Impact of pre-diagnostic triglycerides and HDL-cholesterol on breast cancer recurrence and survival by breast cancer subtypes.

BACKGROUND: High triglycerides and low levels of high density lipoprotein (HDL)-cholesterol are observed to promote tumor growth. However, whether breast cancer heterogeneity may explain the contradictory influence of triglycerides and cholesterol observed on breast cancer prognosis remains unclear. METHODS: A population-based survival study among 464 breast cancer cases identified within the Tromsø study was conducted. Pre-diagnostic triglycerides, total-cholesterol and HDL-cholesterol were measured, and detailed clinical and histopathological data were obtained. Using tissue microarray, all breast cancer cases were reclassified into the following subtypes: Luminal A, Luminal B, HER2-enriched, and triple negative breast cancer (TNBC). Multivariable Cox proportional hazards regression models were used to study the associations between pre-diagnostic lipids and breast cancer recurrence, mortality, and survival. RESULTS: A total of 464 breast cancer patients, with mean age at diagnosis of 57.9 years, were followed for a mean 8.4 years. TNBC patients in the highest tertile of triglycerides (≥ 1.23 mmol/l) had 3 times higher overall mortality compared to TNBC patients in the lowest tertile (≤ 0.82 mmol/l) (HR 2.99, 95% CI 1.17-7.63), and the 5-year overall survival was 19% lower for TNBC patients in the highest vs. lowest tertile of triglycerides (65% vs. 84%). TNBC patients in the highest tertile of the HDL-cholesterol/total-cholesterol ratio (≥0.35), compared to those in the lowest tertile (≤0.27), had a 67% reduced overall mortality risk (HR 0.33, 95% CI 0.12-0.89). No associations were observed between lipids and prognostic outcome among breast cancer patients overall, or among patients with luminal A and luminal B subtypes. Among HER2-enriched patients, pre-diagnostic triglyceride level was inversely associated with overall mortality. CONCLUSION: Our study suggests that pre-diagnostic triglycerides and the HDL-cholesterol/total-cholesterol ratio may independently provide unique information regarding prognostic outcome among triple negative breast cancer patients. However, a small sample size underlines the need for additional studies.

GRK3 is essential for metastatic cells and promotes prostate tumor progression.

The biochemical mechanisms that regulate the process of cancer metastasis are still poorly understood. Because kinases, and the signaling pathways they comprise, play key roles in regulation of many cellular processes, we used an unbiased RNAi in vitro screen and a focused cDNA in vivo screen against human kinases to identify those with previously undocumented roles in metastasis. We discovered that G-protein-coupled receptor kinase 3 (GRK3; or ß-adrenergic receptor kinase 2) was not only necessary for survival and proliferation of metastatic cells, but also sufficient to promote primary prostate tumor growth and metastasis upon exogenous expression in poorly metastatic cells in mouse xenograft models. Mechanistically, we found that GRK3 stimulated angiogenesis, at least in part through down-regulation of thrombospondin-1 and plasminogen activator inhibitor type 2. Furthermore, GRK3 was found to be overexpressed in human prostate cancers, especially in metastatic tumors. Taken together, these data suggest that GRK3 plays an important role in prostate cancer progression and metastasis.

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.

Vascular proliferation is increased in basal-like breast cancer.

Based on molecular sub-classification, basal-like breast cancer is associated with aggressive behavior. These tumors are frequently triple negative and lack traditional treatment targets. Angiogenesis, one of the hallmarks of cancer, is important for the local growth and spread of malignant tumors and is now a treatment target. The aim of this study was to explore whether angiogenesis is increased in relation to certain molecular subtypes of breast cancer with special focus on the basal-like category. Altogether, we analyzed a total of 431 breast cancers from two independent series after dual immunohistochemical staining of Factor VIII for endothelial cells and Ki-67 for proliferating cells. We then determined vascular proliferation in the most vascularized areas of the tumor. In both Series I and II, high vascular proliferation index (VPI) was significantly associated with expression of cytokeratin 5/6 (P = 0.001, 0.010), P-cadherin (P < 0.0005, <0.0005), epidermal growth factor receptor (P = 0.003, 0.001), the basal-like subtype (P = 0.001, 0.011), and the core basal phenotype (P = 0.002, 0.002), respectively. In Series I, high VPI was associated with the triple negative phenotype (P = 0.004) and p63 expression (P = 0.008). Tumor angiogenesis, as measured by vascular proliferation, was increased in the basal-like subtype in two independent breast cancer series and may thus be a possible treatment target in this category. Studies are required to evaluate whether this novel angiogenesis marker can be used to stratify patients for anti-angiogenesis treatment.

Expression of EGFR and c-kit is associated with the basal-like phenotype in breast carcinomas of African women.

Epidermal growth factor receptor (EGFR) and c-kit are tyrosine kinase growth factor receptors which are frequently expressed in basal-like breast carcinomas, and tyrosine kinase inhibition is now a promising strategy in treatment of breast cancer. The aim of this study was to evaluate the expression of EGFR and c-kit in breast cancer with special focus on the basal-like phenotype (BLP) and other prognostic factors in an African population. We analyzed 65 archival tissues immunohistologically. EGFR and/or c-kit were expressed in 55% of basal-like tumors. Expression of EGFR and/or c-kit was strongly associated with high histologic grade (P=0.001), high nuclear grade (P=0.017), high mitotic counts (P=0.002), ER negativity (P=0.003), PR negativity (P=0.007), and HER2 negativity (P=0.014). EGFR and/or c-kit positive tumors were more likely to express the BLP (OR 9.1, CI 2.6-32.0, P<0.0005) than the negative tumors. In conclusion, there is a high expression of EGFR and/or c-kit in basal-like breast carcinoma in this series from Uganda and their expression is associated with features of poor prognosis. More studies are required to assess the clinical significance of EGFR and c-kit in breast cancer patients in Uganda.

Frequency of the basal-like phenotype in African breast cancer.

Basal-like breast carcinoma has been recognized as a subtype with specific prognostic implications. However, there is a lack of reports about this category of breast tumors in African women. The aim of this study was to explore the basal-like phenotype in breast cancer patients in an African population, and a registry-based series was included from the well-defined Kyadondo County in Uganda (1.7 millions). We studied a total of 65 archival paraffin blocks of invasive breast cancer using antibodies against cytokeratin 5/6 and P-cadherin, and these markers were expressed in 34% of all cases and in 52% of ER (estrogen receptor)-negative tumors. All basal-like tumors were ER negative (p<0.0005) and PR (progesterone receptor) negative (p=0.002). Basal-like breast carcinomas were of a higher histologic grade (p=0.001), had high mitotic counts (p=0.002), and marked nuclear pleomorphism (p=0.002). P-cadherin-positive tumors had a high Ki-67 proliferative rate (p=0.039). In conclusion, the basal-like phenotype is frequent in this African series of breast cancer and is strongly associated with poor prognostic factors. Our findings might be significant in relation to clinical management of these patients, including novel targeted therapy.

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.

Strong Expression of Hypoxia-Inducible Factor-1α (HIF-1α) Is Associated with Axl Expression and Features of Aggressive Tumors in African Breast Cancer.

PURPOSE: Inhibition of hypoxia-inducible factor (HIF) and Axl receptor tyrosine kinase is being evaluated for targeted therapy in solid tumors. Both HIF-1α and Axl influence tumor growth and metastatic potential, and they have been linked to treatment failure in many cancers. However, there is a lack of reports on HIF-1α expression in African breast cancer, which has a poor prognosis, and novel treatment targets must therefore be established. Here, we aimed to evaluate HIF-1α in relation to Axl expression, angiogenesis markers, and other tumor characteristics in a series of African breast cancer. METHODS: Using immunohistochemistry, we examined 261 invasive breast cancers on tissue microarrays for HIF-1α and Axl as well as several other markers, and a subset of 185 cases had information on VEGF (vascular endothelial growth factor) expression, microvessel density (MVD), proliferating microvessel density (pMVD) and vascular proliferation index (VPI) for important comparisons. RESULTS: Strong HIF-1α expression was associated with increased Axl (p = 0.007), VEGF (p<0.0005), and p53 (p = 0.032) expression, as well as high tumor cell proliferation by Ki-67 (p = 0.006), and high tumor grade (p = 0.003). Tumors with strong HIF-1α expression had significantly higher MVD (p = 0.019) and higher pMVD (p = 0.027) than tumors with weak expression. CONCLUSIONS: High HIF-1α expression is significantly associated with Axl and VEGF expression, and with markers of poor prognosis in this series of breast cancer, suggesting HIF-1α and Axl as potential therapeutic targets in African breast cancer.

Increased tumor cell expression of Axl is a marker of aggressive features in breast cancer among African women.

Axl, a receptor tyrosine kinase belonging to the Tyro/Axl/Mer (TAM) family, has been shown to be overexpressed in breast cancer with poor outcome. Moreover, Axl was associated with a basal-like phenotype (BLP) in these tumors. Our aim was to investigate Axl expression in breast cancers from an African population since these tumors are known to be aggressive and have a high frequency of the basal-like phenotype. We studied 170 paraffin-embedded breast carcinoma cases by tissue microarrays and immunohistochemical methods. In total, 128 tumor cases (75%) had strong Axl expression and 42 cases (25%) had weak or negative staining. Strong expression of Axl was associated with high tumor grade (p < 0.0005), estrogen receptor (ER) negativity (p = 0.024), p53 expression (p = 0.004), P-cadherin positivity (p = 0.017), and basal-like phenotypic profiles BLP2 (p = 0.033) and BLP3 (p = 0.022). In addition, Axl overexpression also showed an association with markers of tumor cell proliferation and tumor angiogenesis. In conclusion, our findings indicate strong expression of Axl in a high proportion of breast cancer cases among African women and associations with markers of aggressive features, indicating poor prognosis. These findings suggest Axl as a potential therapeutic target in this population.

Decreased serum opsonic activity against Streptococcus pneumoniae in human immunodeficiency virus-infected Ugandan adults.

Type-specific immunoglobulin G (IgG) to pneumococcal capsular polysaccharide (CPS) and opsonic activity against Streptococcus pneumoniae were evaluated in serum samples from 36 Ugandan adults with community-acquired pneumonia and 58 asymptomatic Ugandan adults with or without human immunodeficiency virus type 1 (HIV-1) infection. The levels of serum IgG to CPS were significantly higher in HIV-1-infected subjects than in HIV-uninfected subjects. Serum samples from HIV-1-infected subjects that had lower IgG titers demonstrated higher opsonic activity against type 3 (titers of 7) and type 9 (titers of 7-11) pneumococcal strains. Plasma HIV-1 load also correlated inversely with serum opsonic activity against these strains, and peripheral blood CD4+ lymphocyte numbers also tended to correlate with serum opsonic activity in asymptomatic HIV-1-infected adults. Our findings suggest that the opsonic activity of type-specific IgG is impaired in the serum of HIV-1-infected African adults, which may expose them to a serious risk of invasive pneumococcal infections.

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.