Age-related phenotypes in breast cancer: A population-based study.

Breast cancer in young (<40 years) is associated with a higher frequency of aggressive tumor types and poor prognosis. It remains unclear if there is an underlying age-related biology that contributes to the unfavorable outcome. We aim to investigate the relationship between age and breast cancer biology, with emphasis on proliferation. Clinico-pathologic information, immunohistochemical markers and follow-up data were obtained for all patients aged <50 (Bergen cohort-1; n = 355, not part of a breast screening program) and compared to previously obtained information on patients aged 50 to 69 years (Bergen cohort-2; n = 540), who participated in the Norwegian Breast Cancer Screening Program. Young breast cancer patients presented more aggressive tumor features such as hormone receptor negativity, HER2 positivity, lymph-node metastasis, the HER2-enriched and triple-negative subtypes and shorter survival. Age <40 was significantly associated with higher proliferation (by Ki67). Ki67 showed weaker prognostic value in young patients. We point to aggressive phenotypes and increased tumor cell proliferation in breast cancer of the young. Hence, tumors of young breast cancer patients may present unique biological features, also when accounting for screen/interval differences, that may open for new clinical opportunities, stratifying treatment by age.

In vivo MRI and histopathological assessment of tumor microenvironment in luminal-like and basal-like breast cancer xenografts.

PURPOSE: To explore tumor pathophysiology with special attention to the microenvironment in two molecular subtypes of human breast cancer using in vivo magnetic resonance imaging (MRI) and histopathology. The impact of tumor growth, size, and the influence of estradiol were also investigated. MATERIALS AND METHODS: Two orthotopic and directly transplanted human breast cancer models representing luminal-like and basal-like molecular subtypes were characterized by dynamic contrast-enhanced MRI and diffusion-weighted MRI. Ex vivo measurements of vascularization, hypoxia, mitoses, and the level of VEGF activations were associated with the calculated in vivo MRI parameters of the tumors. RESULTS: The vascular permeability and perfusion (K(trans) ) was significantly higher in basal-like compared to luminal-like tumors. These findings were confirmed by a 4-fold higher proliferating microvessel density (pMVD) in basal-like tumors, reflecting the difference in aggressiveness between the subtypes. No effect of tumor growth was observed during 6 days of growth in any of the models; however, large tumors had lower K(trans) , higher extracellular extravascular volume fraction (v(e) ), and more hypoxia than medium-sized tumors. Estradiol withdrawal induced increased K(trans) , v(e) , and tumor water diffusion (ADC) in luminal-like tumors, corresponding to increased VEGFR2 activation, which is likely to cause increased tumor vessel permeability. CONCLUSION: These novel data confirm the potential of functional MRI methods to map histopathologically proven changes in breast tumor vasculature and microenvironment in vivo.

Baseline microvessel density predicts response to neoadjuvant bevacizumab treatment of locally advanced breast cancer.

A subset of breast cancer patients benefits from preoperative bevacizumab and chemotherapy, but validated predictive biomarkers are lacking. Here, we aimed to evaluate tissue-based angiogenesis markers for potential predictive value regarding response to neoadjuvant bevacizumab treatment in breast cancer. In this randomized 1:1 phase II clinical trial, 132 patients with large or locally advanced HER2-negative tumors received chemotherapy ± bevacizumab. Dual Factor VIII/Ki-67 immunohistochemical staining was performed on core needle biopsies at baseline and week 12. Microvessel density (MVD), proliferative microvessel density (pMVD; Factor VIII/Ki-67 co-expression), glomeruloid microvascular proliferation (GMP), and a gene expression angiogenesis signature score, were studied in relation to pathologic complete response (pCR), clinico-pathologic features and intrinsic molecular subtype. We found that high baseline MVD (by median) significantly predicted pCR in the bevacizumab-arm (odds ratio 4.9, P = 0.012). High pMVD, presence of GMP, and the angiogenesis signature score did not predict pCR, but were associated with basal-like (P ≤ 0.009) and triple negative phenotypes (P ≤ 0.041). pMVD and GMP did also associate with high-grade tumors (P ≤ 0.048). To conclude, high baseline MVD significantly predicted response to bevacizumab treatment. In contrast, pMVD, GMP, and the angiogenesis signature score, did not predict response, but associated with aggressive tumor features, including basal-like and triple-negative phenotypes.

Stathmin expression associates with vascular and immune responses in aggressive breast cancer subgroups.

Studies indicate that stathmin expression associates with PI3K activation in breast cancer, suggesting stathmin as a marker for targetable patient subgroups. Here we assessed stathmin in relation to tumour proliferation, vascular and immune responses, BRCA1 germline status, basal-like differentiation, clinico-pathologic features, and survival. Immunohistochemical staining was performed on breast cancers from two series (cohort 1, n = 187; cohort 2, n = 198), and mass spectrometry data from 24 cases and 12 breast cancer cell lines was examined for proteomic profiles. Open databases were also explored (TCGA, METABRIC, Oslo2 Landscape cohort, Cancer Cell Line Encyclopedia). High stathmin expression associated with tumour proliferation, p53 status, basal-like differentiation, BRCA1 genotype, and high-grade histology. These patterns were confirmed using mRNA data. Stathmin mRNA further associated with tumour angiogenesis, immune responses and reduced survival. By logistic regression, stathmin protein independently predicted a BRCA1 genotype (OR 10.0, p = 0.015) among ER negative tumours. Cell line analysis (Connectivity Map) implied PI3K inhibition in tumours with high stathmin. Altogether, our findings indicate that stathmin might be involved in the regulation of tumour angiogenesis and immune responses in breast cancer, in addition to tumour proliferation. Cell data point to potential effects of PI3K inhibition in tumours with high stathmin expression.

Expression of Nestin associates with BRCA1 mutations, a basal-like phenotype and aggressive breast cancer.

We here examined whether Nestin, by protein and mRNA levels, could be a predictor of BRCA1 related breast cancer, a basal-like phenotype, and aggressive tumours. Immunohistochemical staining of Nestin was done in independent breast cancer hospital cohorts (Series I-V, total 1257 cases). Also, TCGA proteomic data (n = 103), mRNA microarray data from TCGA (n = 520), METABRIC (n = 1992), and 6 open access breast cancer datasets (n = 1908) were analysed. Patients with Nestin protein expression in tumour cells more often had BRCA1 germline mutations (OR 8.7, p < 0.0005, Series III), especially among younger patients (<40 years at diagnosis) (OR 16.5, p = 0.003). Nestin protein positivity, observed in 9-28% of our hospital cases (Series I-IV), was independently associated with reduced breast cancer specific survival (HR = 2.0, p = 0.035) and was consistently related to basal-like differentiation (by Cytokeratin 5, OR 8.7-13.8, p < 0.0005; P-cadherin OR 7.0-8.9, p < 0.0005; EGFR staining, OR 3.7-8.2, p ≤ 0.05). Nestin mRNA correlated significantly with Nestin protein expression (ρ = 0.6, p < 0.0005), and high levels were seen in the basal-like intrinsic subtype. Gene expression signalling pathways linked to high Nestin were explored, and revealed associations with stem-like tumour features. In summary, Nestin was strongly associated with germline BRCA1 related breast cancer, a basal-like phenotype, reduced survival, and stemness characteristics.

Microvascular proliferation in luminal A and basal-like breast cancer subtypes.

AIMS: The aims of this study were to examine microvessel density (MVD), proliferating MVD (pMVD) and Vascular Proliferation Index (VPI) in basal-like phenotype (BP) and luminal A subtypes of breast cancer and to study their prognostic value. METHODS: Dual-colour immunohistochemistry for von Willebrand factor and Ki67 was done on sections from 62 luminal A and 62 BP tumours matched for grade and selected from 909 breast cancers previously reclassified into molecular subtypes. Associations between MVD, pMVD and VPI, molecular subtypes and breast cancer prognosis were estimated using linear regression and survival analyses. RESULTS: Both pMVD (difference 1.9 microvessels/mm(2) (p=0.002)) and VPI (difference 1.7 percentage points (p=0.014)) were higher in BP tumours compared with luminal A. No clear difference between subtypes was found for MVD. However, only MVD was associated with prognosis. HR for breast cancer death for all cases was 1.10 (95% CI 1.02 to 1.18) per 10 vessels increase. Among luminal A tumours, HR was 1.22 per 10 vessels increase (p<0.001) and in BP it was 1.04 (p=0.37). CONCLUSIONS: High MVD was associated with poor prognosis in luminal A, but not in BP cancers. Vascular proliferation was higher in BP, indicating a more active angiogenesis than in luminal A tumours. The luminal A subgroup comprised mostly histopathological grade 3 cancers in this selected series, and further studies are needed to clarify whether MVD provides additional prognostic information for luminal A tumours irrespective of grade. This may contribute to stratification of this large group of patients and may aid in identifying tumours with a particularly good prognosis.

Effect of antiangiogenic therapy on tumor growth, vasculature and kinase activity in basal- and luminal-like breast cancer xenografts.

Several clinical trials have investigated the efficacy of bevacizumab in breast cancer, and even if growth inhibiting effects have been registered when antiangiogenic treatment is given in combination with chemotherapy no gain in overall survival has been observed. One reason for the lack of overall survival benefit might be that appropriate criteria for selection of patients likely to respond to antiangiogenic therapy in combination with chemotherapy, are not available. To determine factors of importance for antiangiogenic treatment response and/or resistance, two representative human basal- and luminal-like breast cancer xenografts were treated with bevacizumab and doxorubicin alone or in combination. In vivo growth inhibition, microvessel density (MVD) and proliferating tumor vessels (pMVD = proliferative microvessel density) were analysed, while kinase activity was determined using the PamChip Tyrosine kinase microarray system. Results showed that both doxorubicin and bevacizumab inhibited basal-like tumor growth significantly, but with a superior effect when given in combination. In contrast, doxorubicin inhibited luminal-like tumor growth most effectively, and with no additional benefit of adding antiangiogenic therapy. In agreement with the growth inhibition data, vascular characterization verified a more pronounced effect of the antiangiogenic treatment in the basal-like compared to the luminal-like tumors, demonstrating total inhibition of pMVD and a significant reduction in MVD at early time points (three days after treatment) and sustained inhibitory effects until the end of the experiment (day 18). In contrast, luminal-like tumors only showed significant effect on the vasculature at day 10 in the tumors having received both doxorubicin and bevacizumab. Kinase activity profiling in both tumor models demonstrated that the most effective treatment in vivo was accompanied with increased phosphorylation of kinase substrates of growth control and angiogenesis, like EGFR, VEGFR2 and PLCγ1. This may be a result of regulatory feedback mechanisms contributing to treatment resistance, and may suggest response markers of value for the prediction of antiangiogenic treatment efficacy.