Analytical techniques for multiplex analysis of protein biomarkers

Introduction: The importance of biomarkers for pharmaceutical drug development and clinical diagnostics is more significant than ever in the current shift toward personalized medicine. Biomarkers have taken a central position either as companion markers to support drug development and patient selection, or as indicators aiming to detect the earliest perturbations indicative of disease, minimizing therapeutic intervention or even enabling disease reversal. Protein biomarkers are of particular interest given their central role in biochemical pathways. Hence, capabilities to analyze multiple protein biomarkers in one assay are highly interesting for biomedical research. Areas covered: We here review multiple methods that are suitable for robust, high throughput, standardized, and affordable analysis of protein biomarkers in a multiplex format. We describe innovative developments in immunoassays, the vanguard of methods in clinical laboratories, and mass spectrometry, increasingly implemented for protein biomarker analysis. Moreover, emerging techniques are discussed with potentially improved protein capture, separation, and detection that will further boost multiplex analyses. Expert commentary: The development of clinically applied multiplex protein biomarker assays is essential as multi-protein signatures provide more comprehensive information about biological systems than single biomarkers, leading to improved insights in mechanisms of disease, diagnostics, and the effect of personalized medicine

An investigation of breast cancer risk factors in Cyprus: a case control study

Background: Breast cancer is the most common form of malignancy affecting women worldwide. It is also the leading cancer in females in Cyprus, with approximately 400 new cases diagnosed annually. It is well recognized that genetic variation as well as environmental factors modulate breast cancer risk. The main aim of this study was to assess the strength of associations between recognized risk factors and breast cancer among Cypriot women. This is the first epidemiological investigation on risk factors of breast cancer among the Cypriot female population.Methods: We carried out a case-control study, involving 1,109 breast cancer patients and a group of 1,177 controls who were recruited while participating in the National screening programme for breast cancer. Information on demographic characteristics and potential risk factors were collected from both groups during a standardized interview. Logistic regression analysis was used to assess the strength of the association between each risk factor and breast cancer risk, before and after adjusting for the possible confounding effect of other factors.Results: In multivariable models, family history of breast cancer (OR 1.64, 95% CI 1.23, 2.19) was the strongest predictor of breast cancer risk in the Cypriot population. Late menarche (OR 0.64, 95% CI 0.45, 0.92 among women reaching menarche after the age of 15 vs. before the age of 12) and breastfeeding (OR 0.74, 95% CI 0.59, 0.92) exhibited a strong protective effect. In the case of breastfeeding, the observed effect appeared stronger than the effect of pregnancy alone. Surprisingly, we also observed an inverse association between hormone replacement therapy (HRT) although this may be a product of the retrospective nature of this study.Conclusion: Overall the findings of our study corroborate with the results of previous investigations on descriptive epidemiology of risk factors for breast cancer. This investigation provides important background information for designing detailed studies that aim to improve our understanding of the epidemiology of breast cancer in the Cypriot population, including the study of gene-environment interactions. Furthermore, our study provides the first scientific evidence for formulating targeted campaigns for prevention and early diagnosis of breast cancer in Cyprus

Identification of Stage-Specific Breast Markers using Quantitative Proteomics

YesMatched healthy and diseased tissues from breast cancer patients were analyzed by quantitative proteomics. By comparing proteomic profiles of fibroadenoma (benign tumors, three patients), DCIS (noninvasive cancer, three patients), and invasive ductal carcinoma (four patients), we identified protein alterations that correlated with breast cancer progression. Three 8-plex iTRAQ experiments generated an average of 826 protein identifications, of which 402 were common. After excluding those originating from blood, 59 proteins were significantly changed in tumor compared with normal tissues, with the majority associated with invasive carcinomas. Bioinformatics analysis identified relationships between proteins in this subset including roles in redox regulation, lipid transport, protein folding, and proteasomal degradation, with a substantial number increased in expression due to Myc oncogene activation. Three target proteins, cofilin-1 and p23 (increased in invasive carcinoma) and membrane copper amine oxidase 3 (decreased in invasive carcinoma), were subjected to further validation. All three were observed in phenotype-specific breast cancer cell lines, normal (nontransformed) breast cell lines, and primary breast epithelial cells by Western blotting, but only cofilin-1 and p23 were detected by multiple reaction monitoring mass spectrometry analysis. All three proteins were detected by both analytical approaches in matched tissue biopsies emulating the response observed with proteomics analysis. Tissue microarray analysis (361 patients) indicated cofilin-1 staining positively correlating with tumor grade and p23 staining with ER positive status; both therefore merit further investigation as potential biomarkers.Cyprus Research Promotion Foundation, Yorkshire Cancer Researc

Methylome Analysis and Epigenetic Changes Associated with Menarcheal Age

CAD received funding from EU-Europe aid grant CRIS 2009/223–507.The EPIC cohort is supported by the Europe Against Cancer Program of the European Commission (SANCO). The individual centres also received funding from: Denmark (Danish Cancer Society); France (Ligue centre le Cancer, Institut Gustave Roussy, Mutuelle Ge´ne´rale de l’Education Nationale, and Institut National de la Sante´ et de la Recherche Me´dicale (INSERM)); Greece (Hellenic Ministry of Health, the Stavros Niarchos Foundation and the Hellenic Health Foundation); Germany (German Cancer Aid, German Cancer Research Center, and Federal Ministry of Education and Research (Grant 01-EA-9401)); Italy (Italian Association for Research on Cancer and the National Research Council); The Netherlands (Dutch Ministry of Public Health, Welfare and Sports (VWS), Netherlands Cancer Registry (NKR), LK Research Funds, Dutch Prevention Funds, and Dutch ZON (Zorg Onderzoek Nederland), World Cancer Research Fund (WCRF)); Spain (Health Research Fund (FIS) of the Spanish Ministry of Health (Exp 96/0032) and the participating regional governments and institutions); Sweden (Swedish Cancer Society, Swedish Scientific Council, and Regional Government of Skane); and the United Kingdom (Cancer Research UK and Medical Research Council UK and Breast Cancer Campaign). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Common variants in breast cancer risk loci predispose to distinct tumor subtypes.

BACKGROUND: Genome-wide association studies (GWAS) have identified multiple common breast cancer susceptibility variants. Many of these variants have differential associations by estrogen receptor (ER) status, but how these variants relate with other tumor features and intrinsic molecular subtypes is unclear. METHODS: Among 106,571 invasive breast cancer cases and 95,762 controls of European ancestry with data on 173 breast cancer variants identified in previous GWAS, we used novel two-stage polytomous logistic regression models to evaluate variants in relation to multiple tumor features (ER, progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and grade) adjusting for each other, and to intrinsic-like subtypes. RESULTS: Eighty-five of 173 variants were associated with at least one tumor feature (false discovery rate < 5%), most commonly ER and grade, followed by PR and HER2. Models for intrinsic-like subtypes found nearly all of these variants (83 of 85) associated at p < 0.05 with risk for at least one luminal-like subtype, and approximately half (41 of 85) of the variants were associated with risk of at least one non-luminal subtype, including 32 variants associated with triple-negative (TN) disease. Ten variants were associated with risk of all subtypes in different magnitude. Five variants were associated with risk of luminal A-like and TN subtypes in opposite directions. CONCLUSION: This report demonstrates a high level of complexity in the etiology heterogeneity of breast cancer susceptibility variants and can inform investigations of subtype-specific risk prediction

The contemporary use of electron microscopy in the diagnosis of ciliary disorders and sperm centriolar abnormalities

Cilia and flagella are hair-like structures which extend from the cell membrane and are involved in a variety of biological functions in eukaryotes. Cilia are found in a wide range of human tissues and cell types including: the epithelium of the upper and lower respiratory tracts, the epithelium of the kidney, bile and pancreatic ducts, the embryonic node, and the ependymal cells in the brain and in the oviduct epithelium

Prevalence of primary ciliary dyskinesia in consecutive referrals of suspect cases and the transmission electron microscopy detection rate: A systematic review and meta-analysis

Diagnostic testing for primary ciliary dyskinesia (PCD) usually includes transmission electron microscopy (TEM), nasal nitric oxide, high-speed video microscopy, and genetics. Diagnostic performance of each test should be assessed toward the development of PCD diagnostic algorithms. We systematically reviewed the literature and quantified PCD prevalence among referrals and TEM detection rate in confirmed PCD patients. Major electronic databases were searched until December 2015 using appropriate terms. Included studies described cohorts of consecutive PCD referrals in which PCD was confirmed by at least TEM and one additional test, in order to compare the index test performance with other test(s). Meta-analyses of pooled PCD prevalence and TEM detection rate across studies were performed. PCD prevalence among referrals was 32% (95% CI: 25-39%, I 2 = 92%). TEM detection rate among PCD patients was 83% (95% CI: 75-90%, I 2 = 90%). Exclusion of studies reporting isolated inner dynein arm defects as PCD, reduced TEM detection rate and explained an important fraction of observed heterogeneity (74%, 95% CI: 66-83%, I 2 = 66%). Approximately, one third of referrals, are diagnosed with PCD. Among PCD patients, a significant percentage, at least as high as 26%, is missed by TEM, a limitation that should be accounted toward the development of an efficacious PCD diagnostic algorithm

Cost-effectiveness analysis of three algorithms for diagnosing primary ciliary dyskinesia: a simulation study

Background: Primary Ciliary Dyskinesia (PCD) diagnosis relies on a combination of tests which may include (a) nasal Nitric Oxide (nNO), (b) High Speed Video Microscopy (HSVM) and (c) Transmission Electron Microscopy (TEM). There is variability in the availability of these tests and lack of universal agreement whether diagnostic tests should be performed in sequence or in parallel. We assessed three combinations of tests for PCD diagnosis and estimated net sensitivity and specificity as well as cost-effectiveness (CE) and incremental cost-effectiveness (ICE) ratios. Methods and results: A hypothetical initial population of 1000 referrals (expected 320 PCD patients) was followed through a probabilistic decision analysis model which was created to assess the CE of three diagnostic algorithms (a) nNO + TEM in sequence, (b) nNO + HSVM in sequence and (c) nNO/HSVM in parallel followed, in cases with conflicting results, by confirmatory TEM (nNO/HSVM+TEM). Number of PCD patients identified, CE and ICE ratios were calculated using Monte Carlo simulations. Out of 320 expected PCD patients, 313 were identified by nNO/HSVM+TEM, 274 with nNO + HSVM and 198 with nNO + TEM. The nNO/HSVM+TEM had the highest mean annual cost (209 K) followed by nNO + TEM (150 K) and nNO + HSVM (136 K). The nNO + HSVM algorithm dominated the nNO + TEM algorithm (less costly and more effective). The ICE ratio for nNO/HSVM+TEM was 2.1 K per additional PCD patient identified. Conclusions: The diagnostic algorithm (nNO/HSVM+TEM) with parallel testing outperforms algorithms with tests in sequence. These findings, can inform the dialogue on the development of evidence-based guidelines for PCD diagnostic testing. Future research in understudied aspects of the disease, such as PCD-related quality of life and PCD-associated costs, is needed to help the better implementation of these guidelines across various healthcare systems

Clinical features of primary ciliary dyskinesia in Cyprus with emphasis on lobectomized patients

Background: Despite the manifestations of primary ciliary dyskinesia (PCD) in early life, the diagnosis is often much delayed. Since 1998 in Cyprus, we have established the only national diagnostic and clinical referral center for PCD. Objective: To review the phenotypic features at presentation of PCD patients in Cyprus in relation to age at diagnosis, with emphasis on previously lobectomised patients. Methods: The medical records of the diagnosed PCD patients were retrospectively reviewed to obtain clinical data on presentation. Results: Thirty patients, aged 13.9 years (range 0.1, 58.4 years), were diagnosed with PCD. Twelve of them presented after the age of 18. The most common manifestations were chronic cough (100%), chronic rhinorrhea (96.7%), sputum production (92.9%), laterality defects (63.3%), a history of pneumonia (53.3%) and neonatal respiratory distress (50%). A history of lobectomy in the past was recorded in 16.7% (5 patients). Patients who presented in adulthood had significantly higher frequency of lobectomy (41.7% vs 0%, p-value=0.006) and had more frequently low FEV1 (58.3% vs 0%, p-value=0.015) than those who presented before. Serial measurements of FEV1 and FVC indicated significantly lower intercepts in lobectomised compared to the adult non-lobectomised patients both in terms of FEV1 (-4.90 vs -1.80, p-value=0.022) and FVC (-5.43 vs -1.91, p-value=0.029) z-score levels. Change in FEV1 and FVC across time was not statistically significant in either group. Conclusions: PCD often remains undiagnosed up to adulthood accompanied by appearance of advanced lung disease. Performance of lobectomies seems to be a poor prognostic factor for PCD in adulthood