Tissue microarrays (also TMAs) consist of paraffin blocks in which up to 1000 [1] separate tissue cores are assembled in array fashion to allow multiplex histological analysis.
Tissue microarrays (also TMAs) consist of paraffin blocks in which up to 1000 [1] separate tissue cores are assembled in array fashion to allow multiplex histological analysis.
The major limitations in molecular clinical analysis of tissues include the cumbersome nature of procedures, limited availability of diagnostic reagents and limited patient sample size. The technique of tissue microarray was developed to address these issues.
Multi-tissue blocks were first introduced by H. Battifora in 1986 with his so-called “multitumor (sausage) tissue block" and modified in 1990 with its improvement, "the checkerboard tissue block" . In 1998, J. Kononen and collaborators developed the current technique, which uses a novel sampling approach to produce tissues of regular size and shape that can be more densely and precisely arrayed.
In the tissue microarray technique, a hollow needle is used to remove tissue cores as small as 0.6 mm in diameter from regions of interest in paraffin-embedded tissues such as clinical biopsies or tumor samples. These tissue cores are then inserted in a recipient paraffin block in a precisely spaced, array pattern. Sections from this block are cut using a microtome, mounted on a microscope slide and then analyzed by any method of standard histological analysis. Each microarray block can be cut into 100 – 500 sections, which can be subjected to independent tests. Tests commonly employed in tissue microarray include immunohistochemistry, and fluorescent in situ hybridization. Tissue microarrays are particularly useful in analysis of cancer samples.
One variation is a Frozen tissue array.
The use of tissue microarrays in combination with immunohistochemistry has been a preferred method to study and validate cancer biomarkers in various defined cancer patient cohorts. The possibility to assemble a large number of representative cancer samples from a defined patient cohort that also has a corresponding clinical database, provides a powerful resource to study how different protein expression patterns correlate with different clinical parameters. Since patient samples are assembled into the same block, sections can be stained with the same protocol to avoid experimental variability and technical artefacts. Clinical cancer patient cohorts and corresponding tissue microarray sets have been used to study diagnostic, prognostic and treatment predictive cancer biomarkers in most forms of cancer, including lung, breast, colorectal and renal cell cancer. [2] [3] [4] [5]
Immunohistochemistry combined with tissue microarrays has also been used with success in large scale efforts to create a map of protein expression on a more global scale. [6]
Proteomics is the large-scale study of proteins. Proteins are vital parts of living organisms, with many functions. The proteome is the entire set of proteins that is produced or modified by an organism or system. Proteomics has enabled the identification of ever increasing numbers of protein. This varies with time and distinct requirements, or stresses, that a cell or organism undergoes. Proteomics is an interdisciplinary domain that has benefitted greatly from the genetic information of various genome projects, including the Human Genome Project. It covers the exploration of proteomes from the overall level of protein composition, structure, and activity. It is an important component of functional genomics.
Comparative genomic hybridization(CGH) is a molecular cytogenetic method for analysing copy number variations (CNVs) relative to ploidy level in the DNA of a test sample compared to a reference sample, without the need for culturing cells. The aim of this technique is to quickly and efficiently compare two genomic DNA samples arising from two sources, which are most often closely related, because it is suspected that they contain differences in terms of either gains or losses of either whole chromosomes or subchromosomal regions. This technique was originally developed for the evaluation of the differences between the chromosomal complements of solid tumor and normal tissue, and has an improved resolution of 5–10 megabases compared to the more traditional cytogenetic analysis techniques of giemsa banding and fluorescence in situ hybridization (FISH) which are limited by the resolution of the microscope utilized.
Immunohistochemistry (IHC) is the most common application of immunostaining. It involves the process of selectively identifying antigens (proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues. IHC takes its name from the roots "immuno", in reference to antibodies used in the procedure, and "histo", meaning tissue. Albert Coons conceptualized and first implemented the procedure in 1941.
Personalized medicine, also referred to as precision medicine, is a medical model that separates people into different groups—with medical decisions, practices, interventions and/or products being tailored to the individual patient based on their predicted response or risk of disease. The terms personalized medicine, precision medicine, stratified medicine and P4 medicine are used interchangeably to describe this concept though some authors and organisations use these expressions separately to indicate particular nuances.
MammaPrint is a prognostic and predictive diagnostic test for early stage breast cancer patients that assess the risk that a tumor will metastasize to other parts of the body. It gives a binary result, high-risk or low-risk classification, and helps physicians determine whether or not a patient will benefit from chemotherapy. Women with a low risk result can safely forego chemotherapy without decreasing likelihood of disease free survival. MammaPrint is part of the personalized medicine portfolio marketed by Agendia.
Biomarker discovery is a medical term describing the process by which biomarkers are discovered. Many commonly used blood tests in medicine are biomarkers. There is interest in biomarker discovery on the part of the pharmaceutical industry; blood-test or other biomarkers could serve as intermediate markers of disease in clinical trials, and as possible drug targets.
An antibody microarray is a specific form of protein microarray. In this technology, a collection of capture antibodies are spotted and fixed on a solid surface such as glass, plastic, membrane, or silicon chip, and the interaction between the antibody and its target antigen is detected. Antibody microarrays are often used for detecting protein expression from various biofluids including serum, plasma and cell or tissue lysates. Antibody arrays may be used for both basic research and medical and diagnostic applications.
Molecular cytogenetics combines two disciplines, molecular biology and cytogenetics, and involves the analyzation of chromosome structure to help distinguish normal and cancer-causing cells. Human cytogenetics began in 1956 when it was discovered that normal human cells contain 46 chromosomes. However, the first microscopic observations of chromosomes were reported by Arnold, Flemming, and Hansemann in the late 1800s. Their work was ignored for decades until the actual chromosome number in humans was discovered as 46. In 1879, Arnold examined sarcoma and carcinoma cells having very large nuclei. Today, the study of molecular cytogenetics can be useful in diagnosing and treating various malignancies such as hematological malignancies, brain tumors, and other precursors of cancer. The field is overall focused on studying the evolution of chromosomes, more specifically the number, structure, function, and origin of chromosome abnormalities. It includes a series of techniques referred to as fluorescence in situ hybridization, or FISH, in which DNA probes are labeled with different colored fluorescent tags to visualize one or more specific regions of the genome. Introduced in the 1980s, FISH uses probes with complementary base sequences to locate the presence or absence of the specific DNA regions you are looking for. FISH can either be performed as a direct approach to metaphase chromosomes or interphase nuclei. Alternatively, an indirect approach can be taken in which the entire genome can be assessed for copy number changes using virtual karyotyping. Virtual karyotypes are generated from arrays made of thousands to millions of probes, and computational tools are used to recreate the genome in silico.
The estrogen receptor test (ERT) uses the estrogen receptor (ER) tumor marker that allows for immunohistochemical techniques to be performed for diagnostic purposes. Immunohistochemistry (IHC) techniques involve the selective identification of antigen proteins by exploiting these antigen-antibody relationships to characterize your analyte of interest. Previously, the ligand binding assay has been used in the determination of ER activity, however this method was limited because of the requirement of large quantities of fresh tissue needed for each assay. IHC serves as a more efficient methods as this technique allows for the morphology of the tissue to be observed in a tumor-specific manner. This increases the practicability of this technique as in many cases, patients’ tissue samples are limited in the applications of biomarker analysis. Anti-estrogen receptor antibodies were among the first of biomarkers which introduced a semi-quantitative assessment of the ER activity. Today, ER analysis is one of many routinely performed immunohistochemical assays performed to classify the hormone receptor status and to serve as a means of insight in the determination of cancer prognosis and management.
Rosetta Genomics Ltd. was a molecular diagnostics company with offices in Israel and the United States that uses micro-ribonucleic acid (microRNA) biomarkers to develop diagnostic tests designed to differentiate between various types of cancer. The company expects the first three tests based on its technology to be submitted for regulatory approval in 2008. The diagnostic tests will differentiate between squamous and non-squamous non-small cell lung cancer (NSCLC); differentiate between adenocarcinoma and peritoneal mesothelioma; and seek to identify the origin of tumors in patients representing cancer of unknown primary (CUP). Using a single microRNA, the highly sensitive, highly specific test for squamous and non-squamous lung cancer has passed the prevalidation phase and has been submitted for approval to the New York State Department of Health Clinical Laboratory Evaluation Program in April 2008.
Frozen tissue array consists of fresh frozen tissues in which up to 50 separate tissue cores are assembled in array fashion to allow simultaneous histological analysis.
A reverse phase protein lysate microarray (RPMA) is a protein microarray designed as a dot-blot platform that allows measurement of protein expression levels in a large number of biological samples simultaneously in a quantitative manner when high-quality antibodies are available.
Virtual karyotype is the digital information reflecting a karyotype, resulting from the analysis of short sequences of DNA from specific loci all over the genome, which are isolated and enumerated. It detects genomic copy number variations at a higher resolution for level than conventional karyotyping or chromosome-based comparative genomic hybridization (CGH). The main methods used for creating virtual karyotypes are array-comparative genomic hybridization and SNP arrays.
A gene signature or gene expression signature is a single or combined group of genes in a cell with a uniquely characteristic pattern of gene expression that occurs as a result of an altered or unaltered biological process or pathogenic medical condition. This is not to be confused with the concept of gene expression profiling. Activating pathways in a regular physiological process or a physiological response to a stimulus results in a cascade of signal transduction and interactions that elicit altered levels of gene expression, which is classified as the gene signature of that physiological process or response. The clinical applications of gene signatures breakdown into prognostic, diagnostic and predictive signatures. The phenotypes that may theoretically be defined by a gene expression signature range from those that predict the survival or prognosis of an individual with a disease, those that are used to differentiate between different subtypes of a disease, to those that predict activation of a particular pathway. Ideally, gene signatures can be used to select a group of patients for whom a particular treatment will be effective.
Geniom RT Analyzer is an instrument used in molecular biology for diagnostic testing. The Geniom RT Analyzer utilizes the dynamic nature of tissue microRNA levels as a biomarker for disease progression. The Geniom analyzer incorporates microfluidic and biochip microarray technology in order to quantify microRNAs via a Microfluidic Primer Extension Assay (MPEA) technique.
Immunosignaturing is a medical diagnostic test which uses arrays of random-sequence peptides to associate antibodies in a blood sample with a disease.
Cubilin is a protein that in humans is encoded by the CUBN gene.
Secretomics is a type of proteomics which involves the analysis of the secretome—all the secreted proteins of a cell, tissue or organism. Secreted proteins are involved in a variety of physiological processes, including cell signaling and matrix remodeling, but are also integral to invasion and metastasis of malignant cells. Secretomics has thus been especially important in the discovery of biomarkers for cancer and understanding molecular basis of pathogenesis. The analysis of the insoluble fraction of the secretome has been termed matrisomics.
Molecular diagnostics is a collection of techniques used to analyze biological markers in the genome and proteome, and how their cells express their genes as proteins, applying molecular biology to medical testing. The technique is used to diagnose and monitor disease, detect risk, and decide which therapies will work best for individual patients.
A liquid biopsy, also known as fluid biopsy or fluid phase biopsy, is the sampling and analysis of non-solid biological tissue, primarily blood. Like traditional biopsy this type of technique is mainly used as a diagnostic and monitoring tool for diseases such as cancer, with the added benefit of being largely non-invasive. Therefore, it can also be done more frequently which can better track tumors and mutations over a duration of time. It may also be used to validate the efficiency of a cancer treatment drug by taking multiple liquid biopsy samples in the span of a few weeks. The technology may also prove beneficial for patients after treatment to monitor relapse.
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