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 .

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.

Long-term follow-up of early stage HER2-positive breast cancer patients treated with trastuzumab: A population-based real world multicenter cohort study.

Introduction: Since its introduction in standard of care, trastuzumab has revolutionized the treatment of patients with early and late stages of HER2-positive breast cancer. While the initial clinical trials were convincing and lead to major changes in practice, more knowledge on the long-term outcome and tolerability is needed. The present study was designed to assess the survival, prognostic factors and relapse patterns after the implementation of trastuzumab in a real-world cohort. Methods: All cases of HER2-positive breast cancer diagnosed between 2006 and 2014 in the Southeast Healthcare Region of Sweden were retrospectively identified. Medical records were thoroughly reviewed with regard to clinicopathological parameters, treatments, relapse pattern and adverse events. Results: 643 patients were identified and 599 were eligible for analysis. Breast cancer specific survival, distant recurrence free survival and local recurrence free survival were 93.4%, 89.7% and 98.0% for trastuzumab treated patients and 87.4%, 81.6% and 87.4% in patients not treated with trastuzumab, respectively. ER status, nodal status and trastuzumab treatment were all independent prognostic factors in multivariable analysis. No new safety concerns were discovered. Conclusion: The real-world outcome of trastuzumab-treated patients with early HER2-positive breast cancer is similar to what has been previously reported in long-term follow up of prospective clinical trials. ER status, nodal status and trastuzumab treatment are independent prognostic factors for breast cancer specific mortality rate, distant recurrence rate and locoregional recurrence rate in HER2-positive patients in the trastuzumab era.

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.

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.

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.

Assessment of 25-Year Survival of Women With Estrogen Receptor-Positive/ERBB2-Negative Breast Cancer Treated With and Without Tamoxifen Therapy: A Secondary Analysis of Data From the Stockholm Tamoxifen Randomized Clinical Trial.

Importance: 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. Objective: 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. Design, Setting, and Participants: 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. Interventions: 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. Main Outcomes and Measures: 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. Results: 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. Conclusions and Relevance: 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.

IP6K2 predicts favorable clinical outcome of primary breast cancer.

The inositol hexakisphosphate kinase (IP6K) 1 and 2 genes are localized at 3p21.31, a highly altered gene-dense chromosomal region in cancer. The IP6Ks convert IP6 to IP7, which inhibits activation of the tumor-promoting PI3K/Akt/mTOR signaling pathway. IP6K2 has been suggested to be involved in p53-induced apoptosis, while IP6K1 may stimulate tumor growth and migration. The present study aimed to elucidate the role of the two IP6Ks in predicting outcome in patients with breast cancer. To the best of our knowledge, the role of IP6K was analyzed for the first time in tumors from three cohorts of patients with breast cancer; one Swedish low-risk cohort, one Dutch cohort and the TCGA dataset. Analyses of gene -and protein expression and subcellular localization were included. IP6K2 gene expression was associated with ER positivity and nuclear p-Akt. Improved prognosis was detected with high IP6K2 gene expression compared with low IP6K2 gene expression in systemically untreated patients in the Swedish low-risk and Dutch cohorts. In the TCGA dataset, IP6K2 prognostic value was significant when selecting for tumors with wild-type TP53. A multivariable analysis testing IP6K2 against other cancer-related genes at 3p.21.31, including IP6K1 and clinical biomarkers, revealed that IP6K2 was associated with decreased risk of distant recurrence. IP6K1 was associated with increased risk of distant recurrence in the multivariable test and protein analysis revealed trends of worse prognosis with high IP6K1 in the cytoplasm. The expression levels of IP6K1 and IP6K2 were associated to a high extent; however, a diverging prognostic value of the two genes was observed in breast cancer. The present data suggest that IP6K2 can be a favorable prognostic factor, while IP6K1 may not be.

Tumor co-expression of progranulin and sortilin as a prognostic biomarker in breast cancer.

BACKGROUND: The growth factor progranulin has been implicated in numerous biological processes such as wound healing, inflammation and progressive tumorigenesis. Both progranulin and its receptor sortilin are known to be highly expressed in subgroups of breast cancer and have been associated with various clinical properties including tamoxifen resistance. Recent data further suggest that progranulin, via its receptor sortilin, drives breast cancer stem cell propagation in vitro and increases metastasis formation in an in vivo breast cancer xenograft model. In this retrospective biomarker analysis, we aimed to determine whether tumor co-expression of progranulin and sortilin has prognostic and treatment predictive values for breast cancer patients. METHODS: We explored how co-expression of progranulin and sortilin was associated with established clinical markers by analyzing a tissue microarray including 560 randomized premenopausal breast cancer patients receiving either 2 years of tamoxifen treatment or no adjuvant treatment, with a median follow-up time of 28 years. Breast cancer-specific survival was analyzed using Kaplan-Meier and Cox Proportional Hazards regression models to assess the prognostic and predictive value of progranulin and sortilin in relation to known clinical markers. RESULTS: Co-expression of progranulin and sortilin was observed in 20% of the breast cancer samples. In untreated patients, prognostic considerations could be detailed separately from treatment prediction and the high progranulin and sortilin expressing subgroup was significantly associated with breast cancer-specific death in multivariable analyses (HR=2.188, CI: 1.317-3.637, p=0.003) along with tumor size, high tumor grade and lymph node positivity. When comparing the untreated patients with tamoxifen treated patients in the ERα positive subgroup, co-expression of progranulin and sortilin was not linked to tamoxifen resistance. CONCLUSION: Data suggest that co-expression of progranulin and its receptor sortilin is a novel prognostic biomarker combination identifying a highly malignant subgroup of breast cancer. Importantly, this subpopulation could potentially be targeted with anti-sortilin based therapies.

MCM3 upregulation confers endocrine resistance in breast cancer and is a predictive marker of diminished tamoxifen benefit.

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(Ser315,33), CHK1(Ser317), and cdc25b(Ser323), 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.

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 .

Expression of HGF, pMet, and pAkt is related to benefit of radiotherapy after breast-conserving surgery: a long-term follow-up of the SweBCG91-RT randomised trial.

Experimental studies suggest that hepatocyte growth factor (HGF) and its transmembrane tyrosine kinase receptor, Met, in part also relying on Akt kinase activity, mediate radioresistance. We investigated the importance of these biomarkers for the risk of ipsilateral breast tumour recurrence (IBTR) after adjuvant radiotherapy (RT) in primary breast cancer. HGF, phosphorylated Met (pMet) and phosphorylated Akt (pAkt) were evaluated immunohistochemically on tissue microarrays from 1004 patients in the SweBCG91-RT trial, which randomly assigned patients to breast-conserving therapy, with or without adjuvant RT. HGF was evaluated in the stroma (HGFstr ); pMet in the membrane (pMetmem ); HGF, pMet and pAkt in the cytoplasm (HGFcyt , pMetcyt , pAktcyt ); and pAkt in the nucleus (pAktnuc ). The prognostic and treatment predictive effects were evaluated to primary endpoint IBTR as first event during the first 5 years. Patients with tumours expressing low levels of HGFcyt and pMetcyt and high levels of pAktnuc derived a larger benefit from RT [hazard ratio (HR): 0.11 (0.037-0.30), 0.066 (0.016-0.28) and 0.094 (0.028-0.31), respectively] compared to patients with high expression of HGFcyt and pMetcyt , and low pAktnuc [HR: 0.36 (0.19-0.67), 0.35 (0.20-0.64) and 0.47 (0.32-0.71), respectively; interaction analyses: P = 0.052, 0.035 and 0.013, respectively]. These differences remained in multivariable analysis when adjusting for patient age, tumour size, histological grade, St Gallen subtype and systemic treatment (interaction analysis, P-values: 0.085, 0.027, and 0.023, respectively). This study suggests that patients with immunohistochemically low HGFcyt , low pMetcyt and high pAktnuc may derive an increased benefit from RT after breast-conserving surgery concerning the risk of developing IBTR.

Low RAB6C expression is a predictor of tamoxifen benefit in estrogen receptor-positive/progesterone receptor-negative breast cancer.

Over the last few decades, improved and more individualized treatment has contributed to the increased survival rate of patients with breast cancer. However, certain patients may receive excessive treatment resulting in undesired side effects. In a previous study, it was demonstrated that systemically untreated patients with estrogen receptor (ER)-positive/progesterone receptor (PR)-negative tumors with high Ras-related protein Rab-6C (RAB6C) expression levels (RAB6C+) had prolonged distant recurrence-free survival compared with that of patients exhibiting low RAB6C (RAB6C-)-expressing tumors. The aim of the present study was to investigate whether RAB6C predicts the effectiveness of tamoxifen treatment. The present study used a dataset comprising 486 female patients with ER+ tumors from a randomized study conducted by the Stockholm Breast Cancer Study Group between November 1976 and August 1990. The patients were considered as low-risk if their tumor size was ≤30 mm and their lymph node status was negative. Patients were followed up until distant recurrence, mortality or when 25 years after randomization was achieved, whichever occurred first. For patients with ER+/PR-/RAB6C+ tumors, prolonged distant recurrence-free survival could not be observed if the patients were treated with tamoxifen [hazard ratio (HR), 1.82; 95% confidence interval (CI), 0.69-4.79; P=0.23], whereas patients with ER+/PR-/RAB6C- tumors had 75% reduced distant recurrence risk (HR, 0.25; 95% CI, 0.09-0.70; P=0.008). In the ER+/PR+ subgroup, patients with RAB6C- and RAB6C+ tumors benefited from tamoxifen treatment, though it was most evident in the RAB6C+ group (HR, 0.27; 95% CI, 0.13-0.58; P=0.001). The results of the present study indicated that, for patients with ER+/PR- tumors, those with low RAB6C expression benefited from tamoxifen treatment, whereas no benefit was observed in patients with high RAB6C levels.

RAB6C is an independent prognostic factor of estrogen receptor-positive/progesterone receptor-negative breast cancer.

The majority of breast cancer tumors are estrogen receptor-positive (ER+) and can be treated with endocrine therapy. However, certain patients may exhibit a good prognosis without systemic treatment. The aim of the present study was to identify novel prognostic factors for patients with ER+ breast cancer tumors using gene copy data, and to investigate if these factors have prognostic value in subgroups categorized by progesterone receptor status (PR). Public data, including the whole genome gene copy data of 199 systemically untreated patients with ER+ tumors, were utilized in the present study. To assess prognostic value, patients were divided into two groups using the median gene copy number as a cut-off for the SNPs that were the most variable. One SNP was identified, which indicated that the Ras-related protein Rab-6C (RAB6C) gene may exhibit prognostic significance. Therefore, RAB6C protein expression was subsequently investigated in a second independent cohort, consisting of 469 systematically untreated patients (of which 310 were ER+) who received long term follow-up. In the public data set, a distant recurrence risk reduction of 55% was determined for copy numbers above the median value of RAB6C compared with numbers below [multivariable adjusted hazard ratio (HR), 0.45; 95% CI 0.28-0.72; P=0.001)]. It was also more pronounced in the ER+/PR- subgroup (HR, 0.15; 95% CI, 0.05-0.46; P=0.001). In the second cohort, patients of the ER+/PR- subgroup who exhibited high RAB6C expression had a reduced distant recurrence risk (HR, 0.17; 95% CI, 0.05-0.60; P=0.006). However, this was not identified among ER+/PR+ tumors (HR, 1.31; 95% CI, 0.69-2.48; P=0.41). The results of the present study indicated that RAB6C serves as an independent prognostic factor of distant recurrence risk in systemically untreated patients with an ER+/PR- tumor.

Assessment of Long-term Distant Recurrence-Free Survival Associated With Tamoxifen Therapy in Postmenopausal Patients With Luminal A or Luminal B Breast Cancer.

IMPORTANCE: 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. OBJECTIVE: To compare the long-term survival from tamoxifen therapy for patients with luminal A or luminal B tumor subtype. DESIGN, SETTING, AND PARTICIPANTS: 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. INTERVENTIONS: 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. MAIN OUTCOMES AND MEASURES: 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. RESULTS: 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). CONCLUSIONS AND RELEVANCE: 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.

The effects of PTPN2 loss on cell signalling and clinical outcome in relation to breast cancer subtype.

PURPOSE: The 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. METHODS: Protein 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. RESULTS: Low 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. CONCLUSION: We 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.

ERBB2 and PTPN2 gene copy numbers as prognostic factors in HER2-positive metastatic breast cancer treated with trastuzumab.

Trastuzumab has markedly improved the treatment and long-term prognosis of patients with HER2-positive breast cancer. A frequent clinical challenge in patients with relapsing and/or metastatic disease is de novo or acquired trastuzumab resistance, and to date no predictive biomarkers for palliative trastuzumab have been established. In the present study, the prognostic values of factors involved in the HER2-associated PI3K/Akt signalling pathway were explored. The first 46 consecutive patients treated at the Department of Oncology, Linköping University Hospital between 2000 and 2007 with trastuzumab for HER2-positive metastatic breast cancer were retrospectively included. The gene copy number variation and protein expression of several components of the PI3K/Akt pathway were assessed in the tumour tissue and biopsy samples using droplet digital polymerase chain reaction and immunohistochemistry. Patients with tumours displaying a high-grade ERBB2 (HER2) amplification level of ≥6 copies had a significantly improved overall survival hazard ratio [(HR)=0.4; 95%, confidence interval (CI): 0.2-0.9] and progression-free survival (HR=0.3; 95% CI: 0.1-0.7) compared with patients with tumours harbouring fewer ERBB2 copies. High-grade ERBB2 amplification was significantly associated with the development of central nervous system metastases during palliative treatment. Copy gain (≥3 copies) of the gene encoding the tyrosine phosphatase PTPN2 was associated with a shorter overall survival (HR=2.0; 95% CI: 1.0-4.0) and shorter progression-free survival (HR=2.1; 95% CI: 1.0-4.1). In conclusion, high ERBB2 amplification level is a potential positive prognostic factor in trastuzumab-treated HER2-positive metastatic breast cancer, whereas PTPN2 gain is a potential negative prognostic factor. Further studies are warranted on the role of PTPN2 in HER2 signalling.

PTPN2 deficiency along with activation of nuclear Akt predict endocrine resistance in breast cancer.

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.

Non-linear transformations of age at diagnosis, tumor size, and number of positive lymph nodes in prediction of clinical outcome in breast cancer.

BACKGROUND: Prognostic factors in breast cancer are often measured on a continuous scale, but categorized for clinical decision-making. The primary aim of this study was to evaluate if accounting for continuous non-linear effects of the three factors age at diagnosis, tumor size, and number of positive lymph nodes improves prognostication. These factors will most likely be included in the management of breast cancer patients also in the future, after an expected implementation of gene expression profiling for adjuvant treatment decision-making. METHODS: Four thousand four hundred forty seven and 1132 women with primary breast cancer constituted the derivation and validation set, respectively. Potential non-linear effects on the log hazard of distant recurrences of the three factors were evaluated during 10 years of follow-up. Cox-models of successively increasing complexity: dichotomized predictors, predictors categorized into three or four groups, and predictors transformed using fractional polynomials (FPs) or restricted cubic splines (RCS), were used. Predictive performance was evaluated by Harrell's C-index. RESULTS: Using FP-transformations, non-linear effects were detected for tumor size and number of positive lymph nodes in univariable analyses. For age, non-linear transformations did, however, not improve the model fit significantly compared to the linear identity transformation. As expected, the C-index increased with increasing model complexity for multivariable models including the three factors. By allowing more than one cut-point per factor, the C-index increased from 0.628 to 0.674. The additional gain, as measured by the C-index, when using FP- or RCS-transformations was modest (0.695 and 0.696, respectively). The corresponding C-indices for these four models in the validation set, based on the same transformations and parameter estimates from the derivation set, were 0.675, 0.700, 0.706, and 0.701. CONCLUSIONS: Categorization of each factor into three to four groups was found to improve prognostication compared to dichotomization. The additional gain by allowing continuous non-linear effects modeled by FPs or RCS was modest. However, the continuous nature of these transformations has the advantage of making it possible to form risk groups of any size.