Clinical trials frequently include multiple end points that mature at different times. The initial report, typically based on the primary end point, may be published when key planned co-primary or secondary analyses are not yet available. Clinical Trial Updates provide an opportunity to disseminate additional results from studies, published in JCO or elsewhere, for which the primary end point has already been reported.

Although dose-dense adjuvant chemotherapy administered once every 2 weeks leads to superior outcomes compared with standard regimens once every 3 weeks, the observed improvement is largely limited to studies using the suboptimal paclitaxel schedule once every 3 weeks as control. PANTHER is an international phase III trial which compared sequential epirubicin/cyclophosphamide and docetaxel administered either once every 2 or once every 3 weeks, with tailored dosing at the dose-dense schedule according to hematologic toxicity. In this end-of-study analysis, the median follow-up was 10.3 years. Compared with standard adjuvant chemotherapy, dose-dense treatment improved breast cancer recurrence-free survival (hazard ratio [HR], 0.80 [95% CI, 0.65 to 0.98]; P = .030), event-free survival (HR, 0.78 [95% CI, 0.65 to 0.94]; P = .009), and distant disease-free survival (HR, 0.79 [95% CI, 0.64 to 0.98]; P = .030) while the improvement in overall survival was not statistically significant (HR, 0.82 [95% CI, 0.65 to 1.04]; P = .109). To our knowledge, this is the first trial that confirms the benefit of a dose-dense regimen over a control regimen containing docetaxel once every 3 weeks.

Background High mammographic density is an independent risk factor for breast cancer by poorly understood molecular mechanisms. Women with dense breasts often undergo conventional magnetic resonance imaging (MRI) despite its limited specificity, which may be increased by diffusion-weighted imaging (DWI) with apparent diffusion coefficient (ADC) and contrast. How these modalities are affected by breast density per se and their association with the local microenvironment are undetermined. Methods Healthy postmenopausal women attending mammography screen with extremely dense or entirely fatty breasts underwent multiparametric MRI for analyses of lean tissue fraction (LTF), ADC and perfusion dynamics. Microdialysis was used for extracellular proteomics in situ. Results Significantly increased LTF and ADC and delayed perfusion were detected in dense breasts. In total, 270 proteins were quantified, whereof 124 related to inflammation, angiogenesis, and cellular growth were significantly upregulated in dense breasts. Most of these correlated significantly with LTF, ADC and the perfusion data. Conclusions ADC and perfusion characteristics depend on breast density, which should be considered during the implementation of thresholds for malignant lesions. Dense and nondense breasts are two essentially different biological entities, with a pro-tumorigenic microenvironment in dense breasts. Our data reveal several novel pathways that may be explored for breast cancer prevention strategies.

Context: High mammographic density in postmenopausal women is an independent risk factor for breast cancer by undetermined mechanisms. No preventive therapy for this risk group is available. Activated platelets release growth factors that modulate the microenvironment into a protumorigenic state. Estrogens may affect the risk of breast cancer and platelet function. Whether platelets are activated in situ in breast cancer or in normal breast tissue at high risk of breast cancer and the association to estradiol remains elusive. Objective: To investigate whether platelets are activated in situ in breast cancers and in dense breast tissue of postmenopausal women and explore correlations between estradiol, released platelet factors, and inflammatory proteins. Setting and design: Sampling of in vivo proteins was performed using microdialysis in a total of 71 women: 10 with breast cancer, 42 healthy postmenopausal women with different breast densities, and 19 premenopausal women. Results: Our data demonstrate increased levels of coagulation factors in dense breast tissue similar to that found in breast cancers, indicating excessive platelet activation. Premenopausal breasts exhibited similar levels of coagulation factors as postmenopausal dense breasts. Out of 13 coagulations factors that were upregulated in dense breasts, 5 exhibited significant correlations with estradiol, both locally in the breast and systemically. In breast tissue, positive correlations between coagulation factors and key inflammatory proteins and matrix metalloproteinases were detected. Conclusions: Breast density, not estradiol, is the major determinant of local platelet activation. Inactivation of platelets may be a therapeutic strategy for cancer prevention in postmenopausal women with dense breasts.

Although blocking estrogen-dependent signaling is a cornerstone of adjuvant treatment for breast cancer, 25% of patients experience recurrent disease. Stroma events including innate immune responses are key in cancer progression. How different estrogen receptor (ER)-targeting therapies, including the partial agonist tamoxifen and the pure antagonist fulvestrant, affect the tumor stroma has not yet been elucidated. Fulvestrant is used in only postmenopausal patients, and its effects in the presence of estradiol remain undetermined. Here we observe that fulvestrant decreases ER+ breast cancer growth compared with tamoxifen in the presence of physiologic levels of estradiol in human breast cancer in nude mice and in murine breast cancer in immune-competent mice. Fulvestrant significantly inhibited macrophage and neutrophil infiltration in both models. These effects were corroborated in a zebrafish model where fulvestrant inhibited neutrophil- and macrophage-dependent cancer cell dissemination more effectively than tamoxifen. A comprehensive analysis of 234 human proteins released into the cancer microenvironment by the cancer cells sampled via microdialysis in vivo revealed that 38 proteins were altered following both treatments; 25 of these proteins were associated with immune response and were altered by fulvestrant only. Compared with tamoxifen, fulvestrant significantly affected inflammatory proteins released by murine stroma cells. Importantly, in vivo microdialysis of human ER+ breast cancer revealed that the majority of affected proteins in murine models were upregulated in patients. Together, these results suggest that fulvestrant targets ER+ breast cancer more effectively than tamoxifen even in the presence of estradiol, mainly by attenuation of the innate immune response. Significance: These findings demonstrate novel effects of the pure antiestrogen fulvestrant in ERthorn breast cancer and evaluate its effects under physiologic levels of estradiol, representative of premenopausal patients.

Fat is a major tissue component in human breast cancer (BC). Whether breast adipocytes (BAd) affect early stages of BC metastasis is yet unknown. BC progression is dependent on angiogenesis and inflammation, and interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF) are key regulators of these events. Here, we show that BAd increased the dissemination of estrogen receptor positive BC cells (BCC) in vivo in the zebrafish model of metastasis, while dissemination of the more aggressive and metastatic BCC such as estrogen receptor negative was unaffected. While anti-VEGF and anti-IL-8 exhibited equal inhibition of angiogenesis at the primary tumor site, anti-IL-8 reduced BCC dissemination whereas anti-VEGF had minor effects on this early metastatic event. Mechanistically, overexpression of cell-adhesion molecules in BCC and neutrophils via IL-8 increased the dissemination of BCC. Importantly, the extracellular in vivo levels of IL-8 were 40-fold higher than those of VEGF in human BC. Our results suggest that IL-8 is a clinical relevant and promising therapeutic target for human BC.

Estradiol (E2) plays a key role in breast cancer progression. Most breast cancer recurrences express the estrogen receptor (ER), but nearly 50% of patients are resistant to antiestrogen therapy. Novel therapeutic targets of ER-positive breast cancers are needed. Protumoral neutrophils expressing the lymphocyte function-associated antigen 1 (LFA-1) integrin may mediate cancer metastasis, and TGFβ1 is the major chemoattractant for neutrophils. The role of E2 in neutrophil–ER+ breast cancer cell interactions is unknown. We studied this in vivo using murine breast cancers in immunocompetent mice and human breast cancers in nude mice. Cell dissemination was evaluated in a zebrafish model, and microdialysis of breast cancer patients was performed. In vitro studies were done with mammosphere cultures of breast cancer cells and human neutrophils. We found that E2 increased the number of LFA-1+ neutrophils recruited to the invasive edge of mouse tumors, increased TGFβ1 secretion and promoted neutrophil infiltration in mammospheres, and induced overexpression of LFA-1 in neutrophils. In zebrafish, in the presence of E2, neutrophils increased dissemination of ER+ breast cancer cells via LFA-1 and TGFβ1, thus causing noninvasive cancer cells to be highly metastatic. Time-lapse imaging in zebrafish revealed close interactions of neutrophils with cancer cells, which drove breast cancer metastasis. We also found that extracellular TGFβ1 was overproduced in human breast cancer tissue compared with adjacent normal breast tissue. Thus, E2 can regulate immune/cancer cell interactions in tumor microenvironments. Our results indicate that extracellular TGFβ1 is a relevant target in human breast cancer.

Metabolic reprogramming is a hallmark of cancer. Nutrient availability in the tissue microenvironment determines cellular events and may play a role in breast carcinogenesis. High mammographic density is an independent risk factor for breast cancer. Whether nutrient availability differs in normal breast tissues with various densities is unknown. Therefore we investigated whether breast tissues with various densities exhibited differences in nutrient availability. Healthy postmenopausal women from the regular mammographic screening program who had either predominantly fatty breast tissue (nondense), n = 18, or extremely dense breast tissue (dense), n = 20, were included. Microdialysis was performed for the in vivo sampling of amino acids (AAs), analyzed by ultra-high performance liquid chromatography with tandem mass spectroscopy, glucose, lactate and vascular endothelial growth factor (VEGF) in breast tissues and, as a control, in abdominal subcutaneous (s.c.) fat. We found that dense breast tissue exhibited significantly increased levels of 20 proteinogenic AAs and that 18 of these AAs correlated significantly with VEGF. No differences were found in the s.c. fat, except for one AA, suggesting tissue-specific alterations in the breast. Glucose and lactate were unaltered. Our findings provide novel insights into the biology of dense breast tissue that may be explored for breast cancer prevention strategies.

Exposure to sex steroids increases the risk of breast cancer but the exact mechanisms are yet to be elucidated. Events in the microenvironment are important for carcinogenesis. Diet containing phytoestrogens can affect the breast microenvironment and alter the risk of breast cancer. It has previously been shown that estrogen regulates extracellular levels of leptin, adiponectin, and VEGF in normal breast tissue in vivo. Whether these proteins correlate in breast tissue in vivo or if diet addition of flaxseed, a major source of phytoestrogens in Western diets, alters adipokine levels in breast tissue are unknown. We used microdialysis to sample proteins of normal human breast tissue and abdominal subcutaneous fat in situ in 34 pre-and postmenopausal women. In vitro, co-culture of breast cancer cells and primary human adipocytes was used. In vivo, in normal breast tissue, a significant positive correlation between VEGF and leptin was detected. No correlations were found in fat tissue. Co-culture of adipocytes and breast cancer cells per se increased the secretion of VEGF and leptin and enhanced the effects of estradiol compared to culture of either cell type alone. In vitro, inhibition of VEGF diminished the release of leptin while inhibition of leptin had no influence on VEGF secretion. The levels of leptin decreased and adiponectin increased after a dietary addition of 25 g of flaxseed/day for one menstrual cycle. We conclude that VEGF and leptin correlate significantly in normal human breast tissue in vivo and that dietary addition of flaxseed affect adipokine levels in the breast.

Breast cancer is one of the most common malignancies of women in the reproductive years. In the Western world there is a trend towards delaying pregnancy to later in life, and in combination with an increased incidence of breast cancer an increased number of women are diagnosed with breast cancer before they have completed their reproductive plans. In addition, breast cancer during pregnancy may affect an increased number of women as the childbearing years are delayed. The survival rate after breast cancer has improved during the last decades, and many young breast cancer survivors will consider a pregnancy subsequent to the completion of adjuvant breast cancer therapy. Traditionally, many women are advised against a pregnancy due to a fear of increased risk of recurrence, especially women with estrogen receptor-positive breast cancer. Due to feasibility issues, evidence from large prospective randomized trials is missing regarding the safety of pregnancy after breast cancer. Today guidelines are based on cohort studies and population-based registry evidence with its limitations. Overall, data suggest that pregnancy after breast cancer therapy is safe, and the current evidence is summarized in this overview.

Deregulation of angiogenesis - the growth of new blood vessels from an existing vasculature - is a main driving force in many severe human diseases including cancer. As such, tumor angiogenesis is important for delivering oxygen and nutrients to growing tumors, and therefore considered an essential pathologic feature of cancer, while also playing a key role in enabling other aspects of tumor pathology such as metabolic deregulation and tumor dissemination/metastasis. Recently, inhibition of tumor angiogenesis has become a clinical anti-cancer strategy in line with chemotherapy, radiotherapy and surgery, which underscore the critical importance of the angiogenic switch during early tumor development. Unfortunately the clinically approved anti-angiogenic drugs in use today are only effective in a subset of the patients, and many who initially respond develop resistance over time. Also, some of the anti-angiogenic drugs are toxic and it would be of great importance to identify alternative compounds, which could overcome these drawbacks and limitations of the currently available therapy. Finding "the most important target" may, however, prove a very challenging approach as the tumor environment is highly diverse, consisting of many different cell types, all of which may contribute to tumor angiogenesis. Furthermore, the tumor cells themselves are genetically unstable, leading to a progressive increase in the number of different angiogenic factors produced as the cancer progresses to advanced stages. As an alternative approach to targeted therapy, options to broadly interfere with angiogenic signals by a mixture of non-toxic natural compound with pleiotropic actions were viewed by this team as an opportunity to develop a complementary anti-angiogenesis treatment option. As a part of the "Halifax Project" within the "Getting to know cancer" framework, we have here, based on a thorough review of the literature, identified 10 important aspects of tumor angiogenesis and the pathological tumor vasculature which would be well suited as targets for anti-angiogenic therapy: (1) endothelial cell migration/tip cell formation, (2) structural abnormalities of tumor vessels, (3) hypoxia, (4) lymphangiogenesis, (5) elevated interstitial fluid pressure, (6) poor perfusion, (7) disrupted circadian rhythms, (8) tumor promoting inflammation, (9) tumor promoting fibroblasts and (10) tumor cell metabolism/acidosis. Following this analysis, we scrutinized the available literature on broadly acting anti-angiogenic natural products, with a focus on finding qualitative information on phytochemicals which could inhibit these targets and came up with 10 prototypical phytochemical compounds: (1) oleic acid, (2) tripterine, (3) silibinin, (4) curcumin, (5) epigallocatechin-gallate, (6) kaempferol, (7) melatonin, (8) enterolactone, (9) withaferin A and (10) resveratrol. We suggest that these plant-derived compounds could be combined to constitute a broader acting and more effective inhibitory cocktail at doses that would not be likely to cause excessive toxicity. All the targets and phytochemical approaches were further cross-validated against their effects on other essential tumorigenic pathways (based on the "hallmarks" of cancer) in order to discover possible synergies or potentially harmful interactions, and were found to generally also have positive involvement in/effects on these other aspects of tumor biology. The aim is that this discussion could lead to the selection of combinations of such anti-angiogenic compounds which could be used in potent anti-tumor cocktails, for enhanced therapeutic efficacy, reduced toxicity and circumvention of single-agent anti-angiogenic resistance, as well as for possible use in primary or secondary cancer prevention strategies.