Biomarker Validation for Aging: Lessons from mtDNA Heteroplasmy Analyses in Early Cancer Detection.

The anticipated biological and clinical utility of biomarkers has attracted significant interest recently. Aging and early cancer detection represent areas active in the search for predictive and prognostic biomarkers. While applications differ, overlapping biological features, analytical technologies and specific biomarker analytes bear comparison.

Quantitative infrared spectroscopy of formalin-fixed, paraffin-embedded tissue specimens: paraffin wax removal with organic solvents.

Formalin-fixed, paraffin-embedded tissue specimens form the basis for diagnostic histopathology. Although adequate for morphologic visualization, clinical variability in preparation of formalin-fixed, paraffin-embedded clinical specimens represents an obstacle to quantitative molecular genetic analysis in areas such as genomics and proteomics. A quantitative reexamination of classical histopathology tissue preparation methods was initiated to determine which protocol steps might improve molecular analysis, beginning with deparaffinization.

Performance of mitochondrial DNA mutations detecting early stage cancer.

Background: Mutations in the mitochondrial genome (mtgenome) have been associated with cancer and many other disorders. These mutations can be point mutations or deletions, or admixtures (heteroplasmy). The detection of mtDNA mutations in body fluids using resequencing microarrays, which are more sensitive than other sequencing methods, could provide a strategy to measure mutation loads in remote anatomical sites.

Quantitation of HER2 and telomerase biomarkers in solid tumors with IgY antibodies and nanocrystal detection.

In an effort to improve affinity biomarker validation in fixed patient tissue specimens, we have developed a novel quantum dot-based bioimaging system that utilizes chicken IgY antibody for high sensitivity and specificity relative quantitation of cancer proteins. Monospecific, polyclonal IgYs were generated against human HER2 and telomerase, and analytically validated for specificity by western blot and immunohistochemistry on tumor and normal cells and for relative affinity by layered peptide array (LPA). IgYs bound desired targets in cell lines and fixed tissues and showed greater affinity than commercial mammalian antibodies for both HER2 and telomerase proteins.

Validation of novel optical imaging technologies: the pathologists' view.

Noninvasive optical imaging technology has the potential to improve the accuracy of disease detection and predict treatment response. Pathology provides the critical link between the biological basis of an image or spectral signature and clinical outcomes obtained through optical imaging. The validation of optical images and spectra requires both morphologic diagnosis from histopathology and parametric analysis of tissue features above and beyond the declared pathologic "diagnosis.

Inhibition of sulfur mustard-induced cytotoxicity and inflammation by the macrolide antibiotic roxithromycin in human respiratory epithelial cells.

Background: Sulfur mustard (SM) is a potent chemical vesicant warfare agent that remains a significant military and civilian threat. Inhalation of SM gas causes airway inflammation and injury. In recent years, there has been increasing evidence of the effectiveness of macrolide antibiotics in treating chronic airway inflammatory diseases.

Towards Clinical Application of Methylated DNA Sequences as Cancer Biomarkers: A Joint NCI's EDRN and NIST Workshop on Standards, Methods, Assays, Reagents and Tools.

The workshop report, entitled Towards Clinical Application of Methylated DNA Sequences as Cancer Biomarkers: A Joint National Cancer Institute's Early Detection Research Network and National Institute of Standards and Technology Workshop, presents a summary of the main issues, current challenges, outcomes, and recommendations toward application of methylated DNA sequences as cancer biomarkers.

Standards for plasma and serum proteomics in early cancer detection: a needs assessment report from the national institute of standards and technology--National Cancer Institute Standards, Methods, Assays, Reagents and Technologies Workshop, August 18-19, 2005.

NIST and the National Cancer Institute cosponsored a workshop on August 18-19, 2005, to examine needs for reference materials for early cancer detection. This meeting focused on standards, methods, assays, reagents, and technologies. Needs for plasma and serum proteomics, DNA methylation, and specimen reference collections were discussed, and recommendations from participants were solicited.

Quantum dots--a versatile tool in plant science?

An optically stable, novel class of fluorophores (quantum dots) for in situ hybridisation analysis was tested to investigate their signal stability and intensity in plant chromosome analyses. Detection of hybridisation sites in situ was based on fluorescence from streptavidin-linked inorganic crystals of cadmium selenide. Comparison of quantum dots (QDs) with conventional detection systems (Alexa 488) in immunolabeling experiments demonstrated greater sensitivity than the conventional system.

Data standards for proteomics: mitochondrial two-dimensional polyacrylamide gel electrophoresis data as a model system.

Proteomics has emerged as a major discipline that led to a re-examination of the need for consensus and a nationally sanctioned set of proteomics technology standards. Such standards for databases and data reporting may be applied to two-dimensional polyacrylamide gel electrophoresis (2D PAGE) technology as a pilot project for assessing global and national needs in proteomics, and the role of the National Institute of Standards and Technology (NIST) and other similar standards and measurement organizations. The experience of harmonizing the heterogeneous data included in the Protein Data Bank (PDB) provides a paradigm for technology in an area where significant heterogeneity in technical detail and data storage has evolved.

Standards requirements for systems biology approaches to health care: mitochondrial proteomics.

A review of the standards needs of the mitochondrial proteomics communities is presented based on the presentations and discussions at National Institute of Standards and Technology (NIST) workshop, Systems Biology Approaches to Health Care: Mitochondrial Proteomics, held on September 17-18, 2002. The mitochondrial proteomics areas addressed for standards needs are model systems, methods and data. This review outlines the challenges in the field, proposes standards efforts that the community would like to see pursued to meet those challenges, and is followed by a summary and NIST's planned efforts to address these standards requirements.

Preparation and characterization of candidate reference materials for telomerase assays.

Background: Telomerase has been measured in body fluids of cancer patients, and clinical tests for telomerase may have utility as noninvasive, cost-effective methods for the early detection of cancer. However, telomerase activity measured by common methods such as the telomerase repeat amplification protocol (TRAP) and telomerase reverse transcriptase catalytic subunit (hTERT) mRNA by reverse transcription-PCR (RT-PCR) varies among laboratories.

Methods: We prepared a CHAPS buffer lysate from cultured A549 cells and stored it at -80 degrees C.

Mitochondrial DNA as a cancer biomarker.

As part of a national effort to identify biomarkers for the early detection of cancer, we developed a rapid and high-throughput sequencing protocol for the detection of sequence variants in mitochondrial DNA. Here, we describe the development and implementation of this protocol for clinical samples. Heteroplasmic and homoplasmic sequence variants occur in the mitochondrial genome in patient tumors.

Measurement reproducibility in the early stages of biomarker development.

Biomarker discovery and development requires measurement reproducibility studies in addition to case-control studies. Parallel pursuit of reproducibility studies is especially important for emerging technologies such as protein biomarkers based on time-of-flight mass spectrometry, the case considered in this paper. For parallel studies, a way to improve reproducibility prior to identification of protein species is necessary.

Measurement of DNA biomarkers for the safety of tissue-engineered medical products, using artificial skin as a model.

To test the hypothesis that the process of tissue engineering introduces genetic damage to tissue-engineered medical products, we employed the use of five state-of-the-art measurement technologies to measure a series of DNA biomarkers in commercially available tissue-engineered skin as a model. DNA was extracted from the skin and compared with DNA from cultured human neonatal control cells (dermal fibroblasts and epidermal keratinocytes) and adult human fibroblasts from a 55-year-old donor and a 96-year-old donor. To determine whether tissue engineering caused oxidative DNA damage, gas chromatography/isotope-dilution mass spectrometry and liquid chromatography/isotope-dilution mass spectrometry were used to measure six oxidatively modified DNA bases as biomarkers.

Inter-rater agreement on chromosome 5 breakage in FISH-based mutagen sensitivity assays (MSAs).

In chromosome breakage assays, validated, universal criteria for selection of cells and classification of chromosome aberrations may enhance their utility for cancer susceptibility screening. To standardize a fluorescence in situ hybridization (FISH) modification of the mutagen sensitivity assay (MSA), scoring criteria were evaluated by web-based validation. Two hundred digital FISH images were assigned random identification numbers.

Analytical validation of telomerase activity for cancer early detection: TRAP/PCR-CE and hTERT mRNA quantification assay for high-throughput screening of tumor cells.

Activation of telomerase plays a critical role in unlimited proliferation and immortalization of cells. Telomerase activity has been shown to correlate with tumor progression, indicating that tumors expressing this enzyme possess aggressive clinical behavior and that telomerase activity may be a useful biomarker for early detection of cancer. However, measurements of telomerase activity by current methods such as telomeric repeat amplification protocol (TRAP)/polymerase chain reaction (PCR) or antibody-based radioimmunoassay (RIA) are low-throughput and not robust enough to easily accommodate the required statistical analysis to determine whether telomerase activity is a practical biomarker.

Semiconductor nanocrystal probes for human metaphase chromosomes.

To improve signal stability and quantitation, an optically stable, novel class of fluorophore for hybridization analysis of human metaphase chromosomes is demonstrated. Detection of hybridization sites in situ was based on fluorescence from streptavidin-linked inorganic crystals of cadmium selenide [(CdSe)ZnS]. Fluorescence of nanocrystal fluorophores was significantly brighter and more photostable than organic fluorophores Texas Red and fluorescein.

Cancer biomarker validation: standards and process: roles for the National Institute of Standards and Technology (NIST).

Rigorous validation of biomarkers for early detection of cancer differs at the National Institute of Standards and Technology (NIST) from similar processes common among research laboratories. As a newly discovered biomarker assay makes the transition from a research setting to the clinical diagnostic laboratory, it should progress through defined stages of assay confirmation. The first task of a validation laboratory is evaluation of research assay technology, performance, and specifications (analytical validation).

Nanotechnology: Emerging Developments and Early Detection of Cancer. A two-day workshop sponsored by the National Cancer Institute and the National Institute of Standards and Technology, August 30-31 2001, on the National Institute of Standards and Technology Campus, Gaithersburg, MD, USA.

A recent meeting jointly sponsored by the National Cancer Institute (NCI) and National Institute of Standards and Technology (NIST) brought together researchers active in nanotechnology and cancer molecular biology to discuss and evaluate the interface between disciplines. Emerging areas where nanotechnologies may impact cancer prevention and early cancer detection were elaborated by key researchers who catalyzed interdisciplinary dialogue aimed at fostering cross-discipline communications and future collaboration.

Cytonectin expression in Alzheimer disease.

Cytonectin is a novel 35,000 molecular weight protein that displays remarkable ion-independent adherence properties. This consigns it to a family of well-known adherence molecules essential for cell communication and the development of 3-dimensional tissue structures. Cytonectin is expressed in a variety of organs and tissues, being evolutionarily conserved from human to avian species.

NIST physical standards for DNA-based medical testing.

As DNA and RNA become major targets for clinical laboratory analysis, benchmark reagents will play an increasingly important role in standardization. Reliable national and international nucleic acid standards promote automation and third-party reimbursement for clinical testing. Furthermore, nucleic acid standards provide materials for quality assurance and quality control (QA/QC), and proficiency testing.