The impact of gene expression microarrays in the evaluation of lung carcinoma subtypes and DNA copy number.
Abstract: * 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 background.

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.
Article Type: Report
Subject: Lung cancer (Diagnosis)
Lung cancer (Genetic aspects)
Gene expression (Research)
Gene expression (Physiological aspects)
DNA microarrays (Usage)
DNA (Research)
DNA (Physiological aspects)
Cellular signal transduction (Research)
Cellular signal transduction (Physiological aspects)
Author: Sanchez-Cespedes, Montserrat
Pub Date: 10/01/2008
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
Accession Number: 230246846
Full Text: 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 them.

[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.

Several factors underlie the specific patterns of gene expression identified in tumors, such as the background of gene alterations (alterations in oncogenes and tumor suppressors) and the tumor's cell of origin (Figure 2). However, other contributing factors need to be taken into consideration. Among them is the presence of alterations at the level of microRNA. MicroRNAs are small RNA molecules that regulate gene expression after transcription. MicroRNAs recognize their targets based on sequence complementarity, being partially complementary to one or more messenger RNAs, and cause inhibition of protein translation or degradation of the messenger RNA. MicroRNAs play a key role in diverse biological processes, including cell development, proliferation, differentiation, and apoptosis. (16) In addition, widespread DNA copy number alteration can lead directly to global deregulation of gene expression. (17) Thus, when arranged according to the position of each gene in the chromosome, global expression analysis can unveil clusters of overexpressed genes that are due to increases in DNA copy number. This is important because this approach can then be used to identify novel oncogenes altered by gene amplification. By using this approach, it was observed a cluster of gene overexpression in squamous cell carcinoma around the PIK3CA gene, on the long arm of chromosome 3. (13) Fluorescence in situ hybridization analysis confirmed that the increase in PIK3CA gene expression was due to gene amplification, which had been previously reported (18) in an analysis using comparative genomic hybridization. PIK3CA encodes p110, the catalytic subunit of the phosphatidylinositol 3-kinase (PI3K), which phosphorylates the phosphoinositide-3,4-diphosphate [PtdIns(3,4)P2] to PtdIns(3,4,5)P3, both messenger molecules that regulate the localization and function of multiple effectors by binding to their specific pleckstrin homology domains. PIK3CA acts as an oncogene and is commonly activated by point mutations in many cancers. (18) However, PIK3CA is found infrequently in lung cancer, (8,19) and thus, gene amplification may constitute a mechanism for oncogenic activation of PIK3CA, especially in lung squamous cell carcinomas, offering a candidate marker that is sensitive to therapy with inhibitors of PI3K activity. In support of the oncogenic relevance of PIK3CA amplification in lung cancer, it has previously been reported (20) that PIK3CA-increased copy numbers confer oncogenic properties on the PI3K protein in ovarian cancer. Given the continuing debate over how to select patients for treatment with EGFR tyrosine kinase inhibitors, it is tempting to speculate that the same considerations might apply to other genes.

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: msanchez@cnio.es).
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