Exploring the effects of lifestyle on breast cancer risk, age at diagnosis, and survival: the EBBA-Life study.

PURPOSE: Whether an unfavorable lifestyle not only affects breast cancer risk, but also influences age at onset of breast cancer and survival, is under debate. METHODS: In a population-based cohort, the Energy Balance and Breast Cancer Aspects throughout life (EBBA-Life) study, a total of 17,145 women were included. During follow-up, 574 women developed invasive breast cancer. Breast cancer cases were followed for an additional 9.1 years. Detailed medical records were obtained. Cox's proportional hazard regression models were used to study the association between pre-diagnostic lifestyle factors (weight, physical activity, alcohol use, smoking, and hypertension), breast cancer risk, age at diagnosis, and survival. RESULTS: At study entry, 34.3% of the participating women were overweight and 30.7% were physically inactive. Mean age at breast cancer diagnosis was 58.0 years, and 78.9% of the tumors were estrogen receptor positive. Among menopausal women who did not use hormone therapy and had an unfavorable lifestyle (3-5 unfavorable factors), compared with women who had a favorable lifestyle, we observed a twofold higher risk for postmenopausal breast cancer (hazard ratio [HR] 2.13, 95% confidence interval [CI] 1.23-3.69), and they were 3.4 years younger at diagnosis (64.8 versus 68.2 years, P = 0.032). Breast cancer patients with an unfavorable lifestyle, compared with patients with a favorable lifestyle, had almost a two times higher overall mortality risk (HR 1.96, 95% CI 1.01-3.80). CONCLUSIONS: Our study supports a healthy lifestyle improving breast cancer prevention, postponing onset of disease, and extending life expectancy among breast cancer patients.

ICAM1 expression is induced by proinflammatory cytokines and associated with TLS formation in aggressive breast cancer subtypes.

Intratumoral formation of tertiary lymphoid structures (TLS) within the tumor microenvironment is considered to be a consequence of antigen challenge during anti-tumor responses. Intracellular adhesion molecule 1 (ICAM1) has been implicated in a variety of immune and inflammatory responses, in addition to associate with triple negative breast cancer (TNBC). In this study, we detected TLS in the aggressive tumor phenotypes TNBC, HER2+ and luminal B, whereas the TLS negative group contained solely tumors of the luminal A subtype. We show that ICAM1 is exclusively expressed in TNBC and HER2 enriched subtypes known to be associated with inflammation and the formation of TLS. Furthermore, cell from normal mammary epithelium and breast cancer cell lines expressed ICAM1 upon stimulation with the proinflammatory cytokines TNFα, IL1ß and IFNγ. ICAM1 overexpression was induced in MCF7, MDA-MB-468 and SK-BR-3 cells regardless of hormone receptor status. Taken together, our findings show that ICAM1 is expressed in aggressive subtypes of breast cancer and its expression is inducible by well-known proinflammatory cytokines. ICAM1 may be an attractive molecular target for TNBC, but further investigations elucidating the role of ICAM1 in targeted therapies have to take into consideration selective subtypes of breast cancer.

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.

Tertiary lymphoid structures are associated with higher tumor grade in primary operable breast cancer patients.

BACKGROUND: Tertiary lymphoid structures (TLS) are highly organized immune cell aggregates that develop at sites of inflammation or infection in non-lymphoid organs. Despite the described role of inflammation in tumor progression, it is still unclear whether the process of lymphoid neogenesis and biological function of ectopic lymphoid tissue in tumors are beneficial or detrimental to tumor growth. In this study we analysed if TLS are found in human breast carcinomas and its association with clinicopathological parameters. METHODS: In a patient group (n = 290) who underwent primary surgery between 2011 and 2012 we assessed the interrelationship between the presence of TLS in breast tumors and clinicopathological factors. Prognostic factors were entered into a binary logistic regression model for identifying independent predictors for intratumoral TLS formation. RESULTS: There was a positive association between the grade of immune cell infiltration within the tumor and important prognostic parameters such as hormone receptor status, tumor grade and lymph node involvement. The majority of patients with high grade infiltration of immune cells had TLS positive tumors. In addition to the degree of immune cell infiltration, the presence of TLS was associated with organized immune cell aggregates, hormone receptor status and tumor grade. Tumors with histological grade 3 were the strongest predictor for the presence of TLS in a multivariate regression model. The model also predicted that the odds for having intratumoral TLS formation were ten times higher for patients with high grade of inflammation than low grade. CONCLUSIONS: Human breast carcinomas frequently contain TLS and the presence of these structures is associated with aggressive forms of tumors. Locally generated immune response with potentially antitumor immunity may control tumorigenesis and metastasis. Thus, defining the role of TLS formation in breast carcinomas may lead to alternative therapeutic approaches targeting the immune system.

Impact of the tumor necrosis factor receptor-associated protein 1 (Trap1) on renal DNaseI shutdown and on progression of murine and human lupus nephritis.

Recent findings show that transformation of mild glomerulonephritis into end-stage disease coincides with shutdown of renal DNaseI expression in (NZBxNZW)F1 mice. Down-regulation of DNaseI results in reduced chromatin fragmentation and deposition of extracellular chromatin fragments in glomerular basement membranes where they appear in complex with IgG antibodies. Here, we implicate the anti-apoptotic and survival protein, tumor necrosis factor receptor-associated protein 1 (Trap1) in the disease process, based on the observation that annotated transcripts from this gene overlap with transcripts from the DNaseI gene. Furthermore, we translate these observations to human lupus nephritis. In this study, mouse and human DNaseI and Trap1 mRNA levels were determined by real-time quantitative PCR and compared with protein expression levels and clinical data. Cellular localization was analyzed by immune electron microscopy, IHC, and in situ hybridization. Data indicate that silencing of DNaseI gene expression correlates inversely with expression of the Trap1 gene. Our observations suggest that the mouse model is relevant for the aspects of disease progression in human lupus nephritis. Acquired silencing of the renal DNaseI gene has been shown to be important for progression of disease in both the murine and human forms of lupus nephritis. Early mesangial nephritis initiates a cascade of inflammatory signals that lead to up-regulation of Trap1 and a consequent down-regulation of renal DNaseI by transcriptional interference.

Autoantibodies in lupus: culprits or passive bystanders?

Several autoantibodies are culprits in the pathogenesis of organ damage in systemic lupus erythematosus, by means of established or postulated mechanisms, whereby inducing a perturbation of cell structure and function, with consequent tissue-organ impairment. Common autoantibody-mediated mechanisms of damage include cell surface binding with or without cytolysis, immune complex-mediated damage, penetration into living cells, binding to cross-reactive extracellular molecules. Experimental data from both murine models and humans have recently clarified the key role of autoantibodies in severe organ involvements, including nephritis, neuropsychiatric (NP) dysfunction, and cerebrovascular disease (CVD). In lupus nephritis early and late phases are distinguishable and mediated by different processes in which anti-chromatin antibodies are both inducing and perpetuating agents, by immune-complex formation and massive deposition in mesangial matrix at first, and in glomerular basement membrane at end-stage. Also NP abnormalities occur very early, much earlier than other systemic manifestations, and exacerbate with the increase in autoantibody titers. Among the autoantibodies mainly implicated in neurolupus, anti-ß2 glycoprotein I (ß2GPI) antibodies are preferentially involved in focal NP events which are a consequence of non-inflammatory microangiopathy; otherwise, anti-ribosomal P protein antibodies and N-methyl-d-aspartate receptor (NMDAR) antibodies cause diffuse NP events through a direct cytotoxic effect on neuronal cells at specific brain zones.

Nuclease deficiencies promote end-stage lupus nephritis but not nephritogenic autoimmunity in (NZB × NZW) F1 mice.

New information has profoundly improved our insight into the processes that account for lupus nephritis. This review summarizes the data proving that secondary necrotic chromatin fragments are generated and retained in kidneys at time-points when the major renal nuclease Dnase-1 is selectively and severely downregulated. Second, we discuss data, which may indicate that nuclease deficiencies are not associated with autoimmunity to chromatin. Secondary to downregulation of renal Dnase-1, large chromatin fragment-immunoglobulin G complexes are accumulated in glomerular basement membranes of patients producing anti-chromatin autoantibodies. Exposure of chromatin in situ in glomeruli is the factor that renders anti-chromatin (anti-dsDNA and anti-nucleosome) antibodies nephritogenic. Without exposed chromatin, they circulate as non-pathogenic antibodies. This shows that acquired loss of renal Dnase-1 enzyme activity is a dominant event responsible for the progression of lupus nephritis into end-stage disease. Before the loss of Dnase-1, lupus-prone (NZB × NZW) F1 mice develop mild or silent nephritis with mesangial immune complex deposits, which correlates solely with onset of anti-dsDNA antibody production. The principal cellular and molecular requirements needed to produce these autoantibodies have been explained experimentally, but the mechanism(s) accounting for them in vivo in context of lupus nephritis have not yet been determined. However, published data show that defects in nucleases operational in apoptotic or necrotic cell death are not associated with the induction of nephritogenic anti-dsDNA autoantibodies. The data discussed in this study explain how an unusual exposure of chromatin may be a central factor in the evolution of lupus nephritis in (NZB x NZW) F1 mice, but not in promoting nephritogenic chromatin-specific autoimmunity.

Heparin exerts a dual effect on murine lupus nephritis by enhancing enzymatic chromatin degradation and preventing chromatin binding in glomerular membranes.

OBJECTIVE: Association of nucleosome-IgG immune complexes with glomerular basement membranes (GBMs) is an important event in the development of lupus nephritis. Preventing this binding and/or increasing nuclease sensitivity of nucleosomes may be viable strategies for the prevention of the disease. Theoretically, heparin may alter nucleosomal structure and increase sensitivity to proteinases and nucleases, and may also inhibit binding of nucleosomes and nucleosome-IgG complexes to basement membrane structures. The aim of this study was to investigate whether and eventually how heparin prevents murine lupus nephritis. METHODS: Surface plasmon resonance was used to analyze if heparin inhibits binding of nucleosomes to laminin and collagen. The effect of heparin on nuclease- and proteinase-mediated degradation of nucleosomes was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and agarose gel electrophoresis. In vitro results were compared with analyses in vivo in heparin-treated (NZB × NZW)F(1) mice. Anti-double-stranded DNA antibody production, deposition of nucleosome-IgG complexes in GBMs, and development of proteinuria were monitored, and circulating chromatin fragments were quantified using quantitative polymerase chain reaction. RESULTS: In vitro studies demonstrated that heparin increased enzymatic degradation of nucleosomes and almost completely inhibited binding of nucleosomes to laminin and collagen. (NZB × NZW)F(1) mice treated with heparin demonstrated delayed or no antibody production and higher variation of circulating chromatin levels compared with untreated control mice. This effect was accompanied by highly reduced nucleosome-IgG complexes in GBMs and delayed development of nephritis. CONCLUSION: Increasing the degradation of nucleosomes, reducing their immunogenicity, and preventing binding of nucleosome-IgG complexes in glomeruli together provide an alternative basis for the treatment of lupus nephritis.

Deposition of chromatin-IgG complexes in skin of nephritic MRL-lpr/lpr mice is associated with increased local matrix metalloprotease activities.

Chromatin-IgG complexes appear as electron dense structures (EDS) in glomerular basement membranes in lupus nephritis. Here, we present results of comparative analyses of the composition of EDS in murine lupus dermatitis and nephritis. One focus was to perform an analytical approach to understand why such complex structures bind skin basement membrane components. Transcription of skin membrane-encoding genes was analysed to see if expression of such genes was increased, eventually indicating that binding capacity of immune complexes increased when dermatitis developed. Variations in matrix metalloprotease 2 (MMP2), MMP9 and Dnase1 mRNA levels and enzymatic activities were correlated with circulatory chromatin-IgG complexes and deposition in skin. We also examined if glomerular deposits of EDS predicted similar deposits in skin of (NZB x NZW)F1 or MRL-lpr/lpr mice, as we observed chromatin-IgG complexes in capillary lumina in skin and glomeruli in both strains. EDS consisting of chromatin fragments and IgG were found sub-epidermally in skin with LE-like lesions of end-stage nephritic MRL-lpr/lpr mice. Dermal MMP-encoding genes were up-regulated during disease progression, and gelatinolytic activity was increased in affected skin. Dnase1 mRNA level and total nuclease activity remained stable in skin during the disease, in contrast to progressive loss of renal Dnase1 mRNA and total renal nuclease activity during development of nephritis. Loss of renal Dnase1 may explain release of chromatin fragments, while increased MMP activity may disrupt membranes making them accessible for chromatin fragment-IgG complexes. Circulatory chromatin-IgG complexes, and up-regulated intradermal MMP activity may be crucial for deposition of immune complexes in skin of lupus-prone mice.

Lupus nephritis: the central role of nucleosomes revealed.

Systemic lupus erythematosus (SLE) is an autoimmune syndrome characterized by autoantibodies to nuclear constituents. Some of these antibodies are diagnostically important, whereas others act as disease-modifying factors. One clinically important factor is autoantibodies against dsDNA and nucleosomes, which have overlapping diagnostic and nephritogenic impact in SLE. Although a scientific focus for 5 decades, the molecular and cellular origin of these antibodies, and why they are associated with lupus nephritis, is still not fully understood. A consensus has, however, evolved that antibodies to dsDNA and nucleosomes are central pathogenic factors in the development of lupus nephritis. In contrast, no agreement has been reached as to which glomerular structures are bound by nephritogenic anti-nucleosome antibodies in vivo. Mutually contradictory paradigms and models have evolved simply because we still lack precise and conclusive data to provide definitive insight into how autoantibodies induce lupus nephritis and which specificity is critical in the nephritic process(es). In this review, data demonstrating the central role of nucleosomes in inducing and binding potentially nephritogenic antibodies to DNA and nucleosomes are presented and discussed. These autoimmune-inducing processes are discussed in the context of Matzinger's danger model (Matzinger P: Friendly and dangerous signals: is the tissue in control? Nat Immunol 2007, 8:11-13; Matzinger P: The danger model: a renewed sense of self. Science 2002, 296:301-305; Matzinger P: Tolerance, danger, and the extended family. Annu Rev Immunol 1994, 12:991-1045) and Medzhitov's and Janeway's (Medzhitov R, Janeway CA Jr: Decoding the patterns of self and nonself by the innate immune system. Science 2002, 296:298-300; Medzhitov R, Janeway CA Jr: How does the immune system distinguish self from nonself? Semin Immunol 2000, 12:185-188; Janeway CA Jr, Medzhitov R: Innate immune recognition. Annu Rev Immunol 2002, 20:197-216) distinction of noninfectious self (NIS) and infectious nonself (INS). The mechanisms leading to production of potentially nephritogenic anti-nucleosome antibodies and to overt lupus nephritis are interpreted in the context of these paradigms.

Glomerular expression of large polyomavirus T antigen in binary tet-off regulated transgenic mice induces apoptosis, release of chromatin and initiates a lupus-like nephritis.

Binary tetracycline-regulated polyomavirus large T antigen transgenic mice were generated to study immunological tolerance for nucleosomes. Expression of T antigen resulted in binding of the protein to chromatin, and released T antigen-nucleosome complexes from dying cells maintained anti-dsDNA and anti-nucleosome antibody-production by activating autoimmune nucleosome-specific B cells and CD4+ and CD8+ T antigen specific T cells. Glomerular T antigen expression was observed in these mice. Here, we demonstrate that this expression was linked to glomerular cell apoptosis, release of nucleosomes and association of nucleosomes with glomerulus basement membranes, detected as electron dense structures. Immune electron microscopy (IEM) revealed that these structures were glomerular targets for induced anti-dsDNA and anti-T antigen antibodies. Co-localization IEM demonstrated that in vivo-bound auto-antibodies co-localized with experimental monoclonal antibodies to dsDNA and to T antigen. A comparative analysis of glomeruli from nephritic (NZWxNZB)F1 and T antigen expressing transgenic mice revealed deposition of nucleosomes in glomerular capillary and mesangial matrix membranes and binding of anti-nucleosome antibodies in both mice strains. A controlled experimental model that may elucidate the initial events accounting for nucleosome-mediated nephritis has not been available. The transgenic mouse may be important to describe early immunological and cellular events accounting for the enigmatic lupus nephritis.

Evidence at autopsy of spasm in the distal right coronary artery in persons with coronary heart disease dying suddenly.

The aim of this study was to examine whether there are morphological signs in spasm in the coronary arteries at autopsy in persons with coronary artery disease dying suddenly. From a forensic autopsy service, 48 cases of sudden and unexpected deaths were selected: 24 cases with a preliminary diagnosis of coronary heart disease and 24 cases involving persons dying of noncoronary causes. A complete autopsy according to a preset protocol was followed with particular emphasis on the heart examination. The myocardium and the coronary arteries were sampled and examined without knowledge to which group the case belonged. The degree of folding of the internal elastic lamina of the proximal and distal parts of the coronary arteries was measured by picture analysis of elastin-stained cross sections of the arteries. The degree of folding was significantly greater in the distal section of the right coronary artery in cases of the coronary group compared to the folding in the same section in cases of the noncoronary group. In the proximal part of the right coronary artery and in the left coronary artery with its two branches, there were no differences in the folding of the internal elastic membrane between the groups. Our findings indicate that a spasmic contracture of an artery may be diagnosed postmortem. The spasm of the distal part of the right coronary artery may have caused focal ischemia in the central parts of the cardiac conducting system, precipitating a lethal arrhythmia.