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 < 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 ؋ 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.breast cancer ͉ hematogeneous dissemination of tumor cells ͉ cDNA ͉ prognostic markers ͉ differential display

Human GLI2 and GLI1 are part of a positive feedback mechanism in Basal Cell Carcinoma

“…Ratios were calculated from the mean value of six real-time PCR measurements obtained from three independent PCR runs with duplicate samples. RPLP0 (Martin et al, 2001) and Cyclophilin E (data not shown) were used as a reference to control for the amount of sample material. GLI1 expression in human keratinocytes leads to GLI2 transcription and GLI1 auto-activation…”

“…Standard deviation was below 20 per cent between all replicate experiments. Large ribosomal protein P0 (RPLP0) was used as a reference standard for all analyses to control for the amount of sample material (Martin et al, 2001) simultaneous increase in GLI1 mRNA levels. We therefore constructed a HaCaT cell line expressing His-tagged GLI2b in a tetracycline-regulated manner.…”

“…'Primer only' controls, without template were done to ensure the absence of primer dimers. Large ribosomal protein P0 (RPLP0) was used as a reference standard for all analyses to control for the amount of sample material (Martin et al, 2001) 1998; Mo et al, 1997;Motoyama et al, 1998;Park et al, 2000). Only recently, it was shown that overexpression of mouse Gli2 in the basal epidermal layer of transgenic mice induced the formation of BCC-like tumours (Grachtchouk et al, 2000).…”

Head and neck squamous cell carcinoma transcriptome analysis by comprehensive validated differential display

“…There are other studies combining DD with array analysis that have experienced the same lack of sensitivity of arrays. Martin and collaborators (Martin et al, 2001) found that less than 10% (12/170) of the DD-isolated sequences spotted on a membrane gave useful results upon further analysis of additional patient samples. Several studies using PCR-DD and mAs as complementary approaches detected few genes (0-10) by both approaches (Outinen et al, 1998;Wells, 1999;Cirelli and Tononi, 2000;Oetting, 2000;Heilig and Sommer, 2004;Pascal et al, 2005).…”

Cooperation between GLI and JUN enhances transcription of JUN and selected GLI target genes

“…cDNA synthesis and qRT-PCR analysis were carried out as described in Eichberger et al (2006). Human large ribosomal protein P0 (RPLP0) was used for normalization of sample material in qRT-PCR analysis (Martin et al, 2001). For primer sequences see Supplementary Table S2.…”