Prognostic Significance of Occult Bone Marrow Micrometastases of Breast Cancer Detected by Quantitative Polymerase Chain Reaction for Cytokeratin 19 mRNA

Amplification of cytokeratin 19 (CK19) transcripts by reverse transcriptase‐polymerase chain reaction (RT‐PCR) has been shown to be a highly sensitive assay for the detection of bone marrow micrometastases (BMM) of breast cancer, but recent studies have demonstrated the occurrence of false‐positive results due to low‐level, illegitimately transcribed CK19 in normal bone marrow tissue. One approach to solve this problem is to develop a quantitative CK19 RT‐PCR assay and to introduce a cut‐off value which can distinguish between illegitimate expression and cancer‐specific expression levels. In the present paper, we describe a quantitative CK19 RT‐PCR assay using a real‐time automated PCR system. The number of CK19 transcripts was normalized to that of GAPDH transcripts as an internal control for quality and quantity of cDNA. The cut‐off value for the ratio of CK19 to GAPDH transcripts was set at 10(−4) since the ratio never exceeded this value in the control bone marrow samples (n=12). In total, 117 bone marrow aspirates from stage I‐III patients with invasive breast cancers were subjected to CK19 RT‐PCR assay and immunocytological examination. Forty (34.2%) were found to be BMM‐positive by CK19 RT‐PCR assay whereas only three (2.6%) were found to be BMM‐positive by immunocytology. Multivariate analysis has shown that occult BMM detected by CK19 RT‐PCR is a significant risk factor for relapse, being independent of axillary lymph node metastases.