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 hone marrow micrometastases (BMM) of breast can cer, but recent studies have demonstrated the occurrence of false-positive results due to low-level, illegitimately transcribed CK19 in normal bone ma rrow tissue. One approach to solve this problem is to develop a quantitativ e CK19 RT-PCR assay and to introduce a cut-off value which can distinguish between illegitimate expression and cancer-specific expression levels, In t he present paper, we describe a quantitative CK19 RT-PCR assay using a real -time automated PCR system, The number of CK19 transcripts was normalized t o 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-II I patients with invasive breast cancers were subjected to CK19 RT-PCR assay and immunocytological examination. Forty (34.2%) were found to be BMM-posi tive by CK19 KT-PCR assay whereas only three (2.6%) were found to be BMM-po sitive by immunocytology, Multivariate analysis has shown that occult BMM d etected by CK19 RT-PCR is a significant risk factor for relapse, being inde pendent of axillary lymph node metastases.