cell lines was compared with that in ER alpha-negative cell lines. Raptor protein expression in the nucleus was high in ER/PgR-positive and HER2-negative tumors with low grade, features associated with the luminal A subtype. Presence of raptor in the nucleus was connected with ER alpha signaling, here shown by a coupled increase of ER alpha phosphorylation at S167 and S305 with accumulation of nuclear raptor. In addition, the expression of ER alpha-activated gene products correlated with nuclear raptor. Similarly, in vitro we observed raptor in the nucleus of ER alpha-positive, but not of ER-negative cells. Interestingly, raptor localized to the nucleus could still be seen in tamoxifen-resistant MCF7 cells. The clinical benefit from tamoxifen was inversely associated with an increase of nuclear raptor. High cytoplasmic raptor expression indicated worse prognosis on long-term follow-up. We present a connection between raptor localization to the nucleus and ER alpha-positive breast cancer, suggesting raptor as a player in stimulating the growth of the luminal A subtype and a possible target along with endocrine treatment. Funding Agencies|Swedish Cancer Society; Region of Ostergotland; Cancer Society in Stockholm; King Gustav V Jubilee Clinical Research Foundation; National Cancer Institute (NCI); American Cancer Society; Atol Charitable Trust

Background In published radiotherapy trials, the failure rate in the control arm among patients with one to three positive nodes is high compared with that seen with modern adjuvant treatments. Therefore, the generalizability of the results has been questioned. The aim of the present study was to compare relative survival in breast cancer patients between two Swedish regions with screening mammography programs and adjuvant treatment guidelines similar with the exception of the indication of radiotherapy for patients with one to three positive nodes. Patients and methods Between 1989 and 2006, breast cancer patients were managed very similarly in the west and southeast regions, except for indication for postoperative radiotherapy. In patients with one to three positive nodes, postmastectomy radiotherapy was generally given in the southeast region (89% of all cases) and generally not given in the west region (15% of all cases). For patients with one to three positive nodes who underwent breast-conserving surgery, patients in the west region had breast radiotherapy only, while patients in the southeast region had both breast and lymph nodes irradiated. Results The 10-year relative survival for patients with one to three positive lymph nodes was 78% in the west region and 77% in the southeast region (P = 0.12). Separate analyses depending on type of surgery, as well as number of examined nodes, also revealed similar relative survival. Conclusion Locoregional postoperative radiotherapy has well-known side-effects, but in this population-based study, there was little or no influence of this type of radiotherapy on survival when one to three lymph nodes were involved.

Background In 2011 The Early Breast Cancer Trialists' Collaborative Group (EBCTCG) reported that five years of adjuvant tamoxifen (TAM) significantly reduces the 15-year risks of breast cancer recurrence and death. Moreover, the estrogen receptor (ER) was considered the clinically most relevant predictive factor for efficacy of TAM treatment. In premenopausal patients participating in a randomized trial of 2 years of adjuvant TAM, we have previously reported on increased recurrence free survival in patients with ER- and PR-positive disease and a trend for increased overall survival in patients with PR-positive tumors. The median follow-up time was 13.9 years for patients without events. Since the effect on mortality may increase beyond year 10, we investigated more mature of data. Aims To investigate the long-term effect of 2 years adjuvant TAM in premenopausal patients choosing cumulative mortality (CM) and cumulative breast cancer mortality (CBCM) as end-points. Methods Premenopausal patients (n=564) with stage II breast cancer were included in a Swedish randomized multicenter trial between 1986-1991 and allocated to 2 years of TAM (n=276) or no treatment (n=288). The hormone receptor status of the tumor was known for 541 (96%) of the patients included, determined by immunohistochemistry or cytosol-based methods. Less than 2% were treated with adjuvant chemotherapy and they were equally distributed between the two groups. Median follow-up time was 26.3 years (22.7-29.7). Mortality data was obtained from the Swedish Cause of Death Register. Results Death from any cause was recorded among 312 (55%) of the patients, and 265 (47%) of the deaths were due to breast cancer. Two years of TAM decreased CM and CBCM in all patients irrespective of hormonal receptor status (n=564) (CM: HR 0.81; 95% CI: 0.65-1.02, p=0.070; CBCM: HR 0.79; 95% CI: 0.61-1.01, p=0.058), as well as in patients with ER-positive disease (n=346) (CM: HR 0.77; 95% CI: 0.57-1.03, p=0.077; CBCM: HR 0.72; 95% CI: 0.52-1.01, p=0.051), however not strictly significant. Importantly, in patients with PR-positive tumors, TAM significantly reduced CM and CBCM (CM: HR 0.73; 95% CI: 0.55-0.98, p=0.037; CBCM: HR 0.68; 95% CI: 0.49-0.94, p=0.020). Moreover, in patients with ER- and PR positive tumors TAM decreased CM (HR 0.74; 95% CI: 0.55-1.01, p=0.057) and CBCM (HR 0.69; 95% CI: 0-49-0.97, p=0.033), whereas there was no effect of TAM in patients with ER-positive and PR-negative tumors (p=0.97 and p=0.99, respectively). Conclusions The present study demonstrates that 2 years of adjuvant TAM as monotherapy significantly reduced CM and CBCM in premenopausal women with PR-positive tumors as well as CBCM in ER- and PR-positive tumors, after > 25 years of follow-up. Five years of adjuvant TAM have been proven to reduce mortality rates at 15 years, but we herein show that also 2 years of TAM yields a survival benefit at 25 years. In the latest meta-analysis from EBCTCG, PR did not add predictive information in patients with ER-positive tumors, but according to our results the significance of PR, as a predictive factor for TAM efficacy, should be re-emphasized in premenopausal women. Citation Format: Maria Ekholm, Pär-Ola Bendahl, Mårten Fernö, Bo Nordenskjöld, Olle Stål, Lisa Rydén. Mature data (> 25 years) on 2 years adjuvant tamoxifen treatment in premenopausal women with breast cancer: Time to re-emphasize the progesterone receptor as predictive factor? [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-12-17.

Breast cancer molecular prognostic tools that predict recurrence risk have mainly been established on endocrine-treated patients and thus are not optimal for the evaluation of benefit from endocrine therapy. The Stockholm tamoxifen (STO-3) trial which randomized postmenopausal node-negative patients to 2-year tamoxifen (followed by an optional randomization for an additional 3-year tamoxifen vs nil), versus no adjuvant treatment, provides a unique opportunity to evaluate long-term 20-year benefit of endocrine therapy within prognostic risk classes of the 70-gene prognosis signature that was developed on adjuvantly untreated patients. We assessed by Kaplan-Meier analysis 20-year breast cancer-specific survival (BCSS) and 10-year distant metastasis-free survival (DMFS) for 538 estrogen receptor (ER)-positive, STO-3 trial patients with retrospectively ascertained 70-gene prognosis classification. Multivariable analysis of long-term (20 years) BCSS by STO-3 trial arm in the 70-gene high-risk and low-risk subgroups was performed using Cox proportional hazard modeling adjusting for classical patient and tumor characteristics. Tamoxifen-treated, 70-gene low- and high-risk patients had 20-year BCSS of 90 and 83%, as compared to 80 and 65% for untreated patients, respectively (log-rank p amp;lt; 0.0001). Notably, there is equivalent tamoxifen benefit in both high (HR 0.42 (0.21-0.86), p = 0.018) and low (HR 0.46 (0.25-0.85), p = 0.013) 70-gene risk categories even after adjusting for clinico-pathological factors for BCSS. Limited tamoxifen exposure as given in the STO-3 trial provides persistent benefit for 10-15 years after diagnosis in a time-varying analysis. 10-year DMFS was 93 and 85% for low- and high-risk tamoxifen-treated, versus 83 and 70% for low- and high-risk untreated patients, respectively (log-rank p amp;lt; 0.0001). Patients with ER-positive breast cancer, regardless of high or low 70-gene risk classification, receive significant survival benefit lasting over 10 years from adjuvant tamoxifen therapy, even when given for a relatively short duration. Funding Agencies|California Breast Cancer Research Program BCRP award [180B-0065]; Breast Cancer Research Foundation [BCRF]; Swedish Research Council [521-2014-2057]; FORTE [2014-1962]; Stiftelsen Gosta Miltons Donationsfond [The Gosta Milton Donation Fund]; Cancerforeningen i Stockholm [Stockholm Cancer Society]

Purpose To analyze the impact of postmastectomy radiation therapy (PMRT) for patients with T1-T2 tumors and 1 to 3 positive lymph nodes enrolled on the Breast International Group (BIG) 02-98 trial. Methods and Materials The BIG 02-98 trial randomized patients to receive adjuvant anthracycline with or without taxane chemotherapy. Delivery of PMRT was nonrandomized and performed according to institutional preferences. The present analysis was performed on participants with T1-T2 breast cancer and 1 to 3 positive lymph nodes who had undergone mastectomy and axillary nodal dissection. The primary objective of the present study was to examine the effect of PMRT on risk of locoregional recurrence (LRR), breast cancer–specific survival, and overall survival. Results We identified 684 patients who met the inclusion criteria and were included in the analysis, of whom 337 (49%) had received PMRT. At 10 years, LRR risk was 2.5% in the PMRT group and 6.5% in the no-PMRT group (hazard ratio 0.29, 95% confidence interval 0.12-0.73; P = .005). Lower LRR after PMRT was noted for patients randomized to receive adjuvant chemotherapy with no taxane (10-year LRR: 3.4% vs 9.1%; P = .02). No significant differences in breast cancer–specific survival (84.3% vs 83.9%) or overall survival (81.7% vs 78.3%) were observed according to receipt of PMRT. Conclusion Our analysis of the BIG 02-98 trial shows excellent outcomes in women with T1-T2 tumors and 1 to 3 positive lymph nodes found in axillary dissection. Although PMRT improved LRR in this cohort, the number of events remained low at 10 years. In all groups, 10-year rates of LRR were relatively low compared with historical studies. As such, the use of PMRT in women with 1 to 3 positive nodes should be tailored to individual patient risks.

In an investigation by the Swedish Cancer Society, an expert group described the present status, critical issues and future aspects and potentials for each of nine major areas of radiation therapy research. This report deals with interactions between chemotherapy, endocrine therapy, other anti-tumour drugs and radiation.

Background: Tamoxifen is a well established treatment for breast cancer, but its long-term effects on the incidence of secondary cancers are not fully evaluated.Material and methods: We have studied 4128 postmenopausal patients with early stage breast cancer who were alive and free of breast cancer recurrence after two years of tamoxifen, and who were randomized to receive totally two or five years of therapy.Results: Compared to patients randomized to two years of tamoxifen the incidence of contralateral breast cancer [hazard ratio (HR) 0.73; 95% CI 0.56–0.96] and of lung cancer (HR 0.45; 95% CI 0.27–0.77), especially squamous cell and small cell lung cancer, were reduced in the five-year group, and similar results were seen when restricting the analysis to the 10-year period after treatment stopped. An increased incidence of endometrial cancer was observed in the five-year group, but the excess risk decreased over time.Conclusion: Further studies of the effects of tamoxifen on the risk of differ...

To investigate the expression levels of CXCL10 and CXCR3 in tumors from breast cancer patients randomized to adjuvant tamoxifen treatment or no endocrine treatment, in order to further study the connection to prognosis and prediction of tamoxifen treatment outcome. Immunohistochemistry on tissue microarrays from 912 breast cancer patients randomized to tamoxifen or no endocrine treatment. CXCR3 status was found to be a prognostic tool in predicting distant recurrence, as well as reduced breast cancer-specific survival. In patients with estrogen receptor (ER)-positive tumors, tumors with strong CXCL10 levels had improved effect of tamoxifen treatment in terms of local recurrence-free survival [risk ratio (RR) 0.46 (95 % CI 0.25-0.85, P = 0.01)] compared with patients with tumors expressing weak CXCL10 expression. Further, patients with ER-positive tumors with strong CXCR3 expression had an improved effect of tamoxifen in terms of breast cancer-specific survival [RR 0.34 (95 % CI 0.19-0.62, P less than 0.001)] compared with the group with weak CXCR3 levels [RR 1.33 (95 % CI 0.38-4.79, P = 0.65)]. We show here for the first time that CXCL10 and CXCR3 expression are both predictors of favorable outcome in patients treated with tamoxifen.

Patients with estrogen receptor (ER)-positive breast cancer have a long-term risk for fatal disease. However, the tumor biological factors that influence the long-term risk and the benefit associated with endocrine therapy are not well understood.To compare the long-term survival from tamoxifen therapy for patients with luminal A or luminal B tumor subtype.Secondary analysis of patients from the Stockholm Tamoxifen (STO-3) trial conducted from 1976 to 1990, which randomized postmenopausal patients with lymph node-negative breast cancer to receive adjuvant tamoxifen or no endocrine therapy. Tumor tissue sections were assessed in 2014 using immunohistochemistry and Agilent microarrays. Only patients with luminal A or B subtype tumors were evaluated. Complete long-term follow-up data up to the end of the STO-3 trial on December 31, 2012, were obtained from the Swedish National registers. Data analysis for the secondary analysis was conducted in 2017 and 2018.Patients were randomized to receive at least 2 years of tamoxifen therapy or no endocrine therapy; patients without recurrence who reconsented were further randomized to 3 additional years of tamoxifen therapy or no endocrine therapy.Distant recurrence-free interval (DRFI) by luminal A and luminal B subtype and trial arm was assessed by Kaplan-Meier analyses and time-dependent flexible parametric models to estimate time-varying hazard ratios (HRs) that were adjusted for patient and tumor characteristics.In the STO-3 treated trial arm, 183 patients had luminal A tumors and 64 patients had luminal B tumors. In the untreated arm, 153 patients had luminal A tumors and 62 had luminal B tumors. Age at diagnosis ranged from 45 to 73 years. A statistically significant difference in DRFI by trial arm was observed (log rank, P .001 [luminal A subtype, n = 336], P = .04 [luminal B subtype, n = 126]): the 25-year DRFI for luminal A vs luminal B subtypes was 87% (95% CI, 82%-93%) vs 67% (95% CI, 56%-82%) for treated patients, and 70% (95% CI, 62%-79%) vs 54% (95% CI, 42%-70%) for untreated patients, respectively. Patients with luminal A tumors significantly benefited from tamoxifen therapy for 15 years after diagnosis (HR, 0.57; 95% CI, 0.35-0.94), and those with luminal B tumors benefited from tamoxifen therapy for 5 years (HR, 0.38; 95% CI, 0.24-0.59).Patients with luminal A subtype tumors had a long-term risk of distant metastatic disease, which was reduced by tamoxifen treatment, whereas patients with luminal B tumors had an early risk of distant metastatic disease, and tamoxifen benefit attenuated over time.

Background: G protein-coupled estrogen receptor (GPER), also known as GPR30, is a novel putative estrogen receptor. Although contradictory results have been presented e.g. regarding the subcellular localization and function of the receptor, previous studies have shown a prognostic value in breast cancer and proposed treatment predictive information for tamoxifen (Tam). This study aimed at clarifying the prognostic and treatment predictive value for Tam of GPER, in different subcellular localizations, by using samples from a randomized clinical trial - the ideal population for assessing treatment prediction. Material and Methods: GPER levels were assessed semi-quantitatively by immunohistochemistry in tissue microarrays from 742 postmenopausal breast cancer patients with no lymph node metastasis and tumor size ≤ 30mm. Patients were originally included in the STO-3 trial 1976-1990. After surgery, they were randomized to Tam treatment (40mg for 2 years or no systemic treatment), regardless of classical estrogen receptor α (ER) status. GPER staining was evaluated in carcinoma both as intensity in 5 levels regardless of subcellular localization, and in the plasma membrane in 3 levels. Due to statistical considerations regarding group size, the final analysis was made with intensity in 3 levels and plasma membrane as positive or negative. The Kaplan-Meier method and logrank test (for trend when applicable) were used for survival analysis and Cox regression analysis for obtaining hazard ratios (HR), interaction testing and multivariate modeling. Distant disease-free survival (DDFS) was used as endpoint. Results: Analyzing all patients, we found no association between DDFS and GPER intensity. However, positive plasma membrane staining showed a strong correlation with poor prognosis (HR 1.8 p = 0.002). This was only observed in the ER+ subgroup (ER+ patients HR 2.1, p No obvious difference in tamoxifen response was observed across plasma membrane or intensity groups, and tests for interaction were not significant. A multivariate model including GPER in plasma membrane, ER, histological grade, HER2, tamoxifen and tumor size showed that GPER was an independent prognostic factor (HR 1.6 p = 0.01). Finally we created a group with ER+, progesterone receptor (PR) + patients treated with Tam, as this group today is treated with Tam and thought to have a good response. GPER in the plasma membrane significantly separated this group into an excellent prognosis group and a poor prognosis group (HR 3.3, p = 0.01). The excellent prognosis group, which constitutes more than half of ER+ patients, had a 20 year DDFS of 91% (95% CI 84-95). Conclusion: We found no treatment predictive value of GPER for Tam. However, GPER expressed in the plasma membrane was a strong independent prognostic factor for a poor prognosis in ER+ breast cancer. Used in ER+, PR+, tamoxifen treated patients, it can distinguish patients with an excellent prognosis from patients with a poor outcome that may benefit from additional treatment. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-08-12.

Background: In women with node-positive breast cancer, the Breast International Group (BIG) 02-98 tested the incorporation of docetaxel (Taxotere) into doxorubicin (Adriamycin)-based chemotherapy, and compared sequential and concurrent docetaxel. At 5 years, there was a trend for improved disease-free survival (DFS) with docetaxel. We present results at 8-year median follow-up and exploratory analyses within biologically defined subtypes. less thanbrgreater than less thanbrgreater thanMethods: Patients were randomly assigned to one of four treatments: (i) sequential control: doxorubicin (A) (75 mg/m(2)) x 4 -andgt; classical cyclophosphamide, methotrexate, 5-fluorouracil (CMF); (ii) concurrent control: doxorubicin, cyclophosphamide (AC)(60/600 mg/m(2)) x 4 -andgt; CMF; (iii) sequential docetaxel: A (75 mg/m(2)) x3 -andgt; docetaxel (T) (100 mg/m(2)) x3. CMF and (iv) concurrent docetaxel: AT(50/75 mg/m(2)) x 4 -andgt; CMF. The primary comparison evaluated docetaxel efficacy regardless of the schedule. Exploratory analyses were undertaken within biologically defined subtypes. less thanbrgreater than less thanbrgreater thanResults: Two thousand eight hundred and eighty-seven patients were enrolled. After 93.4 months of median follow-up, there were 916 DFS events. For the primary comparison, there was no significant improvement in DFS from docetaxel [hazard ratio (HR) = 0.91, 95% confidence interval (CI) = 0.80-1.05, P = 0.187]. In secondary comparisons, sequential docetaxel significantly improved DFS compared with sequential control (HR = 0.81, 95% CI = 0.67-0.99, P = 0.036), and significantly improved DFS (HR = 0.84, 95% CI = 0.72-0.99, P = 0.035) and overall survival (OS) (HR = 0.79, 95% CI = 0.65-0.98, P = 0.028) compared with concurrent doxorubicin-docetaxel. Luminal-A disease had the best prognosis. HRs favored addition of sequential docetaxel in all subtypes, except luminal-A; but this observation was not statistically supported because of limited numbers. less thanbrgreater than less thanbrgreater thanConclusion: With further follow-up, the sequential docetaxel schedule resulted in significantly better OS than concurrent doxorubicin-docetaxel, and continued to show better DFS than sequential doxorubicin-based control.

Reduced or absent Wwox expression has recently been associated with tamoxifen resistance in breast cancer and has also been proposed as a candidate predictive marker for treatment. We aimed to investigate the correlation of Wwox expression with the outcome of tamoxifen treatment by examining tissues from 912 randomized breast cancer patients. Paraffin-embedded tissues from patient tumors were arranged on tissue microarray, and Wwox protein was stained using immunohistochemistry. After microscopic examination, the results were analyzed with Cox regression, Kaplan-Meier survival curves and the log-rank test. In the group of cases having a tumor absent for Wwox expression, there was no difference in recurrence-free survival between treated and untreated patients (P=0.81). For treated cases with a tumor expressing moderate or strong Wwox protein, recurrence-free survival was improved (P=0.001 and P=0.003, respectively). The test for interaction between Wwox and treatment response demonstrated a decreased risk of recurrence for treated patients with a moderate or strong Wwox expression (HR=0.31, 95% CI 0.10-0.98 and HR=0.28, 95% CI 0.08-0.97, respectively). Our results indicate that patients with high expression of Wwox may gain more benefit from treatment with tamoxifen.

Background: Although the androgen receptor (AR) is frequently expressed in breast cancer, its relevance in the disease is not fully understood. In addition, the relevance of AR in determining tamoxifen treatment efficiency requires evaluation. Purpose: To investigate the tamoxifen predictive relevance of the AR protein expression in breast cancer. Methods Patients were randomised to tamoxifen 40 mg daily for 2 or 5 years or to no endocrine treatment. Mean follow-up was 15 years. Hazard ratios were calculated with recurrence-free survival as end point. Results: In patients with oestrogen receptor (ER)-negative tumours, expression of AR predicted decreased recurrence rate with tamoxifen (hazard ratio (HR) = 0.34; 95% confidence interval (CI) = 0.14-0.81; P = 0.015), whereas the opposite was seen in the AR- group (HR = 2.92; 95% CI = 1.16-7.31; P = 0.022). Interaction test was significant P < 0.001. Patients with triple-negative and AR+ tumours benefitted from tamoxifen treatment (HR = 0.12; 95% CI = 0.014-0.95 P = 0.044), whereas patients with AR- tumours had worse outcome when treated with tamoxifen (HR = 3.98; 95% CI = 1.32-12.03; P = 0.014). Interaction test was significant P = 0.003. Patients with ER+ tumours showed benefit from tamoxifen treatment regardless of AR expression. Conclusions: AR can predict tamoxifen treatment benefit in patients with ER- tumours and triple-negative breast cancer. Funding Agencies|Swedish research council [A0346701]; Swedish cancer foundation [13 0435]

Purpose The aim of this study was to evaluate the long-term effect of 2 years of adjuvant tamoxifen compared with no systemic treatment (control) in premenopausal patients with breast cancer over different time periods through long-term (> 25 years) follow-up. Patients and Methods Premenopausal patients with primary breast cancer (N = 564) were randomly assigned to 2 years of tamoxifen (n = 276) or no systemic treatment (n = 288). Data regarding date and cause of death were obtained from the Swedish Cause of Death Register. End points were cumulative mortality (CM) and cumulative breast cancer–related mortality (CBCM). The median follow-up for the 250 patients still alive in April 2014 was 26.3 years (range, 22.7 to 29.7 years). Results In patients with estrogen receptor–positive tumors (n = 362), tamoxifen was associated with a marginal reduction in CM (hazard ratio [HR], 0.77; 95% CI, 0.58 to 1.03; P = .075) and a significant reduction in CBCM (HR, 0.73; 95% CI, 0.53 to 0.99; P = .046). The effect seemed to vary over time (CM years 0 to 5: HR, 1.05; 95% CI, 0.64 to 1.73; years > 5 to 15: HR, 0.58; 95% CI, 0.37 to 0.91; and after 15 years: HR, 0.82; 95% CI, 0.48 to 1.42; CBCM years 0 to 5: HR, 1.09; 95% CI, 0.65 to 1.82; years > 5 to 15: HR, 0.53; 95% CI, 0.33 to 0.86; and after 15 years: HR, 0.72; 95% CI, 0.36 to 1.44). Conclusion Two years of adjuvant tamoxifen resulted in a long-term survival benefit in premenopausal patients with estrogen receptor–positive primary breast cancer.

Background: CXCL10 is a chemokine with chemo attractant properties which signals through G protein-coupled receptor CXCR3. At least two reported isoforms exists; CXCR3-A and CXCR3-B. CXCL10 is involved in the recruitment of leucocytes, immune modulation and angiogenesis. In animal models CXCL10 inhibits tumour growth, metastase formation and inhibits angiogenesis. The receptors have opposite features; CXCR3-A mediates proliferative or anti-apoptotic response and CXCR3-B mediates growth inhibition and apoptosis in response to ligand binding. The aim of this study was to investigate the role of CXCL10 and CXCR3-A as prognostic or tamoxifen treatment predictive factors in breast cancer. Material and Methods: A randomized tamoxifen trial comprising 1,780 breast cancer patients was conducted in Stockholm, Sweden, 1976–1990. All patients had lymph node negative primary breast cancer and were postmenopausal at the time of diagnosis. The patients were randomized to tamoxifen or no endocrine treatment. We analyzed the protein expression of CXCL10 and the receptor CXCR3A with immunohistochemistry using tissue microarrays, which were constructed from paraffin blocks originating from 912 patients. CXCL10 and CXCR3A could be scored in 793 (87%) and 735 (81%), respectively. Results: High expression of CXCL10 was associated with significant benefit of tamoxifen treatment concerning local recurrence and breast cancer survival (P=0.02 and P=0.008). For patients with high CXCL10 expression, tamoxifen decreased the risk concerning local recurrence (rate ratio (RR) = 0.56, 95% C.I. 0.33−0.97, P=0.0029) and breast cancer survival (RR 0.6 95% C.I. 0.39−0.97, P=0.023). High expression of CXCR3-A was associated with significant benefit of tamoxifen treatment concerning total recurrence and breast cancer survival (P=0.00001 and P=0.004, respectively). For patients with high CXCR3-A expression, tamoxifen decreased the risk of total recurrence (RR 0.54 95% C.I. 0.35−0.83, P=0.005) and breast cancer survival (RR 0.49 95% C.I. 0.35−0.69, P=0.00003). On the opposite, in the cohort of patients with no endocrine treatment and high CXCR3-A expression, there was an increased risk of total recurrence (P=0.003). (RR 2.07 95% C.I. 1.18−3.61, P=0.011). Conclusions: This study indicates that high expression of CXCL10 can be used as predictive markers for tamoxifen treatment. Further, high expression of CXCR3-A is related to bad prognosis and predicts tamoxifen treatment in breast cancer patients. This needs to be further evaluated. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-06-06.

Previous retrospective studies have shown that high intratumoural levels of vascular endothelial growth factor (VEGF) correlate with an inferior outcome for patients treated with adjuvant tamoxifen. Our objectives were to validate the impact of VEGF on survival after adjuvant tamoxifen and to investigate the interaction between VEGF and treatment duration. For this purpose tumour homogenates from 402 patients with operable oestrogen receptor positive breast cancer (BC), treated with tamoxifen for 2 (n=149) or 5 years (n=253) as the only systemic adjuvant therapy were included. The median follow-up time for surviving patients was 9.8 years (range 0.5-14.8 years). Expression of VEGF was assessed by an enzyme-linked immunosorbent assay and investigated in relation to the standard BC parameters and survival. In the total population, higher VEGF was significantly correlated with shorter recurrence-free survival (RFS) (HR=1.63, 95%CI=1.11-2.39, p=0.010), breast cancer corrected survival (BCCS) (HR=1.82, 95%CI=1.13-2.93, p=0.014) and overall survival (OS) (HR=1.51, 95%CI=1.11-2.05, p=0.009). High VEGF was significantly associated with reduced RFS (HR=2.61, 95%CI=1.45-4.70, p=0.001) after two years of tamoxifen, whilst no difference was seen in patients treated for five years (HR=1.09, 95%CI=0.64-1.84, p=0.760). A statistically significant interaction was observed between high VEGF expression and improved RFS after 5-year tamoxifen (p=0.034). In concordance with previous studies, high VEGF was significantly correlated with shorter survival. We present data not reported previously revealing that patients expressing high levels of VEGF display a better outcome provided that tamoxifen is given for five years. Further studies on the impact of VEGF on a 5-year regimen are motivated.