Julia Tutzauer

Background: Patients with estrogen receptor–positive breast cancer have a substantial late risk of distant recurrence, but the long-term subtype-specific tamoxifen benefit remains poorly understood. Methods: A secondary analysis of the Stockholm Tamoxifen randomized trials (1976-1997, n = 3930) with 20-year follow-up was conducted. Formalin-fixed, paraffin-embedded blocks were available for 2250 patients. A total of 952 patients with estrogen receptor–positive and HER2-negative tumors, classified as luminal A (n = 688) and luminal B (n = 264) using Agilent microarrays, were analyzed. Patients were randomly assigned to at least 2 years of tamoxifen therapy or no endocrine therapy. Distant recurrence-free interval was assessed by Kaplan–Meier analysis and multivariable Cox proportional hazards regression. Results: Patients with luminal A tumors had low early risk and modest early benefit from tamoxifen therapy (5-year distant recurrence-free interval treated vs control: 93% vs 89%; absolute difference = 4%) that increased over the 20-year follow-up (20-year distant recurrence-free interval: 76% vs 66%; absolute difference = 10%), highlighting long-term benefit. In contrast, patients with luminal B tumors had larger early risk and treatment benefit (5-year distant recurrence-free interval: 72% vs 57%; absolute difference = 15%), which remained stable over time (20-year distant recurrence-free interval: 55% vs 37%; absolute difference = 18%). Multivariable analyses showed that luminal patients benefited from tamoxifen therapy (luminal A: adjusted hazard ratio [HR] = 0.57, 95% confidence interval [CI] = 0.42 to 0.78; luminal B: adjusted HR = 0.68, 95% CI = 0.46 to 0.99). Patients with favorable tumor characteristics benefited regardless of luminal subtype. Conclusions: Tamoxifen reduces the long-term risk of distant recurrence in luminal A and B tumors, although timing of benefit varies by subtype. Even after treatment, patients with luminal tumors have a substantial late risk, highlighting the need for long-term follow-up. Clinical Trial Registration: The trial center for the Stockholm Tamoxifen trials was the Regional Cancer Center Stockholm-Gotland, in Stockholm, Sweden. The start of the Stockholm Tamoxifen trials in 1976 was before the custom of trial registration started in Sweden, therefore no trial number is available.

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.

Background: Metastatic breast cancer (MBC) is the main cause of breast cancer-related death. The outcome of MBC varies, and there is a lack of biomarkers to aid in prognostication. The primary aim of this study was to evaluate the prognostic value of gene expression (GEX) signatures in the primary tumor (PT) and distant metastasis (DM) for progression-free survival (PFS) and overall survival (OS). The secondary aim was to describe GEX changes through MBC evolution and to identify MBC subtypes.Methods: RNA was extracted from the PT, lymph node metastasis (LNM), and DM from MBC patients in a prospective observational study (n = 142; CTC-MBC NCT01322893) and was subjected to GEX analysis retrospectively using the NanoString Breast Cancer 360™ panel. 31 continuous GEX variables in DMs and PTs were analyzed for PFS and OS by Cox regression analysis and Kaplan-Meier estimates. Multivariable Cox regressions were adjusted for number of DM sites and CTCs, visceral metastasis, ECOG status, age at MBC diagnosis and, in additional analyses, PAM50 subtype. Differential GEX analyses and Euclidean distances were used to describe subgroup differences and visualize within-patient heterogeneity.Results: Compared to DM GEX, GEX of the PT was at least equally useful for predicting MBC outcome. The strongest marker for a favorable PFS, both when expressed in the PT and the DM was AR, even after adjustment for prognostic markers including PAM50. GEX signatures related to hormone responsiveness, including ESR1, FOXA1, PGR, and AR were favorable prognostic markers, and the p53 signature was unfavorable for PFS when expressed in PT or DM. The previously published PAM50MET signature was prognostic for both PFS and OS. We established five distinct DM GEX profiles where two associated with liver and bone metastases, respectively. Finally, we identified four DM GEX profiles able to identify MBCs with poor OS in this cohort.Conclusion: GEX of both DM and PT are useful in MBC prognostication. GEX of AR adds prognostic information for MBC. Our descriptive analyses illuminate the biological differences between MBCs in relation to outcome and metastatic site.

Background Metastatic breast cancer (MBC) is the main cause of breast cancer-related death. The outcome of MBC varies, and there is a lack of biomarkers to aid in prognostication. The primary aim of this study was to evaluate the prognostic value of gene expression (GEX) signatures in the primary tumor (PT) and distant metastasis (DM) for progression-free survival (PFS) and overall survival (OS). The secondary aim was to describe GEX changes through MBC evolution and to identify MBC subtypes. Methods RNA was extracted from the PT, lymph node metastases (LNM), and DM from MBC patients in a prospective observational study (n = 142; CTC-MBC NCT01322893) and was subjected to GEX analysis retrospectively using the NanoString Breast Cancer 360™ panel. 31 continuous GEX variables in DMs and PTs were analyzed for PFS and OS by Cox regression analysis and Kaplan-Meier estimates. Multivariable Cox regressions were adjusted for number of DM sites and CTCs, visceral metastasis, ECOG status, age at MBC diagnosis and, in additional analyses, PAM50 subtype. Differential GEX analyses and Euclidean distances were used to describe subgroup differences and visualize within-patient heterogeneity. Results Compared to DM GEX, GEX of the PT was at least equally useful for prognosticating MBC outcome. The strongest marker for a favorable PFS, both when expressed in the PT and the DM was AR, even after adjustment for prognostic markers including PAM50. GEX signatures related to hormone responsiveness, including ESR1, FOXA1, PGR, and AR were favorable prognostic markers, and the p53 signature was unfavorable for PFS when expressed in PT or DM. The previously published PAM50MET signature was prognostic for both PFS and OS. We established five distinct DM GEX profiles where two associated with liver and bone metastases, respectively. Finally, we identified four DM GEX profiles able to identify MBCs with poor OS in this cohort. Conclusion GEX of both DM and PT are useful in MBC prognostication. GEX of AR adds prognostic information for MBC. Our descriptive analyses illuminate the biological differences between MBCs in relation to outcome and metastatic site.

Background 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 randomized tamoxifen trial, to determine if any method is superior at identifying patients that benefit from tamoxifen. Methods Premenopausal patients (n = 564) were randomized to two 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+) tumors (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. Conclusion Cytosol-based methods, IHC, and GEX analysis can all identify patients that benefit from two years of tamoxifen with equal performance, indicating that GEX data might be used to guide adjuvant tamoxifen therapy. The trial is registered on ISRCTN: ISRCTN12474687

Background: Breast-conserving surgery followed by radiotherapy is part of standard treatment for early-stage breast cancer. Hypoxia is common in cancer and may affect the benefit of radiotherapy. Cells adapt to hypoxic stress largely via the transcriptional activity of hypoxia-inducible factor (HIF)-1α. Here, we aim to determine whether tumour HIF-1α-positivity and hypoxic gene-expression signatures associated with the benefit of radiotherapy, and outcome. Methods: Tumour HIF-1α-status and expression of hypoxic gene signatures were retrospectively analysed in a clinical trial where 1178 women with primary T1-2N0M0 breast cancer were randomised to receive postoperative radiotherapy or not and followed 15 years for recurrence and 20 years for breast cancer death. Results: The benefit from radiotherapy was similar in patients with HIF-1α-positive and -negative primary tumours. Both ipsilateral and any breast cancer recurrence were more frequent in women with HIF-1α-positive primary tumours (hazard ratio, HR_{0–5 yrs}1.9 [1.3–2.9], p = 0.003 and HR_{0–5 yrs} = 2.0 [1.5–2.8], p < 0.0001). Tumour HIF-1α-positivity is also associated with increased breast cancer death (HR_{0–10 years} 1.9 [1.2–2.9], p = 0.004). Ten of the 11 investigated hypoxic gene signatures correlated positively to HIF-1α-positivity, and 5 to increased rate/risk of recurrence. Conclusions: The benefit of postoperative radiotherapy persisted in patients with hypoxic primary tumours. Patients with hypoxic primary breast tumours had an increased risk of recurrence and breast cancer death.

Chronic respiratory diseases are often characterized by impaired epithelial function and remodeling. Mast cells (MCs) are known to home into the epithelium in respiratory diseases, but the MC-epithelial interactions remain less understood. Therefore, this study aimed to investigate the effect of MC proteases on bronchial epithelial morphology and function. Bronchial epithelial cells were stimulated with MC tryptase and/or chymase. Morphology and epithelial function were performed using cell tracking analysis and holographic live-cell imaging. Samples were also analyzed for motility-associated gene expression. Immunocytochemistry was performed to compare cytoskeletal arrangement. Stimulated cells showed strong alterations on gene, protein and functional levels in several parameters important for maintaining epithelial function. The most significant increases were found in cell motility, cellular speed and cell elongation compared to non-stimulated cells. Also, cell morphology was significantly altered in chymase treated compared to non-stimulated cells. In the current study, we show that MC proteases can induce cell migration and morphological and proliferative alterations in epithelial cells. Thus, our data imply that MC release of proteases may play a critical role in airway epithelial remodeling and disruption of epithelial function.