Artikel
Disseminated tumor cells in bone marrow of breast cancer patients - Comparison of immunocytochemistry and RT-PCR
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Veröffentlicht: | 20. März 2006 |
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Gliederung
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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 299 bone marrow aspirates from breast cancer patients.
Methods: 299 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 for both procedures.
Results: Disseminated tumor cells could be detected in 53% (157 out of 299) of the aspirates by at least one of the methods. The positivity rates of ICC and RT-PCR were 37% and 39%, respectively. The concordance rate between ICC and RT-PCR was 71%. Discordant results were found in 87 patients.
46 BM aspirates were positive by RT-PCR but negative by ICC whereas 41 samples were positive only by ICC.
Conclusions: Both methods have their advantages. 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 but it lacks a standard protocol. We conclude that RT-PCR-based assays have a potential to improve diagnostics in this field. Further collaborative efforts must be made to establish a standard protocol for RT-PCR for the detection of disseminated tumor cells in bone marrow.