Application of immunomagnetic beads in combination with RT-PCR for the detection of circulating prostate cancer cells

Abstract

Recently published protocols using Reverse Transcriptase Polymerase Chain reaction (RT‐PCR) for prostate specific antigen (PSA) provide a sensitive means for detecting circulating prostate cancer cells. Attempts to use these assays for staging of prostate cancer have produced conflicting results. As a first step towards rectifying these discrepancies, a modified immunobead‐RT‐PCR assay capable of detecting as few as 10 prostate cancer cells in 8cc of blood was developed. This 10 fold increase in sensitivity was achieved in part by introducing two target cell enrichment steps. As a model system to assess sensitivity of the modified assay, template RNA was extracted from PSA positive human carcinoma cells suspended in human blood and isolated with immunomagnetic beads following incubation with an epithelium specific antibody. After 45 cycles of PCR, product from as few as 10 target cells could be readily detected when displayed on a 2% agarose gel stained with SYBR Green fluorescent dye. The identity of amplified DNA fragments was confirmed by Southern blot hybridization. When applied to blood samples from patients with proven metastatic disease, the immuno‐bead RT‐PCR assay was successful in detecting circulating PSA positive epithelial cells, suggesting this assay may be useful for assessment of disease progression or recurrence. J. Clin. Lab. Anal. 11:346–350, 1997. © 1997 Wiley‐Liss, Inc.

Immunobead RT-PCR versus regular RT-PCR amplification of CEA mRNA in peripheral blood

“…Makarovskiy et al (1997) were able to detect ten T-47D human breast cancer cells in 8 ml of blood. Sakaguchi et al (1999) were able to detect ten breast cancer cells in 10 7 cytologically negative eusion cells.…”

“…As 1 ml of normal blood contains 4´10 6 to 1´10 7 white blood cells, the sensitivity of immunobead RT-PCR was 10 1 tumor cells in 4´10 6 to 1´10 7 white blood cells. This sensitivity was comparable to those of previous reports (Hardingham et al 1993;Makarovskiy et al 1997;Ko et al 1998;Sakaguchi et al 1999). In some previous reports, density gradient centrifugation was used to remove erythrocytes before the immunomagnetic separation.…”

“…Sakaguchi et al (1999) established a sensitive and speci®c method for the detection of epithelial cancer cells in eusions using a two-stage molecular-based assay that combines the enrichment for cancer cells by immunomagnetic bead selection and the RT-PCR-based detection of epithelial glycoprotein2 RNA. Circulating prostatic carcinoma cells were detected using a three-step immunobead RT-PCR assay for prostate speci®c antigen (PSA) mRNA (Makarovskiy et al 1997;Ghossein et al 1999).…”

Dissecting the Heterogeneity of Circulating Tumor Cells in Metastatic Breast Cancer: Going Far Beyond the Needle in the Haystack

“…CTC have been identified in many solid tumors including breast [8], prostate [9], lung [10], bladder [11], gastric [12], and colon cancer [13], while they are rarely found in healthy people or in people with nonmalignant tumors [10]. …”

Molecular detection of low-level disease in patients with cancer

“…Although some investigators have published encouraging results using epithelial cell targets, many studies have been uninformative, as their target RNA has been detected in a large number of negative controls46–49. More recently, a number of studies have described a combined approach, involving the enrichment of blood samples with tumour cells using immunomagnetic beads and then assaying by RT‐PCR, which may overcome some of the challenges associated with target gene expression in control samples50, 51. Furthermore, the heterogeneity of tumour cells means that for many cancers a panel of several markers may be more reliable than a single target for the detection of tumour cells.…”