Disseminated tumor cells in bone marrow of breast cancer patients–Comparison of immunocytochemistry and RT-PCR

• Main Authors: Fehm, T, Becker, S, Banys, MJ, Becker-Pergola, G, Duerr-Stoerzer, S, Krawczyk, N, Solomayer, EF
• Format: Conference Proceeding
• Language: eng ; ger
• ISSN: 0016-5751; 1438-8804
• DOI: 10.1055/s-2006-952799

Background Since 1999 immunocytochemistry (ICC) has been regarded as the standard method of choice for detection of disseminated tumor cells (DTC) in bone marrow (BM). In the meantime molecular-based assays were developed, such as RT-PCR. The purpose of our investigation was to evaluate both techniques with respect to their sensitivities on the basis of 417 bone marrow aspirates from breast cancer patients. Methods 417 bone marrow aspirates from primary breast cancer patients were processed with both methods. Immunocytochemistry After Ficoll enrichment of 10ml bone marrow, cytospins were prepared and stained using the A45-B/B3 primary antibody for pCK. Cytospins were analyzed using the ACIS system (Chromavision) according to the ISHAGE evaluation criteria. RT-PCR mRNA was extracted and purified using the mRNA isolation for blood and bone marrow kit (Roche® Molecular Biochemicals). RT-PCR was performed on the LightCycler® system, using the RNAMaster Hybridization Probes kit and custom primers and probes. Primers were selected to amplify a 380bp fragment of the CK19 gene. Positive and negative controls were included with each batch of samples. Results Altogether, in 50% (207 out of 417) of the aspirates, disseminated tumor cells could be detected by at least one method. Concordance rate of 72% between ICC and RT-PCR was observed. On the contrary, in 117 patients discordant results were found. 60 BM aspirates were positive by RT-PCR but negative by ICC whereas 57 samples were positive only by ICC. The positivity rates of ICC and RT-PCR were 35% and 36%, respectively. Conclusions ICC allows an interpretation based on morphological criteria and is a standardized procedure with well-known correlation to the prognosis but it is observer-dependent and labor intensive. RT-PCR is time-efficient and may increase the sensitivity. We conclude that RT-PCR-based assays have a potential to improve diagnostics in this field.