Breast cancer hormone receptor levels and benefit from adjuvant tamoxifen in a randomized trial with long-term follow-up.

BACKGROUND: Hormone receptor positivity predicts benefit from endocrine therapy but the knowledge about the long-term survival of patients with different tumor receptor levels is limited. In this study, we describe the 25 years outcome of tamoxifen (TAM) treated patients. PATIENTS AND METHODS: Between 1983 and 1992, a total of 4,610 postmenopausal patients with early-stage breast cancer were randomized to receive totally 2 or 5 years of TAM therapy. After 2 years, 4,124 were alive and free of breast cancer recurrence. Among these, 2,481 had demonstrated estrogen receptor positive (ER+) disease. From 1988, the Abbot enzyme immunoassay became available and provided quantitative receptor levels for 1,210 patients, for which our analyses were done. RESULTS: After 5 years of follow-up, when all TAM treatment was finished, until 15 years of follow-up, breast cancer mortality for patients with ER+ disease was significantly reduced in the 5-year group as compared with the 2-year group (hazard ratios [HR] 0.67, 95% confidence intervals [CI] 0.55-0.83, p < 0.001). After 15 years, the difference between the groups remained but did not increase further. A substantial benefit from prolonged TAM therapy was only observed for the subgroup of patients with ER levels below the median (HR = 0.62, 95% CI 0.46-0.84, p = 0.002). Similarly, patients with progesterone receptor negative (PR-) disease did benefit from prolonged TAM treatment. For patients with progesterone receptor positive (PR+) disease, there was no statistically significant benefit from more than 2 years of TAM.  Interpretation: As compared with 2 years of adjuvant TAM, 5 years significantly prolonged breast cancer-specific survival. The benefit from prolonged TAM therapy was statistically significant for patients with ER levels below median or PR-negative disease. There was no evident benefit from prolonged TAM for patients with high ER levels or with PR+ tumors.

Hormone receptor mRNA and protein levels as predictors of premenopausal tamoxifen benefit.

BACKGROUND AND PURPOSE: Tamoxifen remains an important adjuvant treatment in premenopausal patients with hormone receptor-positive breast cancer. Thus, determination of hormone receptors is important. Here, we compare cytosol-based methods, immunohistochemistry (IHC), and gene expression (GEX) analysis for determining hormone receptor status in premenopausal breast cancer patients from a randomised tamoxifen trial, to evaluate their performance in identifying patients that benefit from tamoxifen. PATIENTS AND METHODS: Premenopausal patients (n=564) were randomised to 2 years of tamoxifen or no systemic treatment. Estrogen receptor (ER) and progesterone receptor (PR) status by protein expression measured by cytosol-based methods and IHC, and mRNA by GEX analysis were compared in 313 patients with available data from all methods. Kaplan Meier estimates and Cox regression were used to evaluate the treatment-predictive value for recurrence-free interval (RFi) and overall survival (OS). Median follow-up for event-free patients was 26 (RFi) and 33 (OS) years. RESULTS: The mRNA data of ESR1 and PGR distributed bimodally, patterns confirmed in an independent cohort. Kappa-values between all methods were 0.76 and 0.79 for ER and PR, respectively. Tamoxifen improved RFi in patients with ER-positive (ER+) or PR-positive (PR+) tumours (Hazard Ratio [HR] and 95% confidence interval [CI]), cytosol-ER+ 0.53 [0.36-0.79]; IHC-ER+ 0.55 [0.38-0.79]; GEX-ER+ 0.54 [0.37-0.77]; cytosol-PR+ 0.49 [0.34-0.72]; IHC-PR+ 0.58 [0.40-0.85]; GEX-PR+ 0.55 [0.38-0.80]). Results were similar for OS. INTERPRETATION: These methods can all identify patients that benefit from 2 years of tamoxifen with equal performance, indicating that GEX data might be used to guide adjuvant tamoxifen therapy.

Tamoxifen-predictive value of gene expression signatures in premenopausal breast cancer: data from the randomized SBII:2 trial.

BACKGROUND: Gene expression (GEX) signatures in breast cancer provide prognostic information, but little is known about their predictive value for tamoxifen treatment. We examined the tamoxifen-predictive value and prognostic effects of different GEX signatures in premenopausal women with early breast cancer. METHODS: RNA from formalin-fixed paraffin-embedded tumor tissue from premenopausal women randomized between two years of tamoxifen treatment and no systemic treatment was extracted and successfully subjected to GEX profiling (n = 437, NanoString Breast Cancer 360™ panel). The median follow-up periods for a recurrence-free interval (RFi) and overall survival (OS) were 28 and 33 years, respectively. Associations between GEX signatures and tamoxifen effect were assessed in patients with estrogen receptor-positive/human epidermal growth factor receptor 2-negative (ER+ /HER2-) tumors using Kaplan-Meier estimates and Cox regression. The prognostic effects of GEX signatures were studied in the entire cohort. False discovery rate adjustments (q-values) were applied to account for multiple hypothesis testing. RESULTS: In patients with ER+/HER2- tumors, FOXA1 expression below the median was associated with an improved effect of tamoxifen after 10 years with regard to RFi (hazard ratio [HR]FOXA1(high) = 1.04, 95% CI = 0.61-1.76, HRFOXA1(low) = 0.30, 95% CI = 0.14-0.67, qinteraction = 0.0013), and a resembling trend was observed for AR (HRAR(high) = 1.15, 95% CI = 0.60-2.20, HRAR(low) = 0.42, 95% CI = 0.24-0.75, qinteraction = 0.87). Similar patterns were observed for OS. Tamoxifen was in the same subgroup most beneficial for RFi in patients with low ESR1 expression (HRRFi ESR1(high) = 0.76, 95% CI = 0.43-1.35, HRRFi, ESR1(low) = 0.56, 95% CI = 0.29-1.06, qinteraction = 0.37). Irrespective of molecular subtype, higher levels of ESR1, Mast cells, and PGR on a continuous scale were correlated with improved 10 years RFi (HRESR1 = 0.80, 95% CI = 0.69-0.92, q = 0.005; HRMast cells = 0.74, 95% CI = 0.65-0.85, q < 0.0001; and HRPGR = 0.78, 95% CI = 0.68-0.89, q = 0.002). For BC proliferation and Hypoxia, higher scores associated with worse outcomes (HRBCproliferation = 1.54, 95% CI = 1.33-1.79, q < 0.0001; HRHypoxia = 1.38, 95% CI = 1.20-1.58, q < 0.0001). The results were similar for OS. CONCLUSIONS: Expression of FOXA1 is a promising predictive biomarker for tamoxifen effect in ER+/HER2- premenopausal breast cancer. In addition, each of the signatures BC proliferation, Hypoxia, Mast cells, and the GEX of AR, ESR1, and PGR had prognostic value, also after adjusting for established prognostic factors. Trial registration This trial was retrospectively registered in the ISRCTN database the 6th of December 2019, trial ID: https://clinicaltrials.gov/ct2/show/ISRCTN12474687 .

Clinical and molecular characteristics of estrogen receptor-positive ultralow risk breast cancer tumors identified by the 70-gene signature.

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 (Fisher's 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.

Tumour-infiltrating lymphocytes as a prognostic and tamoxifen predictive marker in premenopausal breast cancer: data from a randomised trial with long-term follow-up.

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 < 50% (HRBCFi 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 .

Raptor localization predicts prognosis and tamoxifen response in estrogen receptor-positive breast cancer.

PURPOSE: 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. METHODS: 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 ERα-positive cell lines was compared with that in ERα-negative cell lines. RESULTS: 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 ERα signaling, here shown by a coupled increase of ERα phosphorylation at S167 and S305 with accumulation of nuclear raptor. In addition, the expression of ERα-activated gene products correlated with nuclear raptor. Similarly, in vitro we observed raptor in the nucleus of ERα-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. CONCLUSION: We present a connection between raptor localization to the nucleus and ERα-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.

Tamoxifen therapy benefit for patients with 70-gene signature high and low risk.

BACKGROUND: 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. METHODS: 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. RESULTS: 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 < 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 < 0.0001). CONCLUSIONS: 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.

Long-term effects on the incidence of second primary cancers in a randomized trial of two and five years of adjuvant tamoxifen.

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 different histological types of lung cancer are needed.

Androgen receptor expression predicts beneficial tamoxifen response in oestrogen receptor-α-negative breast cancer.

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.

Loss of protein tyrosine phosphatase, non-receptor type 2 is associated with activation of AKT and tamoxifen resistance in breast cancer.

Breast cancer is a heterogeneous disease and new clinical markers are needed to individualise disease management and therapy further. Alterations in the PI3K/AKT pathway, mainly PIK3CA mutations, have been shown frequently especially in the luminal breast cancer subtypes, suggesting a cross-talk between ER and PI3K/AKT. Aberrant PI3K/AKT signalling has been connected to poor response to anti-oestrogen therapies. In vitro studies have shown protein tyrosine phosphatase, non-receptor type 2 (PTPN2) as a previously unknown negative regulator of the PI3K/AKT pathway. Here, we evaluate possible genomic alterations in the PTPN2 gene and its potential as a new prognostic and treatment predictive marker for endocrine therapy benefit in breast cancer. PTPN2 gene copy number was assessed by real-time PCR in 215 tumour samples from a treatment randomised study consisting of postmenopausal patients diagnosed with stage II breast cancer 1976-1990. Corresponding mRNA expression levels of PTPN2 were evaluated in 86 available samples by the same methodology. Gene copy loss of PTPN2 was detected in 16% (34/215) of the tumours and this was significantly correlated with lower levels of PTPN2 mRNA. PTPN2 gene loss and lower mRNA levels were associated with activation of AKT and a poor prognosis. Furthermore, PTPN2 gene loss was a significant predictive marker of poor benefit from tamoxifen treatment. In conclusion, genomic loss of PTPN2 may be a previously unknown mechanism of PI3K/AKT upregulation in breast cancer. PTPN2 status is a potential new clinical marker of endocrine treatment benefit which could guide further individualised therapies in breast cancer.

C-X-C ligand 10 and C-X-C receptor 3 status can predict tamoxifen treatment response in breast cancer patients.

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 < 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.

Lack of G protein-coupled estrogen receptor (GPER) in the plasma membrane is associated with excellent long-term prognosis in breast cancer.

G protein-coupled estrogen receptor (GPER), or GPR30, is a membrane receptor reported to mediate non-genomic estrogen responses. Tamoxifen is a partial agonist at GPER in vitro. Here, we investigated if GPER expression is prognostic in primary breast cancer, if the receptor is treatment-predictive for adjuvant tamoxifen, and if receptor subcellular localization has any impact on the prognostic value. Total and plasma membrane (PM) GPER expression was analyzed by immunohistochemistry in breast tumors from 742 postmenopausal lymph node-negative patients subsequently randomized for tamoxifen treatment for 2-5 years versus no systemic treatment, regardless of estrogen receptor (ER) status, and with a median follow-up of 17 years for patients free of event. PM GPER expression was a strong independent prognostic factor for poor prognosis in breast cancer without treatment-predictive information for tamoxifen. In the tamoxifen-treated ER-positive and progesterone receptor (PgR)-positive patient subgroup, the absence of PM GPER (53 % of all ER-positive tumors) predicted 91 % 20-year distant disease-free survival, compared to 73 % in the presence of GPER (p = 0.001). Total GPER expression showed positive correlations with ER and PgR and negative correlation with histological grade, but the correlations were biphasic. On the other hand, PM GPER expression showed strong negative correlations with ER and PgR, and strong positive correlation with HER2 overexpression and high histological grade. GPER overexpression and PM localization are critical events in breast cancer progression, and lack of GPER in the PM is associated with excellent long-term prognosis in ER-positive and PgR-positive tamoxifen-treated primary breast cancer.

The mTOR effectors 4EBP1 and S6K2 are frequently coexpressed, and associated with a poor prognosis and endocrine resistance in breast cancer: a retrospective study including patients from the randomised Stockholm tamoxifen trials.

INTRODUCTION: mTOR and its downstream effectors the 4E-binding protein 1 (4EBP1) and the p70 ribosomal S6 kinases (S6K1 and S6K2) are frequently upregulated in breast cancer, and assumed to be driving forces in tumourigenesis, in close connection with oestrogen receptor (ER) networks. Here, we investigated these factors as clinical markers in five different cohorts of breast cancer patients. METHODS: The prognostic significance of 4EBP1, S6K1 and S6K2 mRNA expression was assessed with real-time PCR in 93 tumours from the treatment randomised Stockholm trials, encompassing postmenopausal patients enrolled between 1976 and 1990. Three publicly available breast cancer cohorts were used to confirm the results. Furthermore, the predictive values of 4EBP1 and p4EBP1_S65 protein expression for both prognosis and endocrine treatment benefit were assessed by immunohistochemical analysis of 912 node-negative breast cancers from the Stockholm trials. RESULTS: S6K2 and 4EBP1 mRNA expression levels showed significant correlation and were associated with a poor outcome in all cohorts investigated. 4EBP1 protein was confirmed as an independent prognostic factor, especially in progesterone receptor (PgR)-expressing cancers. 4EBP1 protein expression was also associated with a poor response to endocrine treatment in the ER/PgR positive group. Cross-talk to genomic as well as non-genomic ER/PgR signalling may be involved and the results further support a combination of ER and mTOR signalling targeted therapies. CONCLUSION: This study suggests S6K2 and 4EBP1 as important factors for breast tumourigenesis, interplaying with hormone receptor signalling. We propose S6K2 and 4EBP1 as new potential clinical markers for prognosis and endocrine therapy response in breast cancer.

Activation of Akt, mTOR, and the estrogen receptor as a signature to predict tamoxifen treatment benefit.

The frequent alterations of the PI3K/Akt/mTOR-growth signaling pathway are proposed mechanisms for resistance to endocrine therapy in breast cancer, partly through regulation of estrogen receptor α (ER) activity. Reliable biomarkers for treatment prediction are required for improved individualized treatment. We performed a retrospective immunohistochemical analysis of primary tumors from 912 postmenopausal patients with node-negative breast cancer, randomized to either tamoxifen or no adjuvant treatment. Phosphorylated (p) Akt-serine (s) 473, p-mTOR-s2448, and ER phosphorylations-s167 and -s305 were evaluated as potential biomarkers of prognosis and tamoxifen treatment efficacy. High expression of p-mTOR indicated a reduced response to tamoxifen, most pronounced in the ER+/progesterone receptor (PgR) + subgroup (tamoxifen vs. no tamoxifen: hazard ratio (HR), 0.86; 95 % confidence interval (CI), 0.31-2.38; P = 0.78), whereas low p-mTOR expression predicted tamoxifen benefit (HR, 0.29; 95 % CI, 0.18-0.49; P = 0.000002). In addition, nuclear p-Akt-s473 as well as p-ER at -s167 and/or -s305 showed interaction with tamoxifen efficacy with borderline statistical significance. A combination score of positive pathway markers including p-Akt, p-mTOR, and p-ER showed significant association with tamoxifen benefit (test for interaction; P = 0.029). Cross-talk between growth signaling pathways and ER-signaling has been proposed to affect tamoxifen response in hormone receptor-positive breast cancer. The results support this hypothesis, as an overactive pathway was significantly associated with reduced response to tamoxifen. A clinical pre-treatment test for cross-talk markers would be a step toward individualized adjuvant endocrine treatment with or without the addition of PI3K/Akt/mTOR pathway inhibitors.

Effects of adjuvant tamoxifen therapy on cardiac disease: results from a randomized trial with long-term follow-up.

Tamoxifen is associated with a reduced risk of coronary heart disease (CHD). However, there are few reports on long-term effects. Using data from a large Swedish randomized trial of 5 and 2 years of adjuvant tamoxifen in women with early breast cancer, we here present results on morbidity and mortality from cardiac diseases during treatment and long-term after treatment. A total of 4,150 patients were breast cancer recurrence-free after 2 years. Data from the Swedish National Hospital Discharge Registry combined with information from the Swedish Cause of Death Registry were used to define events of disease. Hazard ratios were estimated using Cox regression. Patients assigned to 5 years in comparison with 2 years of postoperative tamoxifen experienced a reduced incidence of CHD [hazard ratio (HR), 0.83; 95 % CI 0.70-1.00], especially apparent during the active treatment period (HR 0.65; 95 % CI 0.43-1.00). The mortality from CHD was significantly reduced (HR 0.72; 95 % CI 0.53-0.97). During the active treatment, the morbidity of other heart diseases was also significantly reduced (HR 0.40; 95 % CI 0.25-0.64) but not after treatment stopped (HR 1.06; 95 % CI 0.87-1.30). Similar results were seen for both heart failure and atrial fibrillation/flutter. As compared to 2 years of therapy, 5 years of postoperative tamoxifen therapy prevents CHD as well as other heart diseases. The risk reduction is most apparent during the active treatment period, and later tends to diminish.

Breast cancer survival and incidence of second primary cancers after 30 years in a randomized study of two versus five years of adjuvant tamoxifen therapy.

BACKGROUND: Tamoxifen is an established treatment for breast cancer, but its long-term effects on survival and on secondary cancers are not fully evaluated. MATERIAL AND METHODS: We studied 30 years outcome of 4124 postmenopausal patients who were randomized to receive (totally) two or five years of adjuvant tamoxifen. RESULTS: After 5 years of follow-up, when tamoxifen treatment was finished in both groups, until 15 years of follow-up, overall mortality (HR 0.80, 95% CI 0.72-0.90, p < 0.001), breast cancer mortality for all patients (HR 0.80, 95% CI 0.68-0.94, p = 0.006) and breast cancer mortality for patients with estrogen receptor positive disease (HR 0.67, 95% CI 0.55-0.83, p < 0.001) were significantly reduced in the five-year group as compared to the two-year group. After 15 years, the difference remained but did not further increase. In the five-year group, the incidence of contralateral breast cancer was gradually reduced during the entire period of observation. The incidence of lung cancer was also reduced in the five-year group. In contrast there was an increased endometrial cancer incidence in the five-year group and for those receiving 40 mg of tamoxifen this incidence was further increased. CONCLUSION: Three more years of tamoxifen therapy reduced the risk of breast cancer mortality. The difference was established during the first 15 years after randomization. Moreover, the incidence of contralateral breast cancer gradually decreased for 30 years. The incidence of lung cancer was reduced in the five-year group. In contrast the incidence of endometrial cancer was increased.

PAM50 subtyping and ROR score add long-term prognostic information in premenopausal breast cancer patients.

PAM50 intrinsic subtyping and risk of recurrence (ROR) score are approved for risk profiling in postmenopausal women. We aimed to examine their long-term prognostic value in terms of breast cancer-free interval (BCFi) and overall survival (OS) (n = 437) in premenopausal women randomised to 2 years of tamoxifen versus no systemic treatment irrespective of hormone-receptor status. Intrinsic subtyping added independent prognostic information in patients with oestrogen receptor-positive/human epidermal growth factor 2-negative tumours for BCFi and OS after maximum follow-up (overall P-value 0.02 and 0.006, respectively) and those with high versus low ROR had worse prognosis (maximum follow-up: hazard ratio (HR)BCFi: 1.70, P = 0.04). The prognostic information by ROR was similar regarding OS and in multivariable analysis. These results support that PAM50 subtyping and ROR score provide long-term prognostic information in premenopausal women. Moreover, tamoxifen reduced the incidence of breast cancer events only in patients with Luminal APAM50 tumours (0-10 years: HRBCFi(Luminal A): 0.41, HRBCFi(Luminal B): 1.19, Pinteraction = 0.02).Trial registration: This trial is registered in the ISRCTN database, trial ID: ISRCTN12474687.

Twenty-Year Benefit From Adjuvant Goserelin and Tamoxifen in Premenopausal Patients With Breast Cancer in a Controlled Randomized Clinical Trial.

PURPOSE: To assess the long-term (20-year) endocrine therapy benefit in premenopausal patients with breast cancer. METHODS: 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. RESULTS: 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 (P = .016). Genomic low-risk patients (n = 305) significantly benefitted from tamoxifen (HR, 0.24; 95% CI, 0.10 to 0.60), and genomic high-risk patients (n = 158) from goserelin (HR, 0.24; 95% CI, 0.10 to 0.54). Increased risk from the addition of tamoxifen to goserelin was seen in genomic high-risk patients (HR, 3.36; 95% CI, 1.39 to 8.07). Moreover, long-lasting 20-year tamoxifen benefit was seen in genomic low-risk patients, whereas genomic high-risk patients had early goserelin benefit. CONCLUSION: 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.

Clinical potential of the mTOR targets S6K1 and S6K2 in breast cancer.

The mammalian target of rapamycin (mTOR) and its substrates S6K1 and S6K2 regulate cell growth, proliferation, and metabolism through translational control. RPS6KB1 (S6K1) and RPS6KB2 (S6K2) are situated in the commonly amplified 17q21-23 and 11q13 regions. S6K1 amplification and protein overexpression have earlier been associated with a worse outcome in breast cancer, but information regarding S6K2 is scarce. The aim of this study was to evaluate the prognostic and treatment predictive relevance of S6K1/S6K2 gene amplification, as well as S6K2 protein expression in breast cancer. S6K1/S6K2 gene copy number was determined by real-time PCR in 207 stage II breast tumors and S6K2 protein expression was investigated by immunohistochemistry in 792 node-negative breast cancers. S6K1 amplification/gain was detected in 10.7%/21.4% and S6K2 amplification/gain in 4.3%/21.3% of the tumors. S6K2 protein was detected in the nucleus (38%) and cytoplasm (76%) of the tumor cells. S6K1 amplification was significantly associated with HER2 gene amplification and protein expression. S6K2 amplification correlated significantly with high S6K2 mRNA levels, ER+ status and CCND1 amplification. S6K1 and S6K2 gene amplification was associated with a worse prognosis independent of HER2 and CCND1. S6K2 gain and nuclear S6K2 expression was related to an improved benefit from tamoxifen among patients with ER+, respectively ER+/PgR+ tumors. In the ER+/PgR- subgroup, nuclear S6K2 rather indicated decreased tamoxifen responsiveness. S6K1 amplification predicted reduced benefit from radiotherapy. This is the first study showing that S6K2 amplification and overexpression, like S6K1 amplification, have prognostic and treatment predictive significance in breast cancer.

Immunohistochemistry compared to cytosol assays for determination of estrogen receptor and prediction of the long-term effect of adjuvant tamoxifen.

The purpose of this study is to compare immunohistochemistry (IHC) and cytosol-based assays for determination of estrogen receptor (ER) and prediction of response to adjuvant tamoxifen treatment in postmenopausal women with early-stage invasive breast cancer. The Stockholm Breast Cancer Study Group conducted a randomized trial during 1976 through 1990 comparing adjuvant tamoxifen versus control. The patients were stratified according to tumor size and lymph node status in high-risk and low-risk groups. In this study we evaluated 683 patients with "low risk" breast cancer (size ≤30 mm, lymph node-negative) for whom ER status had been determined by both the cytosol assays and IHC at one pathology laboratory. The median follow-up was 17 years. Six hundred eighty-three patients had tumors with ER determined by both methods, 536 (78.5%) were ER-positive by cytosol assays using the cutoff level at ≥0.05 fmol/µg DNA and 539 patients were ER-positive (79%) by IHC using the cutoff level at ≥10% cell stained. Thirty-nine tumors (5.7%) were ER-positive by cytosol but not by IHC, whereas the opposite pattern was found for 42 cases (6.1%). Only seven tumors had stained cells between 0 and 9% by IHC. The concordance between IHC and cytosol assays was high (88%). The kappa statistic was 0.65, 95% CI 0.58-0.72. Among patients classified as ER-negative no therapeutic benefit from tamoxifen was observed. Among patients with ER-expressing tumors, tamoxifen resulted in significantly better recurrence-free survival irrespective of the method (IHC: HR, 0.53, P < 0.001; cytosol: HR, 0.53, P < 0.001). The effect on overall survival was not statistically significant probably due to the limited sample size. Both IHC and cytosol assay accurately predict long-term response to adjuvant tamoxifen.