Systematic Review: Gene Expression Profiling Assays in Early-Stage Breast Cancer

Currently, 3 commercially available prognostic breast cancer tests based on gene expression (see Glossary) technology are available: Oncotype DX (Genomic Health, Redwood City, California), MammaPrint (Agendia BV, Amsterdam, the Netherlands), and H/I (AvariaDX, Carlsbad, California). Although measurement of gene expression is now a core research method, these commercial assays represent the first introduction of these technologies into clinical application. Gene expression is the technical term to describe how active a particular gene isthat is, how many times it is expressed, or transcribed, to produce the protein it encodes (Figure 1). The transcription (see Glossary) of the gene's DNA into messenger RNA (mRNA) is the first step in this process; modern molecular biological tools measure this activity by counting the number of mRNA molecules in a given cell type or tissue. Because the mRNA molecule is translated within the ribosome to produce a complete protein, counting mRNA transcripts provides an estimate of the number of corresponding proteins. High-throughput technologies, such as DNA microarray (see Glossary) and real-time reverse transcriptase polymerase chain reaction (RT-PCR) (see Glossary), allow simultaneous counting of many gene transcriptions (up to tens of thousands). This creates a snapshot of a tissue's global gene activity, called the transcriptome. Figure 1. Technologies used for high-throughput gene expression analysis. A. Breast cancer tumors are sampled at the treatment location and shipped to the central laboratory doing the assay, where pathologic review is done to assess cancer cell contents, followed by RNA preparation and integrity evaluation. Suitable samples are used to quantify RNA levels, thus assessing gene expression. When a gene is expressed, the transcription complex copies its DNA sequence into complementary RNA transcripts that are translated into proteins. High-throughput gene expression analysis aims to quantify messenger RNA (mRNA) populations in a given tissue. B. DNA microarray is the molecular biology technique enabling gene expression analysis in MammaPrint. RNA is labeled with fluorescent dye and hybridized against thousands of different nucleotide sequences corresponding to different genes and arrayed on a solid surface (that is, a modified microscope glass slide). On hybridization, fluorescence emitted by single locations on the microarray is used to estimate gene expression levels. In MammaPrint, a 2-color design is used, and RNA expression is estimated as a relative ratio between the sample and a reference RNA. For each patient, triplicate measurements are obtained from 2 microarrays inverting the labeling scheme. C. Real-time reverse transcriptase polymerase chain reaction (PCR) is the enabling technology to assess gene expression in Oncotype DX and H/I. This technique is based on reverse transcription (RT) (see Glossary) of a specific mRNA into the complementary DNA (cDNA) molecule, which is used as a template in PCR. The production of double-stranded DNA is accompanied by emission of light, which is recorded throughout the process and correlates to the amount of DNA that is produced. The higher the initial amount of RNA, the earlier light is emitted during RT-PCR, a measurable difference that allows gene expression to be quantitated. D. Gene expression levels are mathematically transformed into indexes predicting disease recurrence. Gene expression measurements have been used to develop new biological concepts, refine disease classification, improve diagnostic and prognostic accuracy, and identify new molecular targets for drugs, especially in cancer research (19). Results are commonly reported in the form of a list of genes that are differentially expressed between normal and diseased patients or that correlate with different prognoses or phenotypes. These lists are called gene expression profiles or signatures (see Glossary). Breast cancer is increasingly understood as an umbrella designation for various tumor subtypes that differ in their prognoses and responses to therapy. An important decision for many patients with early-stage breast cancer, especially patients who have tumors that express hormone receptors and will be given antiestrogen therapy, is whether they should also be treated with systemic chemotherapy. Although adjuvant chemotherapy is frequently recommended in this setting, many women will remain recurrence-free at 10 years without it, especially those with small, estrogen receptorpositive tumors without axillary nodal involvement. Patients and their physicians must weigh the possible benefit of chemotherapy in reducing recurrence against its toxicity and other attendant costs. Practicing oncologists frequently base their decisions about therapy on prognostic clinical algorithms that include demographic data; tumor stage; and other tumor characteristics, such as grade and estrogen receptor expression. These conventional combination predictors include the National Institutes of Health (NIH) Consensus Development criteria (10, 11); the St. Gallen expert opinion criteria (12, 13); the National Comprehensive Cancer Network guideline (1416); and a Web-based algorithm, Adjuvant! Online (17, 18). Gene expression profiling has been proposed to potentially augment or replace these prognostic tools. Oncotype DX is based on a 21-gene profile developed by Paik and colleagues (19), MammaPrint is based on a 70-gene prognostic signature developed by van't Veer and colleagues (8), and H/I is based on a 2-gene signature (HOXB13IL17BR) developed by Ma and colleagues (20). The gene sets on which these tests are based have minimal overlap. The 21-gene and the 70-gene expression signatures that form the basis of Oncotype DX and MammaPrints, respectively, share only 1 gene in common. Two technologies are used to determine gene expression: real-time RT-PCR (Oncotype DX and H/I) and DNA microarray (MammaPrint). All 3 tests use pathologic review of specimens to check tumor content and evaluate RNA preparation and quality. The 2 RT-PCRbased assays (Oncotype DX and H/I) are done in formalin-fixed, paraffin-embedded tumor tissues, whereas fresh unfixed tumor tissue is required for MammaPrint. We review evidence on the prognostic accuracy of these 3 tests and their ability to predict treatment benefit. Methods The Agency for Healthcare Research and Quality commissioned the review for the Centers of Disease Control and Prevention's Evaluation of Genomic Applications in Practice and Prevention program. Additional details about the methods and results are found in a comprehensive evidence report that is available through the Agency for Healthcare Research and Quality (www.ahrq.gov/clinic/epcindex.htm). Data Sources On 9 January 2007, we searched the MEDLINE and EMBASE databases by using Medical Subject Headings and other terms relevant to breast cancer, gene expression profiling, and Oncotype DX or MammaPrint. On 7 February 2007, we searched the Cochrane database, including Cochrane Reviews, CENTRAL, and CINAHL. We supplemented this search by updating searches in MEDLINE and by hand-searching added publications that appeared after the initial search (January 2007 to July 2007) and studies related to H/I. The test manufacturers were also asked to provide any published or unpublished data relating to our study questions. Searches were limited to publications in English. Study Selection Two investigators independently reviewed titles and abstracts to identify original data studies that involved the use of any of the 3 assays in women with breast cancer. Data Extraction We extracted and double-checked information on the clinical characteristics of the study population, tumor characteristics, and whether the marketed test or underlying signature was evaluated. To assess the quality of studies, we applied (where appropriate) the general principles of the REMARK (REporting recommendations for tumor MARKer prognostic studies) (21, 22) and Standards for Reporting of Diagnostic Accuracy (23, 24) guidelines. We synthesized data on the ability of a test to accurately predict recurrence risk (clinical validity) and treatment benefit (clinical utility). We distinguished between gene expression signatures and the gene expressionbased marketed tests. The gene signature is the collection of genes whose expression levels are measured in a given test. A gene signature can be measured by using various technologies (RT-PCR or complementary DNA [cDNA] array) and procedures (for example, different reagents, controls, sample acquisition, preparation, and transport procedures), which may not be identical to those used in the marketed test. We report limited information on the technical performance characteristics of the tests, sometimes called analytic validity. The analytic validity of a test usually is assessed by determining how observed measurements differ from standard reference values. However, no reference standard exists for gene expression measurements outside of the technologies used for these tests. Because analytic validity affects predictive ability, our assessment of predictive ability incorporates the effect of less-than-perfect analytic validity. In the full evidence report (available at www.ahrq.gov/clinic/epcindex.htm), we summarize data on the reproducibility of a test when applied repeatedly to the same patient or when repeated over time, as well as variability as a function of tumor sampling and handling, specimen preparation, and biological variation within tumor samples. Appendix Tables 1, 2, and 3 show the evidence summary. Appendix Table 1. Studies on the Oncotype DX Gene Expression Test Appendix Table 2. Studies on the MammaPrint Gene Expression Test Appendix Table 3. Studies on the H/I Gene Expression Test Role of the Funding Source The Agency for Healthcare Research and Quality and the Centers of Disease Control and Prevention's Evaluation of Genomic Applications in Prac