Impact of Gene Expression Microarrays in the Evaluation of Lung Carcinoma Subtypes and DNA Copy Number, The

Archives of Pathology & Laboratory Medicine, Oct 2008 by Sanchez-Cespedes, Montserrat

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.

(Arch Pathol Lab Med. 2008;132:1562-1565)

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 reported8 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.

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 profiles 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.