Gene expression profiling in cancer
Encyclopedia
Cancer
is a disease characterized by uncontrolled cell growth and proliferation. For cancer to develop, genes
regulating cell growth and differentiation must be altered; these mutations are then maintained through subsequent cell divisions and are thus present in all cancerous cells. Gene expression profiling is a technique used in molecular biology
to query the expression of thousands of genes simultaneously. In the context of cancer, gene expression profiling has been used to more accurately classify tumors. The information derived from gene expression profiling often has an impact on predicting the patient’s clinical outcome.
, allowing the formation of a malignant mass. The cells forming this mass undergo natural selection: as cells acquire mutations that enhance their survivability or reproductive capacity, they dominate the growing tumor as other cells are out-competed (see: somatic evolution in cancer
). Because of these selective
properties, the majority of cells within in a tumor will share a common profile of gene expression.
of the organism, only a small subset of those genes is expressed as messenger RNA
(mRNA) at any given time, and their relative expression
can be evaluated. Techniques include DNA microarray
technology or sequenced-based techniques such as serial analysis of gene expression
(SAGE).
Current cancer research makes use primarily of DNA microarrays in which an arrayed series of microscopic spots of pre-defined DNA
oligonucleotides known as probes
are covalently attached to a solid surface such as glass, forming what is known as a gene chip. DNA labeled with fluorophores (target) is prepared from a sample such as a tumor biopsy and is hybridized to the complementary DNA
(cDNA) sequences on the gene chip. The chip is then scanned for the presence and strength of the fluorescent labels at each spot representing probe-target hybrids. The level of fluorescence at a particular spot provides quantitative information about the expression of the particular gene corresponding to the spotted cDNA sequence. DNA microarrays evolved from Southern blotting which allows for detection of a specific DNA sequence in a sample of DNA.
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Classification of cancers has been dominated by the fields of histology
and histopathology
which aim to leverage morphological markers for accurate identification of a tumor type. Histological methods rely on chemical staining of tissues with pigments such as haematoxylin
and eosin
and microscopy-based visualization by a pathologist. The identification of tumor subtypes is based on established classification schemes such as the International Classification of Diseases published by the World Health Organization
which provides codes to classify diseases and a wide variety of signs, symptoms, abnormal findings, complaints, social circumstances, and external causes of injury or diseases. For some types of cancer, these methods are unable to distinguish between subclasses; for example, defining subgroups of diffuse large B-cell lymphoma (DLBCL) have largely failed due to discrepancies between inter- and intra-observer reproducibility. Furthermore, the clinical outcomes of tumors classified as DLBCLs is highly variable suggesting that there are multiple subtypes of DLBCL that cannot be distinguished based on these histological markers. Breast tumor
classification too has largely failed based on these predictors. Development of effective therapies depends on accurate diagnosis; additionally, poor diagnosis can lead to patient suffering due to needless side-effects
from non-targeted treatments and to increased health care expenditure. Most telling perhaps is that 70-80% of breast cancer patients receiving chemotherapy
based on traditional predictors would have survived without it.
Of note, similar gene expression patterns associated with metastatic behaviour of breast cancer tumor cells have also been found in breast cancer of dog, the most common tumor of the female dog.
Presented below are ways that gene expression profiling has been used to more precisely classify tumors into subgroups, often with clinical impact.
published in Nature Genetics
. The authors measured the relative expression of 9,703 human cDNAs in sixty cancer cell lines previously studied and characterized by the National Cancer Institute
’s Developmental Therapeutics Program. A hierarchical clustering algorithm was used to group cell lines based on the similarity by which the pattern of gene expression varied. In this study by Ross et al., the majority of cell lines with common organs of origin (based on information from the National Institutes of Health
) clustered together at terminal branches, suggesting that cancer cells arising from the same tissue share many molecular characteristics. This allows for reliable identification of tumor type based on gene expression.
In 2000, Stanford researchers published results in Nature
, utilizing expression profiling techniques to stratify DLBCL to two subtypes: germinal center B-like DLBCL and activated B-like DLBCL. The authors developed custom microarrays termed “lymphochips” that were used to query expression of 17,856 genes preferentially expressed in lymphoid cells and those with roles in cancer or immunology for 96 lymphocyte samples. The hierarchical clustering algorithm identified a subset of tumors that would have been labeled DLBCLs by traditional histological methods; however, the expression profiles of these tumors were heterogeneous. When the tumors were re-clustered based on expression of germinal center B-cell genes, a second group of genes characteristic of activated B-cells emerged and were oppositely regulated compared to the first set of genes. Based on these expression patterns, the heterogeneous DLBCL cluster was subclassified to the germinal center B-like DLBCL and activated B-like DLBCL. The distinction of these groups is significant in terms of patient overall survival: the probability of survival for patients with germinal center B-like DLBCLs over 10 years was about 80% while those with activated B-like DLBCLs was lowered to about 40% over a shorter eight-year period.
Breast cancers are also difficult to distinguish based on histological markers. In a 2000 study published in Nature
, Stanford researchers led by Perou, C.M. characterized gene expression patterns across 8,102 genes for 65 biopsies obtained from breast cancers. The goal of the study was to identify patterns of gene expression that could be used to describe the phenotypic diversity of breast tumors by comparing the profiles of the biopsies to those of cultured cell lines and relating this information to clinical data. The tumors were clustered into two major groups that largely reflected the ER
-positive and ER-negative clinical descriptions. The ER-positive tumors were characterized by high expression of genes normally expressed in breast luminal cells. The authors suggest that this higher-order distinction may encompass at least two biologically distinct types of cancer that may each require a unique course of treatment. Within the ER-negative group, additional clusters were identified based on expression of Erb-B2 and keratin 5- and 17-enriched basal epithelial-like genes. These groups reflect distinct molecular features as related to mammary epithelial biology, based on the outcome of disease.
In a 2001 study published in the Proceedings of the National Academy of Sciences
, Sørlie et al. further stratified the classifications described by Perou et al. and explored the clinical value of these breast cancer subtypes. The authors separated the ER-positive tumors into two distinct groups and found that tumor classification based on gene expression was related to patient survival. The expression of 427 genes was measured for 78 cancers and seven non-malignant breast samples. Following hierarchical clustering, the samples formed two groups at the highest level of organization reflecting the ER-positive and ER-negative phenotypes; the ER-negative cluster further stratified to groups identical to those described by Perou et al. In contrast to previous results, Sørlie et al. found that the ER-positive group could also be separated into three distinct subgroups termed luminal subtypes A, B, and, C based on patterns of luminal-specific gene expression with different outcomes. The authors further found once they performed survival analyses that tumors belonging to the various groups showed significantly different outcomes when treated uniformly. Survival analyses are often shown as Kaplan-Meier survival plots, an example of which is shown to the right.
In addition to identifying genes that correlate to survival, microarray analyses have been utilized to establish gene expression profiles associated with prognosis. It is agreed upon that patients with tumors exhibiting poor prognostic features would benefit the most from adjuvant therapy
as these treatments substantially improve overall survival for women with breast cancer. Traditional prognostic factors, however, are inexact as mentioned above. Researchers at the Netherlands Cancer Institute were able to identify "good-prognosis" and "bad-prognosis" signatures based on the expression of 70 genes that was better able to predict the likelihood of metastasis
development within five years for breast cancer patients Metastasis involves the spread of cancer from one organ to others throughout the body and is the principal cause of death in cancer patients. While the study at the Netherlands Cancer Institute applied to breast cancer patients only, researchers at Massachusetts Institute of Technology
identified a molecular signature of metastasis that applied to adenocarcinomas in general.
Cancer
Cancer , known medically as a malignant neoplasm, is a large group of different diseases, all involving unregulated cell growth. In cancer, cells divide and grow uncontrollably, forming malignant tumors, and invade nearby parts of the body. The cancer may also spread to more distant parts of the...
is a disease characterized by uncontrolled cell growth and proliferation. For cancer to develop, genes
Gênes
Gênes is the name of a département of the First French Empire in present Italy, named after the city of Genoa. It was formed in 1805, when Napoleon Bonaparte occupied the Republic of Genoa. Its capital was Genoa, and it was divided in the arrondissements of Genoa, Bobbio, Novi Ligure, Tortona and...
regulating cell growth and differentiation must be altered; these mutations are then maintained through subsequent cell divisions and are thus present in all cancerous cells. Gene expression profiling is a technique used in molecular biology
Molecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...
to query the expression of thousands of genes simultaneously. In the context of cancer, gene expression profiling has been used to more accurately classify tumors. The information derived from gene expression profiling often has an impact on predicting the patient’s clinical outcome.
Oncogenesis
Oncogenesis is the process by which normal cells acquire the properties of cancer cells leading to the formation of a cancer or tumor (see: tumorigenesis). It is characterized by a molecular reprogramming of a cell to undergo uninhibited cell divisionCell division
Cell division is the process by which a parent cell divides into two or more daughter cells . Cell division is usually a small segment of a larger cell cycle. This type of cell division in eukaryotes is known as mitosis, and leaves the daughter cell capable of dividing again. The corresponding sort...
, allowing the formation of a malignant mass. The cells forming this mass undergo natural selection: as cells acquire mutations that enhance their survivability or reproductive capacity, they dominate the growing tumor as other cells are out-competed (see: somatic evolution in cancer
Somatic evolution in cancer
Somatic evolution is the accumulation of mutations in the cells of a body during a lifetime, and the effects of those mutations on the fitness of those cells. Somatic evolution is important in the process of aging as well as the development of some diseases, including cancer.-Natural selection in...
). Because of these selective
Natural selection
Natural selection is the nonrandom process by which biologic traits become either more or less common in a population as a function of differential reproduction of their bearers. It is a key mechanism of evolution....
properties, the majority of cells within in a tumor will share a common profile of gene expression.
Gene expression profiling
Gene expression profiling is a technique used in molecular biology to query the expression of thousands of genes simultaneously. While almost all cells cell in an organism contain the entire genomeGenome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....
of the organism, only a small subset of those genes is expressed as messenger RNA
Messenger RNA
Messenger RNA is a molecule of RNA encoding a chemical "blueprint" for a protein product. mRNA is transcribed from a DNA template, and carries coding information to the sites of protein synthesis: the ribosomes. Here, the nucleic acid polymer is translated into a polymer of amino acids: a protein...
(mRNA) at any given time, and their relative expression
Gene expression
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA , transfer RNA or small nuclear RNA genes, the product is a functional RNA...
can be evaluated. Techniques include DNA microarray
DNA microarray
A DNA microarray is a collection of microscopic DNA spots attached to a solid surface. Scientists use DNA microarrays to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome...
technology or sequenced-based techniques such as serial analysis of gene expression
Serial Analysis of Gene Expression
Serial analysis of gene expression is a technique used by molecular biologists to produce a snapshot of the messenger RNA population in a sample of interest in the form of small tags that correspond to fragments of those transcripts. The original technique was developed by Dr. Victor Velculescu...
(SAGE).
Current cancer research makes use primarily of DNA microarrays in which an arrayed series of microscopic spots of pre-defined DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
oligonucleotides known as probes
Hybridization probe
In molecular biology, a hybridization probe is a fragment of DNA or RNA of variable length , which is used in DNA or RNA samples to detect the presence of nucleotide sequences that are complementary to the sequence in the probe...
are covalently attached to a solid surface such as glass, forming what is known as a gene chip. DNA labeled with fluorophores (target) is prepared from a sample such as a tumor biopsy and is hybridized to the complementary DNA
Complementary DNA
In genetics, complementary DNA is DNA synthesized from a messenger RNA template in a reaction catalyzed by the enzyme reverse transcriptase and the enzyme DNA polymerase. cDNA is often used to clone eukaryotic genes in prokaryotes...
(cDNA) sequences on the gene chip. The chip is then scanned for the presence and strength of the fluorescent labels at each spot representing probe-target hybrids. The level of fluorescence at a particular spot provides quantitative information about the expression of the particular gene corresponding to the spotted cDNA sequence. DNA microarrays evolved from Southern blotting which allows for detection of a specific DNA sequence in a sample of DNA.
Background
Histology
Histology is the study of the microscopic anatomy of cells and tissues of plants and animals. It is performed by examining cells and tissues commonly by sectioning and staining; followed by examination under a light microscope or electron microscope...
and histopathology
Histopathology
Histopathology refers to the microscopic examination of tissue in order to study the manifestations of disease...
which aim to leverage morphological markers for accurate identification of a tumor type. Histological methods rely on chemical staining of tissues with pigments such as haematoxylin
Haematoxylin
Haematoxylin, hematoxylin, Natural Black 1, or C.I. 75290 is extracted from the heartwood of the logwood tree. When oxidized it forms haematein, a compound that forms strongly coloured complexes with certain metal ions, the most notable ones being Fe and Al salts. Metal-haematein complexes are used...
and eosin
Eosin
Eosin is a fluorescent red dye resulting from the action of bromine on fluorescein. It can be used to stain cytoplasm, collagen and muscle fibers for examination under the microscope. Structures that stain readily with eosin are termed eosinophilic....
and microscopy-based visualization by a pathologist. The identification of tumor subtypes is based on established classification schemes such as the International Classification of Diseases published by the World Health Organization
World Health Organization
The World Health Organization is a specialized agency of the United Nations that acts as a coordinating authority on international public health. Established on 7 April 1948, with headquarters in Geneva, Switzerland, the agency inherited the mandate and resources of its predecessor, the Health...
which provides codes to classify diseases and a wide variety of signs, symptoms, abnormal findings, complaints, social circumstances, and external causes of injury or diseases. For some types of cancer, these methods are unable to distinguish between subclasses; for example, defining subgroups of diffuse large B-cell lymphoma (DLBCL) have largely failed due to discrepancies between inter- and intra-observer reproducibility. Furthermore, the clinical outcomes of tumors classified as DLBCLs is highly variable suggesting that there are multiple subtypes of DLBCL that cannot be distinguished based on these histological markers. Breast tumor
Breast cancer
Breast cancer is cancer originating from breast tissue, most commonly from the inner lining of milk ducts or the lobules that supply the ducts with milk. Cancers originating from ducts are known as ductal carcinomas; those originating from lobules are known as lobular carcinomas...
classification too has largely failed based on these predictors. Development of effective therapies depends on accurate diagnosis; additionally, poor diagnosis can lead to patient suffering due to needless side-effects
Adverse effect
In medicine, an adverse effect is a harmful and undesired effect resulting from a medication or other intervention such as surgery.An adverse effect may be termed a "side effect", when judged to be secondary to a main or therapeutic effect. If it results from an unsuitable or incorrect dosage or...
from non-targeted treatments and to increased health care expenditure. Most telling perhaps is that 70-80% of breast cancer patients receiving chemotherapy
Chemotherapy
Chemotherapy is the treatment of cancer with an antineoplastic drug or with a combination of such drugs into a standardized treatment regimen....
based on traditional predictors would have survived without it.
Of note, similar gene expression patterns associated with metastatic behaviour of breast cancer tumor cells have also been found in breast cancer of dog, the most common tumor of the female dog.
Presented below are ways that gene expression profiling has been used to more precisely classify tumors into subgroups, often with clinical impact.
Molecular Signature
In a particular type of cell or tissue, only a small subset of an organism’s genomic DNA will be expressed as mRNAs at any given time. The unique pattern of gene expression for a given cell or tissue is referred to as its molecular signature. For example, the expression of genes in skin cells would be very different compared to those expressed in blood cells. Microarray analysis can provide quantitative gene expression information allowing for the generation of a molecular signature, each unique to a particular class of tumor. This idea was first shown experimentally in 2000 by researchers at Stanford UniversityStanford University
The Leland Stanford Junior University, commonly referred to as Stanford University or Stanford, is a private research university on an campus located near Palo Alto, California. It is situated in the northwestern Santa Clara Valley on the San Francisco Peninsula, approximately northwest of San...
published in Nature Genetics
Nature Genetics
Nature Genetics is a scientific journal concerning genetics. It is published by Nature Publishing Group, and was founded as part of the Nature family of journal in 1992. The 2010 impact factor is 36.377. Its sister journal is Nature Reviews Genetics.- External links :*...
. The authors measured the relative expression of 9,703 human cDNAs in sixty cancer cell lines previously studied and characterized by the National Cancer Institute
National Cancer Institute
The National Cancer Institute is part of the National Institutes of Health , which is one of 11 agencies that are part of the U.S. Department of Health and Human Services. The NCI coordinates the U.S...
’s Developmental Therapeutics Program. A hierarchical clustering algorithm was used to group cell lines based on the similarity by which the pattern of gene expression varied. In this study by Ross et al., the majority of cell lines with common organs of origin (based on information from the National Institutes of Health
National Institutes of Health
The National Institutes of Health are an agency of the United States Department of Health and Human Services and are the primary agency of the United States government responsible for biomedical and health-related research. Its science and engineering counterpart is the National Science Foundation...
) clustered together at terminal branches, suggesting that cancer cells arising from the same tissue share many molecular characteristics. This allows for reliable identification of tumor type based on gene expression.
Tumor subclass
A more powerful result of gene expression profiling is the ability to further classify tumors into subtypes having distinct biological properties and impact on prognoses. For example, some diffuse large B-cell lymphomas (DLBCLs) are indistinguishable based on histological methods yet are clinically heterogeneous: 40% of patients respond well and exhibit prolonged survival while the remaining 60% do not.In 2000, Stanford researchers published results in Nature
Nature (journal)
Nature, first published on 4 November 1869, is ranked the world's most cited interdisciplinary scientific journal by the Science Edition of the 2010 Journal Citation Reports...
, utilizing expression profiling techniques to stratify DLBCL to two subtypes: germinal center B-like DLBCL and activated B-like DLBCL. The authors developed custom microarrays termed “lymphochips” that were used to query expression of 17,856 genes preferentially expressed in lymphoid cells and those with roles in cancer or immunology for 96 lymphocyte samples. The hierarchical clustering algorithm identified a subset of tumors that would have been labeled DLBCLs by traditional histological methods; however, the expression profiles of these tumors were heterogeneous. When the tumors were re-clustered based on expression of germinal center B-cell genes, a second group of genes characteristic of activated B-cells emerged and were oppositely regulated compared to the first set of genes. Based on these expression patterns, the heterogeneous DLBCL cluster was subclassified to the germinal center B-like DLBCL and activated B-like DLBCL. The distinction of these groups is significant in terms of patient overall survival: the probability of survival for patients with germinal center B-like DLBCLs over 10 years was about 80% while those with activated B-like DLBCLs was lowered to about 40% over a shorter eight-year period.
Breast cancers are also difficult to distinguish based on histological markers. In a 2000 study published in Nature
Nature (journal)
Nature, first published on 4 November 1869, is ranked the world's most cited interdisciplinary scientific journal by the Science Edition of the 2010 Journal Citation Reports...
, Stanford researchers led by Perou, C.M. characterized gene expression patterns across 8,102 genes for 65 biopsies obtained from breast cancers. The goal of the study was to identify patterns of gene expression that could be used to describe the phenotypic diversity of breast tumors by comparing the profiles of the biopsies to those of cultured cell lines and relating this information to clinical data. The tumors were clustered into two major groups that largely reflected the ER
Estrogen receptor
Estrogen receptor refers to a group of receptors that are activated by the hormone 17β-estradiol . Two types of estrogen receptor exist: ER, which is a member of the nuclear hormone family of intracellular receptors, and the estrogen G protein-coupled receptor GPR30 , which is a G protein-coupled...
-positive and ER-negative clinical descriptions. The ER-positive tumors were characterized by high expression of genes normally expressed in breast luminal cells. The authors suggest that this higher-order distinction may encompass at least two biologically distinct types of cancer that may each require a unique course of treatment. Within the ER-negative group, additional clusters were identified based on expression of Erb-B2 and keratin 5- and 17-enriched basal epithelial-like genes. These groups reflect distinct molecular features as related to mammary epithelial biology, based on the outcome of disease.
Clinical application
Proceedings of the National Academy of Sciences
The Proceedings of the National Academy of Sciences of the United States of America, usually referred to as PNAS, is the official journal of the United States National Academy of Sciences...
, Sørlie et al. further stratified the classifications described by Perou et al. and explored the clinical value of these breast cancer subtypes. The authors separated the ER-positive tumors into two distinct groups and found that tumor classification based on gene expression was related to patient survival. The expression of 427 genes was measured for 78 cancers and seven non-malignant breast samples. Following hierarchical clustering, the samples formed two groups at the highest level of organization reflecting the ER-positive and ER-negative phenotypes; the ER-negative cluster further stratified to groups identical to those described by Perou et al. In contrast to previous results, Sørlie et al. found that the ER-positive group could also be separated into three distinct subgroups termed luminal subtypes A, B, and, C based on patterns of luminal-specific gene expression with different outcomes. The authors further found once they performed survival analyses that tumors belonging to the various groups showed significantly different outcomes when treated uniformly. Survival analyses are often shown as Kaplan-Meier survival plots, an example of which is shown to the right.
In addition to identifying genes that correlate to survival, microarray analyses have been utilized to establish gene expression profiles associated with prognosis. It is agreed upon that patients with tumors exhibiting poor prognostic features would benefit the most from adjuvant therapy
Adjuvant chemotherapy
Adjuvant therapy, also called adjuvant care, is treatment that is given in addition to the primary, main or initial treatment. The surgeries and complex treatment regimens used in cancer therapy have led the term to be used mainly to describe adjuvant cancer treatments...
as these treatments substantially improve overall survival for women with breast cancer. Traditional prognostic factors, however, are inexact as mentioned above. Researchers at the Netherlands Cancer Institute were able to identify "good-prognosis" and "bad-prognosis" signatures based on the expression of 70 genes that was better able to predict the likelihood of metastasis
Metastasis
Metastasis, or metastatic disease , is the spread of a disease from one organ or part to another non-adjacent organ or part. It was previously thought that only malignant tumor cells and infections have the capacity to metastasize; however, this is being reconsidered due to new research...
development within five years for breast cancer patients Metastasis involves the spread of cancer from one organ to others throughout the body and is the principal cause of death in cancer patients. While the study at the Netherlands Cancer Institute applied to breast cancer patients only, researchers at Massachusetts Institute of Technology
Massachusetts Institute of Technology
The Massachusetts Institute of Technology is a private research university located in Cambridge, Massachusetts. MIT has five schools and one college, containing a total of 32 academic departments, with a strong emphasis on scientific and technological education and research.Founded in 1861 in...
identified a molecular signature of metastasis that applied to adenocarcinomas in general.