Gene expression profiling is a technology for identifying genes potentially associated with disease prognosis and response to therapies. The technology identifies and quantifies cellular messenger RNA (mRNA), thus providing information on the global activity of genes producing the mRNA transcripts. Because mRNA is translated into proteins, differences in mRNA levels are ultimately related to differences in cellular protein composition as well as differences in properties and functions of cells and tissues.

Available Assays

Although molecular profiling is well established in the treatment of breast cancer-for example, in decisions made on the basis of estrogen receptor (ER)/progesterone receptor and HER2 status-use of multiparameter gene profiling assays is relatively new. The first three such assays to become commercially available are the Oncotype DX breast cancer assay (Genomic Health), the MammaPrint test (Agendia), and the Breast Cancer Profiling test (developed by AviaraDx and licensed to Quest Diagnostics).

The Oncotype DX assay is offered to newly diagnosed patients with node-negative, ER-positive disease who are to be treated with tamoxifen. It assigns a recurrence risk score: Low recurrence score (< 18) predicts optimal therapeutic benefit from tamoxifen and suggests that adjuvant chemotherapy may not be required, whereas a high recurrence score (≥ 31) suggests greater likelihood of benefit from adjuvant CMF (cyclophosphamide, methotrexate, fluorouracil) or MF adjuvant chemotherapy (reflecting the adjuvant regimens in the patient population in which the assay was investigated). The assay quantifies expression of 21 genes-16 cancer-related genes and 5 reference genes-in RNA extracted from formalin-fixed, paraffin-embedded tumor tissue using real-time reverse transcription polymerase chain reaction (RT-PCR). The expression of the cancer-related genes is normalized to the expression of the reference genes. The cancer-related genes include those associated with proliferation, HER2, estrogen, and invasion.

The OncotypeDx assay was also shown to be predictive of the need for chemotherapy in a group of women with node-positive breast cancer.

The MammaPrint test is available for newly diagnosed patients aged < 61 years with stage I or II node-negative disease ≤ 5 cm in diameter and is intended to predict the likelihood of rapid recurrence of cancer. The assay measures 70 cancer-related genes and approximately 1,800 reference genes in fresh tissue samples containing a minimum of 30% malignant cells using a DNA microarray technique. Cancer-related genes include those associated with cell signaling, growth factors, transcription, cell cycle, chromatin, nuclear proteins, adhesion, motility, cytoskeleton organization, cellular metabolism, intracellular transport, ubiquitination, apoptosis, and drug resistance.

The Breast Cancer Profiling assay is a quantitative RT-PCR-based assay (also known as the H:I ratio test) that measures the ratio of the estrogen-regulated genes HOXB13 and IL17BR and four reference genes in formalin-fixed, paraffin-embedded samples. It is intended to predict recurrence risk in newly diagnosed patients with ER-positive, node-negative disease.

RT-PCR and DNA Microarray Analysis

The technologies underlying these assays are RT-PCR and DNA microarray analysis (see Figs. 1 and 2). In RT-PCR, quantification of a specific RNA molecule is performed by reverse transcription of the RNA into its complementary DNA and amplification of the DNA by PCR. The DNA is quantified after rounds of amplification through the use of fluorescent dyes that intercalate with double-stranded DNA or through use of modified DNA oligonucleotide probes that fluoresce when hybridized with complementary DNA. By attaching fluorescent dyes with different emission spectra to different probes, repeated implementations of PCR permit multiple DNA species to be quantitated in a single sample. RT-PCR is highly sensitive and is now widely used in quantifying absolute changes in gene expression and in validating results obtained with microarray and other techniques that measure global changes in gene expression.

DNA microarray analysis is based on the natural pairing of complementary nucleic acids. A set of DNA sequences (probes) is arrayed on a miniaturized solid surface (microarray) and used to detect concentrations of corresponding complementary RNA sequences (targets) in a sample. Advances in this technique have made it possible to assess the expression of thousands of different genes in a single reaction. In a basic "two-color" microarray analysis, RNA labeled with a fluorescent dye is hybridized to the microarray and incubated with RNA from another sample labeled with a different dye. Samples can be compared directly or in relation to a reference RNA. The microarray is scanned to produce grayscale images corresponding to fluorescence intensities of each gene being analyzed. The intensities of the signals are compared to detect relative levels of expression, which are quantified through computational/statistical techniques.

Guidelines for Use

These techniques offer considerable promise of producing gene profiles that can provide useful predictive and prognostic information about cancers. For the present, exactly how best to use information provided by existing assays to supplement clinicopathologic information in making treatment decisions remains somewhat uncertain. In part, this uncertainty arises because it is difficult to generalize about performance of the assays beyond the particular patient populations (in terms of both disease characteristics and treatments received) in which they were investigated and initially validated.

The most recent ASCO breast cancer guidelines (2007), in which multiparameter gene expression analysis was a new topic, state that the Oncotype DX assay may be used to identify patients who are predicted to obtain the most benefit from adjuvant tamoxifen and who may not require adjuvant chemotherapy. On the other hand, the guidelines note that patients with high recurrence scores appear to achieve relatively more benefit from adjuvant chemotherapy (specifically CMF or MF) than tamoxifen. The guidelines further state that there are insufficient data to comment on whether these conclusions generalize to hormonal therapies other than tamoxifen or to chemotherapy regimens other than (C)MF and that the precise clinical utility and appropriate application of other multiparameter assays remain under investigation.

The 2010 National Comprehensive Cancer Network (NCCN) guidelines recommend considering the Oncotype DX assay as an option in patients with tumors of 0.6 to 1.0 cm with unfavorable features or > 1.0 cm and node-negative, hormone receptor-positive, and HER2-negative disease. Those with a low score (< 18) can be treated with hormonal therapy, those with an intermediate score (18-30) can be treated with hormonal therapy with/without chemotherapy, and those with a high score (≥ 31) can be treated with both (all recommendations for use of the assay are considered NCCN category 2B). The guidelines emphasize that the recurrence score should be used for decision-making only in the context of other elements of risk stratification for an individual patient.

Randomized Trials

Important information on the potential use of the Oncotype DX assay and the MammaPrint test is expected from two prospective randomized trials. The NCI-sponsored Trial Assigning IndividuaLized Options for Treatment (Rx) (TAILORx) is being conducted by the North American Breast Cancer Intergroup. In this study, women whose ER-positive, node-negative disease meets established clinical guidelines for adjuvant chemotherapy will receive hormonal therapy alone if they have an Oncotype DX recurrence score < 11 and hormonal therapy and chemotherapy if they have a score > 25. Patients with scores of 11 to 25 are being randomized to hormonal therapy alone or in combination with chemotherapy to determine whether hormonal therapy alone offers less benefit than the combination.

The MINDACT trial (Microarray in Node-negative Disease may Avoid ChemoTherapy), conducted by the TRANSBIG network (a translational research arm of the Breast International Group), is comparing the MammaPrint test with a standard clinicopathologic prognostic tool (Adjuvant! Online) in selecting patients with 0 vs 1 to 3 positive nodes (hormone receptor-positive or -negative) for adjuvant chemotherapy. Patients with low risk on both measures do not receive chemotherapy, those with high risk on both measures do receive chemotherapy, and those with discordant risk assessment are randomized to chemotherapy or no chemotherapy. Both trials are also assessing outcomes according to different hormonal therapy and chemotherapy regimens. ■

For more information on multiparameter gene profiling in breast cancer, see:

Albain KS, Barlow WE, Shak S, et al: Prognostic and predictive value of the 21-gene recurrence score assay in postmenopausal women with node-positive, oestrogen-receptor-positive breast cancer on chemotherapy: a retrospective analysis of a randomised trial. Lancet Oncology 11(1):55-65, 2010.

Harris L, Fritsche H, Mennel R, et al: American Society of Clinical Oncology 2007 update of recommendations for the use of tumor markers in breast cancer. J Clin Oncol 33:5287-5312, 2007.

Marchionni L, Wilson RF, Marinopoulos SS, et al: Impact of gene expression profiling tests on breast cancer outcomes. Evidence Report/Technology Assessment no. 160. Prepared by The Johns Hopkins University Evidence-based Practice Center under contract no. 290-02-0018. AHRQ publication no. 08-E002. Rockville, MD; Agency for Healthcare Research and Quality; January 2008. http://www.ahrq.gov/clinic/tp/brcgenetp.htm

NCCN clinical practice guidelines in oncology: Breast cancer, V.2.2010. h ttp://www.nccn.org/professionals/physician_gls/PDF/breast.pdf.

Sotiriou C, Pusztai L: Gene-expression signatures in breast cancer. N Engl J Med 360:790-800, 2009.

Sparano JA, Solin LJ: Defining the clinical utility of gene expression assays in breast cancer: The intersection of science and art in clinical decision making. J Clin Oncol 28:1625-1627, 2010. See, also, references cited therein.

For information on the TAILORx trial, see: http://www.cancer.gov/clinicaltrials/ECOG-PACCT-1

For information on the MINDACT trial, see: http://www.cancer.gov/clinicaltrials/EORTC-10041