MINIMAL ILLEGITIMATE LEVELS OF CYTOKERATIN K19 EXPRESSION IN MONONUCLEATED BLOOD-CELLS DETECTED BY A REVERSE TRANSCRIPTION PCR METHOD (RT-PCR)

Cytokeratin K19 (CK19) expression was evaluated by a reverse transcrip tion PCR method (RT-PCR) in the RNA obtained from peripheral blood ste m cell collections (PBSC) from four patients with breast cancers (BC) and 34 mononucleated blood cell (MBC) negative controls (17 PBMC from normal subjects 12 PBSC from different types of leukaemias - M3, M4Eo, M2, etc. - and two from patients with Hodgkin's lymphoma; and three b one marrow (BM) collections). Two BC tissues were taken as positive co ntrols. The method studied (Datta YH, Paul T, Adams PT, Drobyski WR. S ensitive detection of occult breast cancer by reverse transcription po lymerase chain reaction. J Oncol 1994;12:475-8) is sensitive enough to allow the detection of CK19 transcripts in a 10(-6) dilution of cDNA reverse transcribed from 1 mu g of BC RNA, but CK19 transcripts were a lso detected in 64% of the RNA obtained from the MBC controls. However , the amplified product detected in the control samples represents the transcript of the CK19 gene as confirmed by the results of Mae III di gestion. It should be pointed out that although the CK19 expression wa s detected, the levels of expression in PBMC were almost negligible fo r they disappeared at 1:5 cDNA dilution. Moreover, a direct relationsh ip between the number of BC cells added to PBMC and the increasing dil ution levels of the cDNA necessary to prevent CK19 expression was obse rved. This allows us to conclude that the cDNA dilutions make it possi ble to distinguish the false from the true positive samples and that, in addition, the cDNA dilutions inform about the degree of BC cell con tamination. (C) 1997 Elsevier Science B.V.