[Levels of cytokeratin CK19 expression in mononuclear blood cells evaluated using a reverse PCR (RT-PCR)]

The sensitivity and specificity of a reverse transcription PCR method (RT-PCR) to detect cytokeratin K19 (CK19) expression was evaluated with the purpose of assessing its capability to detect the presence of breast cancer tumour cells in peripheral blood progenitor cell collection that had to be reinfused to breast cancer patients submitted to intensive chemotherapy as haematopoietic support.

Two breast cancers as positive samples were used and 34 samples of mononucleated blood cells as negative controls: 18 peripheral blood samples from normal subjects, 14 from different types of leukaemias (M3, M4Eo, M2, etc.) and two from two patients with Hodgkin's lymphoma. The method studied is a nested RT-PCR that amplifies the CK19 expression from the sample RNA extracted following the method of phenol-chloroform.

The right performance of the method is demonstrated by observing the detection of CK19 transcripts in the breast cancer RNA and by obtaining good blank results both with non transcribed RNA and with DNA. Moreover, the method has an excellent sensitivity such as to allow the detection of CK19 transcripts in a 10(-6) dilution of cDNA reverse transcribed from 1 microgram of breast cancer RNA. The CK19 transcripts were also detected in the 64% of RNA obtained from the mononucleated blood cells controls, although the percentage of positivities was lower (47%) in the RNA from peripheral blood samples. Nevertheless it should be remarked that the levels of CK19 expression in the blood mononucleated cells is almost negligible since it used to extinguish at 1:5 cDNA dilution.

The method studied is specific and has a high sensitivity that explains the detection of CK19 illegitimate expression approximately a half in mononucleated blood cells negative controls. However, the levels of CK19 expression in mononucleated blood cells were almost negligible and it used to extinguish at 1:5 cDNA dilution, therefore it could be concluded that the method might be useful to detect breast cancer occult tumours cells in mononucleated blood cell collection, always provided that a lower amount of cDNA is taken, thus decreasing to nil almost the false positive samples and keeping always a good sensitivity.