Breast cancer in young women and prognosis: How important are proliferation markers?

AIM: Compared to middle-aged women, young women with breast cancer have a higher risk of systemic disease. We studied expression of proliferation markers in relation to age and subtype and their association with long-term prognosis. METHODS: Distant disease-free survival (DDFS) was studied in 504 women aged <40 years and 383 women aged ≥40 years from a population-based cohort. Information on patient characteristics, treatment and follow-up was collected from medical records. Tissue microarrays were produced for analysis of oestrogen receptor, progesterone receptor (PR), Her2, Ki-67 and cyclins. RESULTS: Young women with luminal tumours had significantly higher expression of Ki-67 and cyclins. Proliferation markers were prognostic only within this subtype. Ki-67 was a prognostic indicator only in young women with luminal PR+ tumours. The optimal cut-off for Ki-67 varied by age. High expression of cyclin E1 conferred a better DDFS in women aged <40 years with luminal PR- tumours (hazard ratio [HR] 0.47 [0.24-0.92]). Age <40 years was an independent risk factor of DDFS exclusively in women with luminal B PR+ tumours (HR 2.35 [1.22-4.50]). Young women with luminal B PR- tumours expressing low cyclin E1 had a six-fold risk of distant disease compared with luminal A (HR 6.21 [2.17-17.6]). CONCLUSIONS: The higher expression of proliferation markers in young women does not have a strong impact on prognosis. Ki-67 is only prognostic in the subgroup of young women with luminal PR+ tumours. The only cyclin adding prognostic value beyond subtype is cyclin E1. Age is an independent prognostic factor only in women with luminal B PR+ tumours.

ANLN is a prognostic biomarker independent of Ki-67 and essential for cell cycle progression in primary breast cancer.

BACKGROUND: Anillin (ANLN), an actin-binding protein required for cytokinesis, has recently been presented as part of a prognostic marker panel in breast cancer. The objective of the current study was to further explore the prognostic and functional value of ANLN as a single biomarker in breast cancer. METHODS: Immunohistochemical assessment of ANLN protein expression was performed in two well characterized breast cancer cohorts (n = 484) with long-term clinical follow-up data and the results were further validated at the mRNA level in a publicly available transcriptomics dataset. The functional relevance of ANLN was investigated in two breast cancer cell lines using RNA interference. RESULTS: High nuclear fraction of ANLN in breast tumor cells was significantly associated with large tumor size, high histological grade, high proliferation rate, hormone receptor negative tumors and poor prognosis in both examined cohorts. Multivariable analysis showed that the association between ANLN and survival was significantly independent of age in cohort I and significantly independent of proliferation, as assessed by Ki-67 expression in tumor cells, age, tumor size, ER and PR status, HER2 status and nodal status in cohort II. Analysis of ANLN mRNA expression confirmed that high expression of ANLN was significantly correlated to poor overall survival in breast cancer patients. Consistent with the role of ANLN during cytokinesis, transient knock-down of ANLN protein expression in breast cancer cell lines resulted in an increase of senescent cells and an accumulation of cells in the G2/M phase of the cell cycle with altered cell morphology including large, poly-nucleated cells. Moreover, ANLN siRNA knockdown also resulted in decreased expression of cyclins D1, A2 and B1. CONCLUSIONS: ANLN expression in breast cancer cells plays an important role during cell division and a high fraction of nuclear ANLN expression in tumor cells is correlated to poor prognosis in breast cancer patients, independent of Ki-67, tumor size, hormone receptor status, HER2 status, nodal status and age.

Long-term outcome in young women with breast cancer: a population-based study.

PURPOSE: Whether young age at diagnosis of breast cancer is an independent risk factor for death remains controversial, and the question whether young age should be considered in treatment decisions is still to be answered. METHODS: From a population-based cohort of 22,017 women with breast cancer, all women <35 years (n = 471) were compared to a random sample of 700 women aged 35-69 years from the same cohort. Information on patient and tumor characteristics, treatment, and follow-up was collected from the medical records. Tissue microarrays were produced for analysis of classical biomarkers. Breast cancer-specific survival (BCSS), distant disease-free survival (DDFS), and locoregional recurrence-free survival (LRFS) by age were compared using women 50-69 years as reference. RESULTS: At 10 years follow-up, women <35 years and 35-39 years had a worse BCSS [age <35 years 69 % (HR 2.75, 95 % CI 1.93-3.94), age 35-39 years 76 % (HR 2.33, 95 % CI 1.54-3.52), age 40-49 years 84 % (HR 1.53, 95 % CI 0.97-2.39), and age 50-69 years 89 % (reference)]. The worse BCSS was statistically significant in stages I-IIa and Luminal B tumors. At multivariate analysis age <35 years and 35-39 years confined a risk in LRFS (HR 2.13, 95 % CI 1.21-3.76 and HR 1.97, 95 % CI 1.06-3.68) but not in DDFS and BCSS. In the subgroup of women <40 years with luminal tumors stage I-IIa, low age remained an independent risk factor also in DDFS (HR 1.87, 95 % CI 1.03-3.44). CONCLUSION: Young women have a high risk of systemic disease even when diagnosed in an early stage. The excess risk of relapse is most pronounced in Luminal B tumors, where low age is an independent prognostic factor of DDFS and LRFS.

Specific uptake of an amyloid-ß protofibril-binding antibody-tracer in AßPP transgenic mouse brain.

Evidence suggests that amyloid-ß (Aß) protofibrils/oligomers are pathogenic agents in Alzheimer's disease (AD). Unfortunately, techniques enabling quantitative estimates of these species in patients or patient samples are still rather limited. Here we describe the in vitro and ex vivo characteristics of a new antibody-based radioactive ligand, [125I]mAb158, which binds to Aß protofibrils with high affinity. [125I]mAb158 was specifically taken up in brain of transgenic mice expressing amyloid-ß protein precursor (AßPP) as shown ex vivo. This was in contrast to [125I]mAb-Ly128 which does not bind to Aß. The uptake of intraperitoneally-administered [125I]mAb158 into the brain was age- and time-dependent, and saturable in AßPP transgenic mice with modest Aß deposition. Brain uptake was also found in young AßPP transgenic mice that were devoid of Aß deposits, suggesting that [125I]mAb158 targets soluble Aß protofibrils. The radioligand was diffusely located in the parenchyma, sometimes around senile plaques and only occasionally colocalized with cerebral amyloid angiopathy. A refined iodine-124-labeled version of mAb158 with much improved blood-brain barrier passage and a shorter plasma half-life might be useful for PET imaging of Aß protofibrils.

CDK-mediated activation of the SCF(FBXO) (28) ubiquitin ligase promotes MYC-driven transcription and tumourigenesis and predicts poor survival in breast cancer.

SCF (Skp1/Cul1/F-box) ubiquitin ligases act as master regulators of cellular homeostasis by targeting key proteins for ubiquitylation. Here, we identified a hitherto uncharacterized F-box protein, FBXO28 that controls MYC-dependent transcription by non-proteolytic ubiquitylation. SCF(FBXO28) activity and stability are regulated during the cell cycle by CDK1/2-mediated phosphorylation of FBXO28, which is required for its efficient ubiquitylation of MYC and downsteam enhancement of the MYC pathway. Depletion of FBXO28 or overexpression of an F-box mutant unable to support MYC ubiquitylation results in an impairment of MYC-driven transcription, transformation and tumourigenesis. Finally, in human breast cancer, high FBXO28 expression and phosphorylation are strong and independent predictors of poor outcome. In conclusion, our data suggest that SCF(FBXO28) plays an important role in transmitting CDK activity to MYC function during the cell cycle, emphasizing the CDK-FBXO28-MYC axis as a potential molecular drug target in MYC-driven cancers, including breast cancer.

SATB2 in combination with cytokeratin 20 identifies over 95% of all colorectal carcinomas.

The special AT-rich sequence-binding protein 2 (SATB2), a nuclear matrix-associated transcription factor and epigenetic regulator, was identified as a tissue type-specific protein when screening protein expression patterns in human normal and cancer tissues using an antibody-based proteomics approach. In this respect, the SATB2 protein shows a selective pattern of expression and, within cells of epithelial lineages, SATB2 expression is restricted to glandular cells lining the lower gastrointestinal tract. The expression of SATB2 protein is primarily preserved in cancer cells of colorectal origin, indicating that SATB2 could function as a clinically useful diagnostic marker to distinguish colorectal cancer (CRC) from other types of cancer. The aim of this study was to further explore and validate the specific expression pattern of SATB2 as a clinical biomarker and to compare SATB2 with the well-known cytokeratin 20 (CK20). Immunohistochemistry was used to analyze the extent of SATB2 expression in tissue microarrays with tumors from 9 independent cohorts of patients with primary and metastatic CRCs (n=1882). Our results show that SATB2 is a sensitive and highly specific marker for CRC with distinct positivity in 85% of all CRCs, and that SATB2 and/or CK20 was positive in 97% of CRCs. In conclusion, the specific expression of SATB2 in a large majority of CRCs suggests that SATB2 can be used as an important complementary tool for the differential diagnosis of carcinoma of unknown primary origin.

Human tumors instigate granulin-expressing hematopoietic cells that promote malignancy by activating stromal fibroblasts in mice.

Systemic instigation is a process by which endocrine signals sent from certain tumors (instigators) stimulate BM cells (BMCs), which are mobilized into the circulation and subsequently foster the growth of otherwise indolent carcinoma cells (responders) residing at distant anatomical sites. The identity of the BMCs and their specific contribution or contributions to responder tumor growth have been elusive. Here, we have demonstrated that Sca1+ cKit- hematopoietic BMCs of mouse hosts bearing instigating tumors promote the growth of responding tumors that form with a myofibroblast-rich, desmoplastic stroma. Such stroma is almost always observed in malignant human adenocarcinomas and is an indicator of poor prognosis. We then identified granulin (GRN) as the most upregulated gene in instigating Sca1+ cKit- BMCs relative to counterpart control cells. The GRN+ BMCs that were recruited to the responding tumors induced resident tissue fibroblasts to express genes that promoted malignant tumor progression; indeed, treatment with recombinant GRN alone was sufficient to promote desmoplastic responding tumor growth. Further, analysis of tumor tissues from a cohort of breast cancer patients revealed that high GRN expression correlated with the most aggressive triple-negative, basal-like tumor subtype and reduced patient survival. Our data suggest that GRN and the unique hematopoietic BMCs that produce it might serve as novel therapeutic targets.

Molecular profiling using tissue microarrays as a tool to identify predictive biomarkers in laryngeal cancer treated with radiotherapy.

AIM: To explore the usefulness of the expression of five potential cancer biomarkers in predicting outcome in patients with laryngeal cancer. MATERIALS AND METHODS: In the present study, the Swedish National Cancer Registry databases were used to identify patients with laryngeal cancer diagnosed during the years 1978-2004 in the Uppsala-Orebro region and treated with radiotherapy. The expression of Ki-67, MutS homolog 2, (MSH2), p53, B-cell CLL/lymphoma 2 (Bcl-2) and cyclin D1 in the cancer cells was assessed immunohistochemically using tissue microarrays (TMAs) and its predicitve value on survival and relapse was analyzed using Cox regression models. RESULTS: A total of 39 patients were included in the present study. Nuclear MSH2 staining was statistically significantly correlated to Ki-67 expression (p=0.022). However, univariate and multivariate Cox analyses showed no statistically significant association between the expression of the investigated biomarkers and overall survival or relapse. CONCLUSION: The present exploratory study does not show any significant predictive value of the biomarkers examined with respect to survival or relapse. However, with larger patient cohorts, we believe that protein profiling using TMAs and immunohistochemistry is a feasible strategy for prognostic and predictive biomarker screening in laryngeal cancer.

Nuclear expression of the RNA-binding protein RBM3 is associated with an improved clinical outcome in breast cancer.

Single-strand RNA-binding proteins (RBPs) are involved in many aspects of RNA metabolism and in the regulation of gene transcription. The RBP RBM3 was recently suggested to be a proto-oncogene in colorectal cancer; however, such a role has not been corroborated by previous studies in the colon or other tumor types, and the prognostic implications of tumor-specific RBM3 expression remain unclear. Mono-specific antibodies against RBM3 were generated. Antibody specificity was confirmed using siRNA gene silencing, western blotting and immunohistochemistry on a panel of breast cancer cell lines. Using tissue microarrays and IHC, RBM3 protein expression was examined in 48 normal tissues and in 20 common cancers. Additional analysis in two independent breast cancer cohorts (n=1016) with long-term follow-up was also carried out. RBM3 was upregulated in cancer compared to normal tissues. The nuclear expression of RBM3 in breast cancer was associated with low grade (P<0.001), small tumors (P<0.001), estrogen receptor (ER) positivity (P<0.001) and Ki-67 negativity (P<0.001) in both the breast cancer cohorts. An increased nuclear expression of RBM3 was associated with a prolonged overall and recurrence-free survival. The prognostic value was particularly pronounced in hormone receptor-positive tumors and remained significant in multivariate interaction analysis after controlling for tamoxifen treatment (HR: 0.49, 95% CI: 0.30-0.79, P=0.004). These data strongly indicate that nuclear RBM3 is an independent favorable prognostic factor in breast cancer, and seems to have a specific role in ER-positive tumors.

Nandrolone decanoate administration elevates hippocampal prodynorphin mRNA expression and impairs Morris water maze performance in male rats.

The misuse of anabolic androgenic steroids has in several reports been associated with effects resulting in altered behavior. This study used the Morris water maze task to investigate the effect of high doses of the anabolic androgenic steroid nandrolone on spatial learning and memory in male rats. During the experiment, we observed a significantly impaired Morris water maze performance in the nandrolone-treated rats compared with controls. The hippocampus, a brain region associated with cognitive function, was analyzed for mRNA expression of prodynorphin, the precursor of dynorphinergic peptides. The results indicated that the transcription levels of prodynorphin were significantly elevated in the animals treated with nandrolone compared with controls. Thus, the findings suggest that administration of nandrolone to male rats impairs memory function, possibly via dynorphinergic actions.

Selective expression of Syntaxin-7 protein in benign melanocytes and malignant melanoma.

To search for proteins expressed in human melanocytes and melanoma, we employed an antibody-based proteomics strategy to screen for protein expression in tissue microarrays containing normal tissues, cancer tissues and cell lines. Syntaxin-7 (STX7) was identified as a novel protein, not previously characterized in cells of melanocytic lineage, displaying a cell type-specific protein expression pattern. In tumor tissues, STX7 was expressed in malignant melanoma and lymphoma. The protein was further characterized regarding subcellular localization, specificity, tissue distribution pattern and potential as a diagnostic and prognostic marker using cell lines and tissue microarrays containing normal skin, melanocytic nevi and primary and metastatic melanoma. STX7 was expressed in normal melanocytes, various benign melanocytic nevi, atypical nevi and malignant melanoma. Analysis in two independent melanoma cohorts demonstrated STX7 expression in nearly all investigated tumors, although at varying levels (> 90% positive tumors). The expression level of STX7 protein was inversely correlated to tumor stage, suggesting that decreased expression of STX7 is associated with more aggressive tumors. In conclusion, we present protein profiling data for a novel protein showing high sensitivity and specificity for cells of the melanocytic lineage. The presented antibody-based proteomics approach can be used as an effective strategy to identify novel tumor markers and evaluate their potential clinical relevance.

The Arctic Alzheimer mutation favors intracellular amyloid-beta production by making amyloid precursor protein less available to alpha-secretase.

Mutations within the amyloid-beta (Abeta) domain of the amyloid precursor protein (APP) typically generate hemorrhagic strokes and vascular amyloid angiopathy. In contrast, the Arctic mutation (APP E693G) results in Alzheimer's disease. Little is known about the pathologic mechanisms that result from the Arctic mutation, although increased formation of Abeta protofibrils in vitro and intraneuronal Abeta aggregates in vivo suggest that early steps in the amyloidogenic pathway are facilitated. Here we show that the Arctic mutation favors proamyloidogenic APP processing by increased beta-secretase cleavage, as demonstrated by altered levels of N- and C-terminal APP fragments. Although the Arctic mutation is located close to the alpha-secretase site, APP harboring the Arctic mutation is not an inferior substrate to a disintegrin and metalloprotease-10, a major alpha-secretase. Instead, the localization of Arctic APP is altered, with reduced levels at the cell surface making Arctic APP less available for alpha-secretase cleavage. As a result, the extent and subcellular location of Abeta formation is changed, as revealed by increased Abeta levels, especially at intracellular locations. Our findings suggest that the unique clinical symptomatology and neuropathology associated with the Arctic mutation, but not with other intra-Abeta mutations, could relate to altered APP processing with increased steady-state levels of Arctic Abeta, particularly at intracellular locations.

Increase in [11C]vorozole binding to aromatase in the hypothalamus in rats treated with anabolic androgenic steroids.

In the present study, we investigated the alteration of aromatase expression in the brain by anabolic androgenic steroid treatment in male rats. The rats were given nandrolone decanoate (15 mg/kg/day) for 14 days, and the brains were used for autoradiography with [C]vorozole, which has been developed as a positron emission tomography tracer for aromatase by our group. The results indicated a significant increase of [C]vorozole binding by anabolic androgenic steroids in the bed nucleus of the stria terminalis and preoptic area. In contrast, no significant change of [C]vorozole binding was observed in the medial amygdala. Our results suggest that aromatase is significantly upregulated in the bed nucleus of the stria terminalis and preoptic area by anabolic androgenic steroids and also suggest that androgens regulate aromatase differently in these structures compared with the medial amygdala.

Enzymatic conversion of dynorphin A in the rat brain is affected by administration of nandrolone decanoate.

The misuse of anabolic androgenic steroids (AAS) seems to produce profound effects on the central nervous system, leading to aggressive behavior and increased sensitivity to other drugs of abuse. The present study addresses the effect on the enzymatic transformation, here called dynorphin converting enzyme-like activity. The formation of the mu/delta opioid peptide receptor-preferring Leu-enkephalin-Arg(6) from the kappa opioid peptide receptor-preferring dynorphin A was measured in rats treated with nandrolone decanoate. Significant variations in enzymatic transformation were observed in several brain regions. An altered receptor activation profile in these regions may be one contributory factor behind AAS-induced personality changes.

Administration of the anabolic androgenic steroid nandrolone decanoate affects substance P endopeptidase-like activity in the rat brain.

The effect of the anabolic androgenic steroid, nandrolone decanoate, on substance P endopeptidase-like activity was examined in adult male Sprague-Dawley rats. Nandrolone decanoate (15 mg/kg day) or oil vehicle (sterile arachidis oleum) were administered by intramuscular injections during 14 days. Substance P endopeptidase, a predominantly cytosolic enzyme, generates the bioactive N-terminal fragment substance P(1-7) from the enzyme substrate substance P. Nandrolone decanoate significantly reduced the substance P endopeptidase-like activity compared to control animals in hypothalamus (43% reduction), caudate putamen (44%), substantia nigra (32%) and the ventral tegmental area (27%). It was previously reported that both hypothalamus and caudate putamen contained significantly higher levels of substance P after nandrolone administration. The higher concentration of substance P in these regions could to an extent be attributed to the reduction in substance P endopeptidase-like activity. This result elucidates the important role of peptidase activity in the regulation of the substance P transmitter system. The present study provides additional support for the hypothesis that alterations in the substance P system in certain brain areas may contribute to some of the personality changes reported in connection with AAS abuse.

Nandrolone treatment decreases the alpha1B-adrenoceptor mRNA level in rat kidney, but not the density of alpha1B-adrenoceptors in cultured MDCK-D1 kidney cells.

We have previously shown that treatment of rats with the anabolic androgen steroid nandrolone decreased the density of alpha1B-adrenoceptors in the rat kidney [Uhlén, S., Lindblom, J., Kindlundh, A., Muhisha, P., Nyberg, F., (2003). Nandrolone treatment decreases the level of rat kidney alpha1B-adrenoceptors. Naunyn-Schmiedeberg's Arch. Pharmacol. 368, 91-98]. This effect may have been caused either by decreased de novo synthesis of alpha1B-adrenoceptors or by increased degradation of alpha1B-adrenoceptors [corrected] In the present study, we show that treatment of rats with nandrolone decreases the level of mRNA for the alpha1B-adrenoceptor in the kidneys, implying decreased synthesis of alpha1B-adrenoceptors. On the other hand, nandrolone did not decrease the density of alpha1B-adrenoceptors in Madin-Darby Canine Kidney (MDCK) cells, even though the sub-cell line tested, MDCK-D1, expresses both the androgen receptor and the alpha1B-adrenoceptor. It is concluded that the regulation of alpha1B-adrenoceptor expression by anabolic androgenic steroids is intricate and cell-type specific.

A human protein atlas for normal and cancer tissues based on antibody proteomics.

Antibody-based proteomics provides a powerful approach for the functional study of the human proteome involving the systematic generation of protein-specific affinity reagents. We used this strategy to construct a comprehensive, antibody-based protein atlas for expression and localization profiles in 48 normal human tissues and 20 different cancers. Here we report a new publicly available database containing, in the first version, approximately 400,000 high resolution images corresponding to more than 700 antibodies toward human proteins. Each image has been annotated by a certified pathologist to provide a knowledge base for functional studies and to allow queries about protein profiles in normal and disease tissues. Our results suggest it should be possible to extend this analysis to the majority of all human proteins thus providing a valuable tool for medical and biological research.