High-sensitivity array analysis of gene expression for the early detection of disseminated breast tumor cells in peripheral blood

Abstract

Early detection is an effective means of reducing cancer mortality. Here, we describe a highly sensitive high-throughput screen that can identify panels of markers for the early detection of solid tumor cells disseminated in peripheral blood. The method is a two-step combination of differential display and high-sensitivity cDNA arrays. In a primary screen, differential display identified 170 candidate marker genes differentially expressed between breast tumor cells and normal breast epithelial cells. In a secondary screen, high-sensitivity arrays assessed expression levels of these genes in 48 blood samples, 22 from healthy volunteers and 26 from breast cancer patients. Cluster analysis identified a group of 12 genes that were elevated in the blood of cancer patients. Permutation analysis of individual genes defined five core genes (P < or = 0.05, permax test). As a group, the 12 genes generally distinguished accurately between healthy volunteers and patients with breast cancer. Mean expression levels of the 12 genes were elevated in 77% (10 of 13) untreated invasive cancer patients, whereas cluster analysis correctly classified volunteers and patients (P = 0.0022, Fisher's exact test). Quantitative real-time PCR confirmed array results and indicated that the sensitivity of the assay (1:2 x 10(8) transcripts) was sufficient to detect disseminated solid tumor cells in blood. Expression-based blood assays developed with the screening approach described here have the potential to detect and classify solid tumor cells originating from virtually any primary site in the body.

Figure 1

View of gene cluster I and control gene expression levels for 48 blood samples. Healthy volunteers included 15 women with no history of breast disease; patients included 26 women with a variety of stages of breast cancer. Gene expression levels are color-coded according to key. Blood samples N0a, N0b, and N0c were drawn from the same individual at successive time points, as were samples N2a, N2b, N2c, N4a, and N4b. N2c(1) and N2c(2) represent two different experiments performed with the same sample of N2c RNA, as do N4a(1) and N4a(2). Cancer patients are ordered by stage of disease at the time of blood collection. Patients who had received any chemotherapy treatments before blood collection are indicated. (Middle) Five “housekeeping” genes as nondifferential controls. (Lower) Four genes commonly used as breast tumor markers. Full cluster diagram with all gene identities is available (see Fig. 5, which is published as supplemental data on the PNAS web site).

Figure 2

Real-time PCR confirmed cDNA array expression levels. mdm-2 and gro-alpha expression levels were measured in the blood of breast cancer patients and healthy volunteers. PCR results were normalized to total cDNA determined by OD₂₆₀ and expressed relative to sample N27. Array assay was not performed for sample N27.

Figure 3

Real-time PCR results to determine sensitivity of cDNA arrays. (A) Representative real-time PCR curves for a set of dilutions of mdm-2 primers with N27 normal blood. Threshold level is indicated. Cycle times exceeding the threshold (C_T) for a set of dilutions were used to generate a standard curve. (B) Standard curve generated for mdm-2 dilution series with N27 normal blood RNA. (C) Identical standard curves were generated when maspin plasmid was measured alone or in the presence of normal blood cDNA. (D) Amplification plots used to determine the expression level of maspin in blood sample N21.