Comparison of molecular and immunocytochemical methods for detection of disseminated tumor cells in bone marrow from early breast cancer patients

Bjørnar Gilje, Oddmund Nordgård, Kjersti Tjensvoll, Elin Borgen, Marit Synnestvedt, Rune Smaaland, Bjørn Naume, Bjørnar Gilje, Oddmund Nordgård, Kjersti Tjensvoll, Elin Borgen, Marit Synnestvedt, Rune Smaaland, Bjørn Naume

Abstract

Background: Disseminated tumor cells (DTCs) have potential to predict the effect of adjuvant treatment. The purpose of this study was to compare two methods, reverse transcription quantitative PCR (RT-qPCR) and immunocytochemisty (ICC), for detecting breast cancer DTCs in bone marrow (BM) from early breast cancer patients.

Methods: We investigated a subset (n = 313) of BM samples obtained from 271 early breast cancer patients in the "Secondary Adjuvant Taxotere Treatment" (SATT)-trial. All patients in this study had node positive or intermediate/high-risk node negative non-metastatic disease. The DTCs were detected by ICC using AE1-AE3 anti-cytokeratin monoclonal antibodies. Patients with DTCs detected in their BM by ICC after standard adjuvant fluorouracil, cyclophosphamide, epirubicin (FEC) chemotherapy were offered docetaxel treatment. For comparison, 5 × 106 mononuclear cells from the aliquoted BM samples were also analyzed by RT-qPCR using a multimarker (MM) assay based on the tumor cell mRNA markers keratin 19 (KRT19), mammaglobin A (hMAM), and TWIST1. In the MM-assay, a sample was defined as positive for DTCs if at least one of the mRNA markers was positive.

Results: The MM RT-qPCR assay identified DTCs in 124 (40%) of the 313 BM samples compared with 23/313 (7%) of the samples analyzed by ICC. The concordance between the MM RT-qPCR and ICC was 61% (Kappa value = 0.04) and twelve of the BM samples were positive by both methods. By RT-qPCR, 46/313 (15%) samples were positive for KRT19, 97/313 (31%) for TWIST1, and 3/313 (1%) for hMAM mRNA. There were no statistically significant associations between the individual mRNA markers.

Conclusion: The RT-qPCR based method demonstrated more DTC-positive samples than ICC. The relatively low concordance of positive DTC-status between the two different assessment methods suggests that they may be complementary. The clinical relevance of the methods will be evaluated based on future clinical outcome data.

Trial registration: ClinicalTrials.gov: NCT00248703.

Figures

Figure 1
Figure 1
Relative levels of TWIST1 and KRT19 mRNA in BM samples from 267 early breast cancer patients. The levels were calculated using the 2ΔΔCq method and normalized by dividing by the highest level in the control samples. The horizontal line represents the highest level in the control samples with the relative value of one. hMAM is not shown in the plot because no expression was found in the normal control samples.

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