Serial explant culture provides novel insights into the potential location and phenotype of corneal endothelial progenitor cells

The routine cultivation of human corneal endothelial cells, with the view to treating patients with endothelial dysfunction, remains a challenging task. While progress in this field has been buoyed by the proposed existence of progenitor cells for the corneal endothelium at the corneal limbus, strategies for exploiting this concept remain unclear. In the course of evaluating methods for growing corneal endothelial cells, we have noted a case where remarkable growth was achieved using a serial explant culture technique. Over the course of 7 months, a single explant of corneal endothelium, acquired from cadaveric human tissue, was sequentially seeded into 7 culture plates and on each occasion produced a confluent cell monolayer. Sample cultures were confirmed as endothelial in origin by positive staining for glypican-4. On each occasion, small cells, closest to the tissue explant, developed into a highly compact layer with an almost homogenous structure. This layer was resistant to removal with trypsin and produced continuous cell outgrowth during multiple culture periods. The small cells gave rise to larger cells with phase-bright cell boundaries and prominent immunostaining for both nestin and telomerase. Nestin and telomerase were also strongly expressed in small cells immediately adjacent to the wound site, following transfer of the explant to another culture plate. These findings are consistent with the theory that progenitor cells for the corneal endothelium reside within the limbus and provide new insights into expected expression patterns for nestin and telomerase within the differentiation pathway.

Development of reverse phase protein microarrays for clinical applications and patient-tailored therapy

While genomics provide important information about the somatic genetic changes, and RNA transcript profiling can reveal important expression changes that correlate with outcome and response to therapy, it is the proteins that do the work in the cell. At a functional level, derangements within the proteome, driven by post-translational and epigenetic modifications, such as phosphorylation, is the cause of a vast majority of human diseases. Cancer, for instance, is a manifestation of deranged cellular protein molecular networks and cell signaling pathways that are based on genetic changes at the DNA level. Importantly, the protein pathways contain the drug targets in signaling networks that govern overall cellular survival, proliferation, invasion and cell death. Consequently, the promise of proteomics resides in the ability to extend analysis beyond correlation to causality. A critical gap in the information knowledge base of molecular profiling is an understanding of the ongoing activity of protein signaling in human tissue: what is activated and “in use” within the human body at any given point in time. To address this gap, we have invented a new technology, called reverse phase protein microarrays, that can generate a functional read-out of cell signaling networks or pathways for an individual patient obtained directly from a biopsy specimen. This “wiring diagram” can serve as the basis for both, selection of a therapy and patient stratification.

Glutathione transferase activities in renal carcinomas and adjacent normal renal tissues : factors influencing renal carcinogenesis induced by xenobiotics

In general, the biological activation of nephrocarcinogenic chlorinated hydrocarbons proceeds via conjugatiton with glutathione. It has mostly been assamed that the main site of initial conjugation is the liver, followed by a mandatory transfer of intermediates to the kidney. It was therefore of interest to study the enzyme activities of subgroups of glutathione transferases (GSTs) in renal cancers and the surrounding normal renal tissues of the same individuals (n = 21). For genotyping the individuals with respect to known polymorphic GST isozymes the following substrates with differential specificity were used: 1-chloro-2,4-dinitrobenzene for overall GST activity (except GST θ); 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole for GST α; 1,2-dichloro-4-nitro-benzene for GST μ; ethacrynic acid and 4-vinylpyridine for GST π; and methyl chloride for GST θ. In general, the normal tissues were able to metabolize the test substrates. A general decrease in individual GST enzyme activities was apparent in the course of cancerization, and in some (exceptional) cases individual activities, expressed in the normal renal tissue, were lost in the tumour tissue. The GST enzyme activities in tumours were independent of tumour stage, or the age and gender of the patients. There was little influence of known polymorphisms of GSTM1, GSTM3 and GSTP1 upon the activities towards the test substrates, whereas the influence of GSTT1 polymorphism on the activity towads methyl chloride was straightforward. In general, the present findings support the concept that the initial GST-dependent bioactivation step of nephrocarcinogenic chlorinated hydrocarbons may take place in the kidney itself. This should be a consideration in toxicokinetic modelling.

Species differences in the glutathione transferase GSTT1-1 activity towards the model substrates methyl chloride and dichloromethane in liver and kidney

Glutathione transferase (GST) GSTT1-1 is involved in the biotransformation of several chemicals widely used in industry, such as butadiene and dichloro methane DCM. The polymorphic hGSTT1-1 may well play a role in the development of kidney tumours after high and long-term occupational exposure against trichloroethylene. Although several studies have investigated the association of this polymorphism with malignant diseases little is known about its enzyme activity in potential extrahepatic target tissues. The known theta-specific substrates methyl chloride (MC) dichloromethane and 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) were used to assay GSTT1-1 activity in liver and kidney of rats, mice, hamsters and humans differentiating the three phenotypes (non-conjugators, low conjugators, high conjugators) seen in humans. In addition GSTT1-1 activity towards MC and DCM was determined in human erythrocytes. No GSTT1-1 activity was found in any tissue of non-conjugators (NC). In all organs high conjugators (HC) showed twofold higher activity towards MC and DCM than low conjugators (LC). The activity in human samples towards EPNP was too close to the detection limit to differentiate between the three conjugator phenotypes. GSTT1-1 activity towards MC was two to seven-times higher in liver cytosol than in kidney cytosol. The relation for MC between species was identical in both organs: mouse > HC > rat > LC > hamster > NC. In rats, mice and hamsters GSTT1-1 activity in liver cytosol towards DCM was also two to seven-times higher than in the kidney cytosol. In humans this activity was twice as high in kidney cytosol than in liver cytosol. The relation between species was mouse > rat > HC > LC > hamster > NC for liver, but mouse > HC > LC/rat > hamster/NC for kidney cytosol. The importance to heed the specific environment at potential target sites in risk assessment is emphasized by these results.

DNA methylation at the novel CpG sites in the promoter of MED15/PCQAP gene as a biomarker for head and neck cancers

Head and neck cancers (HNCs) represent a significant and ever-growing burden to the modern society, mainly due to the lack of early diagnostic methods. A significant number of HNCs is often associated with drinking, smoking, chewing beetle nut, and human papilloma virus (HPV) infections. We have analyzed DNA methylation patterns in tumor and normal tissue samples collected from head and neck squamous cell carcinoma (HNSCC) patients who were smokers. We have identified novel methylation sites in the promoter of the mediator complex subunit 15 (MED15/PCQAP) gene (encoing a co-factor important for regulation of transcription initiation for promoters of many genes), hypermethylated specifically in tumor cells. Two clusters of CpG dinucleotides methylated in tumors, but not in normal tissue from the same patients, were identified. These CpG methylation events in saliva samples were further validated in a separate cohort of HNSCC patients (who developed cancer due to smoking or HPV infections) and healthy controls using methylation-specific PCR (MSP). We used saliva as a biological medium because of its non-invasive nature, close proximity to the tumors, easiness and it is an economically viable option for large-scale screening studies. The methylation levels for the two identified CpG clusters were significantly different between the saliva samples collected from healthy controls and HNSCC individuals (Welch's t-test returning P, 0.05 and Mann-Whitney test P, 0.01 for both). The developed MSP assays also provided a good discriminative ability with AUC values of 0.70 (P, 0.01) and 0.63 (P, 0.05). The identified novel CpG methylation sites may serve as potential non-invasive biomarkers for detecting HNSCC. © the authors.

The MyD88+ phenotype is an adverse prognostic factor in epithelial ovarian cancer

The prognosis of epithelial ovarian cancer is poor in part due to the high frequency of chemoresistance. Recent evidence points to the Toll-like receptor-4 (TLR4), and particularly its adaptor protein MyD88, as one potential mediator of this resistance. This study aims to provide further evidence that MyD88 positive cancer cells are clinically significant, stem-like and reproducibly detectable for the purposes of prognostic stratification. Expression of TLR4 and MyD88 was assessed immunohistochemically in 198 paraffin-embedded ovarian tissues and in an embryonal carcinoma model of cancer stemness. In parallel, expression of TLR4 and MyD88 mRNA and regulatory microRNAs (miR-21 and miR-146a) was assessed, as well as in a series of chemosensitive and resistant cancer cells lines. Functional analysis of the pathway was assessed in chemoresistant SKOV-3 ovarian cancer cells. TLR4 and MyD88 expression can be reproducibly assessed via immunohistochemistry using a semi-quantitative scoring system. TLR4 expression was present in all ovarian epithelium (normal and neoplastic), whereas MyD88 was restricted to neoplastic cells, independent of tumour grade and associated with reduced progression-free and overall survival, in an immunohistological specific subset of serous carcinomas, p<0.05. MiR-21 and miR-146a expression was significantly increased in MyD88 negative cancers (p<0.05), indicating their participation in regulation. Significant alterations in MyD88 mRNA expression were observed between chemosensitive and chemoresistant cells and tissue. Knockdown of TLR4 in SKOV-3 ovarian cells recovered chemosensitivity. Knockdown of MyD88 alone did not. MyD88 expression was down-regulated in differentiated embryonal carcinoma (NTera2) cells, supporting the MyD88+ cancer stem cell hypothesis. Our findings demonstrate that expression of MyD88 is associated with significantly reduced patient survival and altered microRNA levels and suggest an intact/functioning TLR4/MyD88 pathway is required for acquisition of the chemoresistant phenotype. Ex vivo manipulation of ovarian cancer stem cell (CSC) differentiation can decrease MyD88 expression, providing a potentially valuable CSC model for ovarian cancer.

Isolation of microvascular endothelial cells from cadaveric corneal limbus

Limbal microvascular endothelial cells (L-MVEC) contribute to formation of the corneal-limbal stem cell niche and to neovascularization of diseased and injuries corneas. Nevertheless, despite these important roles in corneal health and disease, few attempts have been made to isolate L-MVEC with the view to studying their biology in vitro. We therefore explored the feasibility of generating primary cultures of L-MVEC from cadaveric human tissue. We commenced our study by evaluating growth conditions (MesenCult-XF system) that have been previously found to be associated with expression of the endothelial cell surface marker thrombomodulin/CD141, in crude cultures established from collagenase-digests of limbal stroma. The potential presence of L-MVEC in these cultures was examined by flow cytometry using a more specific marker for vascular endothelial cells, CD31/PECAM-1. These studies demonstrated that the presence of CD141 in crude cultures established using the MesenCult-XF system is unrelated to L-MVEC. Thus we subsequently explored the use of magnetic assisted cell sorting (MACS) for CD31 as a tool for generating cultures of L-MVEC, in conjunction with more traditional endothelial cell growth conditions. These conditions consisted of gelatin-coated tissue culture plastic and MCDB-131 medium supplemented with fetal bovine serum (10% v/v), D-glucose (10 mg/mL), epidermal growth factor (10 ng/mL), heparin (50 μg/mL), hydrocortisone (1 μg/mL) and basic fibroblast growth factor (10 ng/mL). Our studies revealed that use of endothelial growth conditions are insufficient to generate significant numbers of L-MVEC in primary cultures established from cadaveric corneal stroma. Nevertheless, through use of positive-MACS selection for CD31 we were able to routinely observe L-MVEC in cultures derived from collagenase-digests of limbal stroma. The presence of L-MVEC in these cultures was confirmed by immunostaining for von Willebrand factor (vWF) and by ingestion of acetylated low-density lipoprotein. Moreover, the vWF+ cells formed aligned cell-to-cell ‘trains’ when grown on Geltrex™. The purity of L-MVEC cultures was found to be unrelated to tissue donor age (32 to 80 years) or duration in eye bank corneal preservation medium prior to use (3 to 10 days in Optisol) (using multiple regression test). Optimal purity of L-MVEC cultures was achieved through use of two rounds of positive-MACS selection for CD31 (mean ± s.e.m, 65.0 ± 20.8%; p<0.05). We propose that human L-MVEC cultures generated through these techniques, in conjunction with other cell types, will provide a useful tool for exploring the mechanisms of blood vessel cell growth in vitro.

A linkage study across the T cell receptor A and T cell receptor B loci in families with rheumatoid arthritis

Objective. To examine whether the T cell receptor (TCR) A or TCRB loci exhibit linkage with disease in multiplex rheumatoid arthritis (RA) families. Methods. A linkage study was performed in 184 RA families from the UK Arthritis and Rheumatism Council Repository, each containing at least 1 affected sibpair. The microsatellites D14S50, TCRA, and D14S64 spanning the TCRA locus and D7S509, Vβ6.7, and D7S688 spanning the TCRB locus were used as DNA markers. The subjects were genotyped using a semiautomated polymerase chain reaction-based method. Two-point and multipoint linkage analyses were performed. Results. Nonparametric single-marker likelihood odds (LOD) scores were 0.49 (P = 0.07) for D14S50, 0.65 (P = 0.04) for TCRA, 0.07 (P = 0.29) for D14S64, 0.01 (P = 0.43) for D7S509, 0.0 (P = 0.50) for Vβ6.7, and 0.0 (P = 0.50) for D7S688. By multipoint analysis, there was no evidence of linkage at TCRB (LOD score 0), and the maximum LOD score at the TCRA locus was 0.37 (at D14S50). The presence of a susceptibility locus (LOD score < -2.0) was excluded, with lambda ≤ 1.8 at TCRA and ≤1.4 at TCRB. Conclusion. These linkage studies provide no significant evidence of a major germline-encoded TCRA or TCRB component of susceptibility to RA.

Brief report: Intestinal dysbiosis in ankylosing spondylitis

Objective Ankylosing spondylitis (AS) is a common, highly heritable immune-mediated arthropathy that occurs in genetically susceptible individuals exposed to an unknown but likely ubiquitous environmental trigger. There is a close relationship between the gut and spondyloarthritis, as exemplified in patients with reactive arthritis, in whom a typically self-limiting arthropathy follows either a gastrointestinal or urogenital infection. Microbial involvement in AS has been suggested; however, no definitive link has been established. The aim of this study was to determine whether the gut in patients with AS carries a distinct microbial signature compared with that in the gut of healthy control subjects. Methods Microbial profiles for terminal ileum biopsy specimens obtained from patients with recent-onset tumor necrosis factor antagonist-naive AS and from healthy control subjects were generated using culture-independent 16S ribosomal RNA gene sequencing and analysis techniques. Results Our results showed that the terminal ileum microbial communities in patients with AS differ significantly (P < 0.001) from those in healthy control subjects, driven by a higher abundance of 5 families of bacteria (Lachnospiraceae [P = 0.001], Ruminococcaceae [P = 0.012], Rikenellaceae [P = 0.004], Porphyromonadaceae [P = 0.001], and Bacteroidaceae [P = 0.001]) and a decrease in the abundance of 2 families of bacteria (Veillonellaceae [P = 0.01] and Prevotellaceae [P = 0.004]). Conclusion We show evidence for a discrete microbial signature in the terminal ileum of patients with AS compared with healthy control subjects. The microbial composition was demonstrated to correlate with disease status, and greater differences were observed between disease groups than within disease groups. These results are consistent with the hypothesis that genes associated with AS act, at least in part, through effects on the gut microbiome.

Unique and cross-reactive T cell epitope peptides of the major bahia grass pollen allergen, pas n 1

Background Bahia grass pollen (BaGP) is a major cause of allergic rhinitis. Subcutaneous allergen-specific immunotherapy is effective for grass pollen allergy, but is unsuitable for patients with moderate to severe asthma due to the risk of anaphylaxis. T cell-reactive but IgE nonreactive peptides provide a safer treatment option. This study aimed to identify and characterize dominant CD4+ T cell epitope peptides of the major BaGP allergen, Pas n 1. Methods Pas n 1-specific T cell lines generated from the peripheral blood of BaGP-allergic subjects were tested for proliferative and cytokine response to overlapping 20-mer Pas n 1 peptides. Cross-reactivity to homologous peptides from Lol p 1 and Cyn d 1 of Ryegrass and Bermuda grass pollen, respectively, was assessed using Pas n 1 peptide-specific T cell clones. MHC class II restriction of Pas n 1 peptide T cell recognition was determined by HLA blocking assays and peptide IgE reactivity tested by dot blotting. Results Three Pas n 1 peptides showed dominant T cell reactivity; 15 of 18 (83%) patients responded to one or more of these peptides. T cell clones specific for dominant Pas n 1 peptides showed evidence of species-specific T cell reactivity as well as cross-reactivity with other group 1 grass pollen allergens. The dominant Pas n 1 T cell epitope peptides showed HLA binding diversity and were non-IgE reactive. Conclusions The immunodominant T cell-reactive Pas n 1 peptides are candidates for safe immunotherapy for individuals, including those with asthma, who are allergic to Bahia and possibly other grass pollens.

Towards the pathogenesis of autoimmune liver disease

There have been recent improvements in the clinical understanding and definition of the major types of autoimmune liver disease. However, still lacking is knowledge of their prevalence and pathogenesis. Three areas of study are in progress in our laboratory. First, in type 1 autoimmune hepatitis, the search continues to identify a liver/disease-specific autoantigenic reactant. Using hepatocyte membrane preparations, immunoblotting has underlined the problem of distinguishing, among multiple reactants, those that may be causally rather than consequentially related to hepatocellular damage. Second, in primary biliary cirrhosis (PBC), the need for population screening to ascertain prevalence and detect preclinical cases can be met by a rapid automated procedure for detection, by specific enzyme inhibition in microtitre wells, of antibody (anti-M2) to the pyruvate dehydrogenase complex E2 subunit (PDC-E2). Third, the structure of the conformational epitope within the inner lipoyl domain of PDC-E2 is being investigated by screening random phage-displayed peptide libraries using PBC sera. This has yielded phage clones in which the sequence of the peptide insert portrays the structure of this epitope, as judged by clustering of PBC-derived sequences to particular branches of a guide-tree that shows relatedness of peptides, and by reactivity of selected phage clones with anti-PDC-E2. Thus phage display identifies a peptide 'mimotope' of the antibody epitope in the inner lipoyl domain of PDC-E2.

Cardiometabolic effects of genetic upregulation of the interleukin 1 receptor antagonist: A Mendelian randomisation analysis

Background To investigate potential cardiovascular and other effects of long-term pharmacological interleukin 1 (IL-1) inhibition, we studied genetic variants that produce inhibition of IL-1, a master regulator of inflammation. Methods We created a genetic score combining the effects of alleles of two common variants (rs6743376 and rs1542176) that are located upstream of IL1RN, the gene encoding the IL-1 receptor antagonist (IL-1Ra; an endogenous inhibitor of both IL-1α and IL-1β); both alleles increase soluble IL-1Ra protein concentration. We compared effects on inflammation biomarkers of this genetic score with those of anakinra, the recombinant form of IL-1Ra, which has previously been studied in randomised trials of rheumatoid arthritis and other inflammatory disorders. In primary analyses, we investigated the score in relation to rheumatoid arthritis and four cardiometabolic diseases (type 2 diabetes, coronary heart disease, ischaemic stroke, and abdominal aortic aneurysm; 453 411 total participants). In exploratory analyses, we studied the relation of the score to many disease traits and to 24 other disorders of proposed relevance to IL-1 signalling (746 171 total participants). Findings For each IL1RN minor allele inherited, serum concentrations of IL-1Ra increased by 0·22 SD (95% CI 0·18–0·25; 12·5%; p=9·3 × 10−33), concentrations of interleukin 6 decreased by 0·02 SD (−0·04 to −0·01; −1·7%; p=3·5 × 10−3), and concentrations of C-reactive protein decreased by 0·03 SD (−0·04 to −0·02; −3·4%; p=7·7 × 10−14). We noted the effects of the genetic score on these inflammation biomarkers to be directionally concordant with those of anakinra. The allele count of the genetic score had roughly log-linear, dose-dependent associations with both IL-1Ra concentration and risk of coronary heart disease. For people who carried four IL-1Ra-raising alleles, the odds ratio for coronary heart disease was 1·15 (1·08–1·22; p=1·8 × 10−6) compared with people who carried no IL-1Ra-raising alleles; the per-allele odds ratio for coronary heart disease was 1·03 (1·02–1·04; p=3·9 × 10−10). Per-allele odds ratios were 0·97 (0·95–0·99; p=9·9 × 10−4) for rheumatoid arthritis, 0·99 (0·97–1·01; p=0·47) for type 2 diabetes, 1·00 (0·98–1·02; p=0·92) for ischaemic stroke, and 1·08 (1·04–1·12; p=1·8 × 10−5) for abdominal aortic aneurysm. In exploratory analyses, we observed per-allele increases in concentrations of proatherogenic lipids, including LDL-cholesterol, but no clear evidence of association for blood pressure, glycaemic traits, or any of the 24 other disorders studied. Modelling suggested that the observed increase in LDL-cholesterol could account for about a third of the association observed between the genetic score and increased coronary risk. Interpretation Human genetic data suggest that long-term dual IL-1α/β inhibition could increase cardiovascular risk and, conversely, reduce the risk of development of rheumatoid arthritis. The cardiovascular risk might, in part, be mediated through an increase in proatherogenic lipid concentrations. Funding UK Medical Research Council, British Heart Foundation, UK National Institute for Health Research, National Institute for Health Research Cambridge Biomedical Research Centre, European Research Council, and European Commission Framework Programme 7.

Utility of temporal artery biopsy samples for genome-wide analysis of giant cell arteritis

Giant Cell Arteritis (GCA) is the most common vasculitis affecting the elderly. Archived formalin-fixed paraffin-embedded (FFPE) temporal artery biopsy (TAB) specimens potentially represent a valuable resource for large-scale genetic analysis of this disease. FFPE TAB samples were obtained from 12 patients with GCA. Extracted TAB DNA was assessed by real time PCR before restoration using the Illumina HD FFPE Restore Kit. Paired FFPE-blood samples were genotyped on the Illumina OmniExpress FFPE microarray. The FFPE samples that passed stringent quality control measures had a mean genotyping success of >97%. When compared with their matching peripheral blood DNA, the mean discordant heterozygote and homozygote single nucleotide polymorphisms calls were 0.0028 and 0.0003, respectively, which is within the accepted tolerance of reproducibility. This work demonstrates that it is possible to successfully obtain high-quality microarray-based genotypes FFPE TAB samples and that this data is similar to that obtained from peripheral blood.

High-resolution SNP microarray investigation of copy number variations on chromosome 18 in a control cohort

Copy number variations (CNVs) as described in the healthy population are purported to contribute significantly to genetic heterogeneity. Recent studies have described CNVs using lymphoblastoid cell lines or by application of specifically developed algorithms to interrogate previously described data. However, the full extent of CNVs remains unclear. Using high-density SNP array, we have undertaken a comprehensive investigation of chromosome 18 for CNV discovery and characterisation of distribution and association with chromosome architecture. We identified 399 CNVs, of which loss represents 98%, 58% are less than 2.5 kb in size and 71% are intergenic. Intronic deletions account for the majority of copy number changes with gene involvement. Furthermore, one-third of CNVs do not have putative breakpoints within repetitive sequences. We conclude that replicative processes, mediated either by repetitive elements or microhomology, account for the majority of CNVs in the healthy population. Genomic instability involving the formation of a non-B structure is demonstrated in one region.

Mapping the regulatory sequences controlling 93 breast cancer-associated miRNA genes leads to the identification of two functional promoters of the Hsa-mir-200b cluster, methylation of which is associated with metastasis or hormone receptor status in advanced breast cancer

MicroRNAs (miRNAs) are small non-coding RNAs of 20 nt in length that are capable of modulating gene expression post-transcriptionally. Although miRNAs have been implicated in cancer, including breast cancer, the regulation of miRNA transcription and the role of defects in this process in cancer is not well understood. In this study we have mapped the promoters of 93 breast cancer-associated miRNAs, and then looked for associations between DNA methylation of 15 of these promoters and miRNA expression in breast cancer cells. The miRNA promoters with clearest association between DNA methylation and expression included a previously described and a novel promoter of the Hsa-mir-200b cluster. The novel promoter of the Hsa-mir-200b cluster, denoted P2, is located 2 kb upstream of the 5′ stemloop and maps within a CpG island. P2 has comparable promoter activity to the previously reported promoter (P1), and is able to drive the expression of miR-200b in its endogenous genomic context. DNA methylation of both P1 and P2 was inversely associated with miR-200b expression in eight out of nine breast cancer cell lines, and in vitro methylation of both promoters repressed their activity in reporter assays. In clinical samples, P1 and P2 were differentially methylated with methylation inversely associated with miR-200b expression. P1 was hypermethylated in metastatic lymph nodes compared with matched primary breast tumours whereas P2 hypermethylation was associated with loss of either oestrogen receptor or progesterone receptor. Hypomethylation of P2 was associated with gain of HER2 and androgen receptor expression. These data suggest an association between miR-200b regulation and breast cancer subtype and a potential use of DNA methylation of miRNA promoters as a component of a suite of breast cancer biomarkers.