The impact of gene expression microarrays in the evaluation of lung carcinoma subtypes and DNA copy number.
* Context.--The development of targeted therapies creates a need to
accurately classify tumors. Among the more pressing needs are the
identification of the complete catalog of genes that are altered in
cancer and the accurate discrimination of tumors based on their genetic
Objectives.--To discuss the use of gene expression profiles to recapitulate the pathology and to distinguish the genetic background of non-small cell lung cancer. Also, to comment on using global analysis of gene expression to identify chromosomal regions carrying clusters of highly expressed genes, likely due to gene amplification. Gene amplification at these regions may target the activation of an oncogene critical to tumor development and potentially important in therapy.
Data Sources.--Review of relevant, recent literature on molecular alterations and expression analysis in lung cancer.
Conclusions.--The complexity of genetic and epigenetic alterations and the cell type of origin confer marked patterns of gene expression to lung tumors, which differentiate different tumor entities.
Lung cancer (Genetic aspects)
Gene expression (Research)
Gene expression (Physiological aspects)
DNA microarrays (Usage)
DNA (Physiological aspects)
Cellular signal transduction (Research)
Cellular signal transduction (Physiological aspects)
|Publication:||Name: Archives of Pathology & Laboratory Medicine Publisher: College of American Pathologists Audience: Academic; Professional Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2008 College of American Pathologists ISSN: 1543-2165|
|Issue:||Date: Oct, 2008 Source Volume: 132 Source Issue: 10|
|Topic:||Event Code: 310 Science & research|
|Product:||Product Code: 2831812 Deoxyribonucleic Acid NAICS Code: 325414 Biological Product (except Diagnostic) Manufacturing|
Cancer is driven by the activation/inactivation of key genes that
are essential point controllers in regulatory pathways, such as signal
transduction, cell cycles, DNA repair, and apoptosis. Because such
alterations are becoming increasingly important in drug development and
in tailored therapies, efforts need to focus on identifying the complete
catalog of genes that are altered in cancer and on accurately
distinguishing tumors based on their genetic background. In the case of
lung cancer, several gene alterations are known to contribute to its
development, including activating mutations; gene amplification of the
oncogenes BRAF, EGFR, ERBB2, KRAS, NRAS, PIK3CA, and MYC; inactivating
point mutations; homozygous deletions; and promoter hypermethylation of
the tumor suppressor genes LKB1, MYC, PTEN, P16, RB,and TP53. 1 Some of
these gene alterations are known to be specific to lung tumor
histologies, (1-6) likely heralding differences in the cell type of
origin. In addition, it is also well established that some gene
alterations are mutually exclusive events, which is the case for genes
that encode proteins that act in the same signaling pathway, such as
KRAS and EGFR or P16 and RB. (2,5-6) It is widely accepted that gene
alterations are not redundant in cancer cells. So, genes found altered
in a mutually exclusive manner suggest that the encoded proteins act in
the same biologic pathway. Figure 1 is a schematic representation of the
proteins genetically altered in lung cancer and the general biologic
pathways to which they belong. The recent identification of activating
somatic mutations in the EGFR gene, especially in lung adenocarcinomas
arising in nonsmokers, and their proposed relevance in predicting EGFR
response to tyrosine kinase inhibitors have had a significant impact in
lung cancer treatment, exemplifying the clinical effect when changes
that underlie tumor development are understood.(5-6) Thus, it is now
critical to unravel the mechanisms and characteristics underlying the
presence of EGFR mutations. In addition to gene mutations, EGFR gene
amplification concomitant with protein overexpression have been observed
in a subset of lung tumors. (7) Activation of EGFR is mediated by
autophosphorylation at key tyrosine residues, leading to the modulation
of downstream signaling, such as Akt and Ras-ERK/MAPK, which are
involved in cell survival and cell proliferation. At present, it is well
established that mutations in EGFR and KRAS are mutually exclusive,
(5-8) indicating that both genes are functionally equivalent; therefore,
alterations at only one of them is enough to trigger constant activation
of the downstream targets. Similarly, it was previously reported (8)
that EGFR and KRAS mutant primary lung tumors have higher levels of
phospho-S6, a ribosomal protein that is phosphorylated by the mTOR
substrate S6 kinase, but not of phospho-ERK as compared with the EGFR
and KRAS wild types. These observations point to selective activation of
mTOR-mediated signal transduction by mutations in EGFR or KRAS. Although
less frequent, alterations at other genes, such as amplification or
point mutations at ERBB2, NRAS, or BRAF, may also contribute to the
activation of the EGFR/KRAS pathway in lung adenocarcinomas. It would be
interesting to understand the differences among lung adenocarcinomas
with and without alterations of these oncogenes. Among the challenges in
the coming years will be identifying the complete set of gene
alterations in lung cancer and unraveling the complex interactions among
[FIGURE 1 OMITTED]
At present, gene expression microarrays allow the evaluation of the expression of thousands of genes simultaneously, thus, providing patterns of gene expression that serve to categorize tumors sharing common characteristics (eg, specific outcome, tumor histology). Gene expression microarrays contain complementary DNA or oligonucleotides printed on glass as high-density hybridization targets. Fluorescent probe mixtures, derived from total messenger RNA, hybridize to cognate elements on the array. A 2-color, fluorescence-detection scheme allows the rapid analysis of the expression levels of the corresponding genes. In lungs, expression profiling has been shown to discriminate between healthy lung tissue and lung tumors, as well as profiling distinct lung tumor histologies and clinical entities. (9-14) Presumably, global analysis of gene expression may also correlate with specific gene-alteration patterns, thereby serving as a tool for selecting patients for targeted therapies. In using expression pro files to discriminate lung cancer histologies, several studies agree that gene expression profiles clearly segregate lung adenocarcinoma from squamous cell carcinoma and from small cell lung cancer. (9-13) Overall, squamous cell carcinomas feature differentially higher levels of expression in more genes than adenocarcinomas, including those markers currently used by pathology departments for differential diagnosis, such as the keratins. Remarkably, DSC3 and PKP1, components of desmosomes, have been reported to be highly upregulated in squamous cell carcinomas. Other genes substantially overexpressed in squamous cell carcinomas, as compared with adenocarcinomas and healthy lung, include SPRR, GPX2, CSTA, FABP,and TP73L/P63, whereas upregulation of ERBB2 was more common in adenocarcinomas. On the other hand, small cell lung cancer expresses many genes consistent with its neuroendocrine differentiation, such as GPCT and ASCL2. (10) The identification of novel markers to differentiate lung tumor histologies, especially when only small biopsies are available for histopathologic diagnosis, is increasingly important because therapeutic agents, such as gefitinib or erlotinib, which have maximum response in tumors with EGFR mutations, are restricted to lung adenocarcinomas.
[FIGURE 2 OMITTED]
On the other hand, unsupervised analysis of global gene expression does not segregate, in any particular branch, lung tumors with other common characteristics, such as sex, smoking status, tumor size, or lymph node metastasis. (13,15) Similarly, unsupervised analysis of gene expression profiles in lung tumors did not discern the presence of specific gene alterations, other than EGFR mutations. (13) Lung tumors with EGFR mutations constitute a closely defined disease entity, very different from other lung adenocarcinomas. In fact, most EGFR-mutant lung adenocarcinomas are etiologically and histopathologically different from the EGFR wild types because they arise in nonsmokers and have bronchioloalveolar or papillary characteristics. (8) Rather than being an inherent fault of the global gene expression analysis, its poor performance in discriminating genetic backgrounds may be caused by other limitations. One technical limitation that hinders the detection of gene alterations is the relatively low sensitivity of the current methods for detecting somatic mutations in primary tumors, which are necessarily contaminated with healthy cells. Hopefully, novel technologies will soon overcome this limitation. Other explanations for the limited accuracy of global gene expression analysis in discriminating tumor subgroups are related to the procedures commonly used to record gene expression data. When analyzing the results obtained from the global gene expression platforms, only very high or very low expression is usually recorded, whereas expression that is within confidence intervals is not further analyzed.
In conclusion, the complexity of the genetic and epigenetic alterations, as well as the cell type of origin, confer lung tumors with marked patterns of gene expression that may allow discrimination of different tumor entities, such as histopathologic types. In addition, global analysis of gene expression serves to identify novel amplified regions.
Accepted for publication March 1 1, 2008.
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Montserrat Sanchez-Cespedes, PhD
From the Lung Cancer Group, Molecular Pathology Program, Spanish National Cancer Centre, Madrid.
The author has no relevant financial interest in the products or companies described in this article.
Reprints: Montserrat Sanchez-Cespedes, PhD, Lung Cancer Group, Molecular Pathology Program, Spanish National Cancer Centre (CNIO), Calle Melchor Fernandez Almagro, 3, 28029 Madrid, Spain (e-mail: email@example.com).
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