Isolated Tumor Cells in Bone Marrow Three Years after Diagnosis in Disease-Free Breast Cancer Patients Predict Unfavorable Clinical Outcome1

Abstract

Purpose: The aim of the study was to explore the value of analyzing bone marrow (BM) for the presence of isolated tumor cell(s) (ITCs) in disease-free breast cancer patients 3 years after diagnosis.Experimental Design: ITCs in BM at operation was found to be an independent prognostic factor in 817 breast cancer patients. Among these, 356 disease-free patients were analyzed with a second BM after 3 years follow-up (median 40 months, SD 3 months, range 29 -52). ITC was detected by immunocytochemistry with anticytokeratine antibodies (AE1/AE3).Results: The population consisted of 70% T 1 and 72% node-negative patients. ITC in BM was detected in 15%. At a median of 25 months since the second BM aspiration (66 months since diagnosis), 32 patients had developed relapse, 12 local and 20 systemic. Of the patients with ITC in BM, 21% relapsed compared with 7% of the ITC-negative patients (P < 0.001). Ten patients died of breast cancer. Survival analyses showed that ITC in BM predicted reduced distant disease-free survival (DDFS) and breast cancer specific survival (BCSS; P < 0.001, log-rank test). Uni-and multivariate analyses of the prognostic value of N, T, estrogen receptor/progesterone receptor, and BM status, histological grade, vascular invasion, p53-, c-erb-B2-, and cathepsin D expression were performed. BM status was the only independent prognostic factor for both DDFS and BCSS, whereas c-erbB-2 and N status were independent for BCSS and vascular invasion and T status for DDFS.Conclusions: ITC in BM 3 years after diagnosis in disease-free breast cancer patients is an independent prognostic factor. Detection of residual disease by BM analysis at follow-up may unravel insufficient adjuvant treatment. The clinical implications should be further explored.

A concept for the standardized detection of disseminated tumor cells in bone marrow from patients with primary breast cancer and its clinical implementation

“…26 Repeated bone marrow aspiration performed under local anesthesia also can be performed to obtain information regarding the persistence of DTCs. [18][19][20][21] Bone marrow aspirate is then transferred into heparinized tubes. 10,[13][14][15]18,23,24,27 Ethylenediamine tetraacetic acid 24 or sodium citrate 28 also may be used as anticoagulant.…”

“…8,10,13,14,19,25,28,30 Negative immunomagnetic separation of epithelial tumor cells has been investigated in a single, large clinical trial but showed no significant advantage over Ficoll density-gradient centrifugation. 8 For this reason and because of the significantly higher costs of immunomagnetic separation, the immunomagnetic separation procedure is not recommended currently for routine use.…”

“…After density-gradient centrifugation, the interphase layer that contains mononucleated cells is washed, the cellularity is counted, and from 0.5 to 1.0 Â 10 6 cells are spun down onto each glass slide. 13,14,19 This procedure ensures reproducible cell numbers for evaluation and provides the basis for comparison of results from different laboratories. The number of mononucleated cells per slide depends on the type of cytospin device used; it is important that the cytospin should not be overloaded (i.e., the cells should not be on top of each other).…”

“…16 Numerous studies (Table 1) have now demonstrated that the detection of DTCs in bone marrow is associated with a poor prognosis, a finding that recently was confirmed in a pooled analysis study of >4700 patients with Stage I, II, or III breast cancer. 17 Beyond the conclusive data on the prognostic relevance of the presence of DTCs at the time of diagnosis, monitoring of therapeutic efficacy and assessment of residual risk during event-free followup [18][19][20][21] represent a promising option for the clinical utility of DTC detection. Acceptable standards for DTC detection need to be defined to allow prospective clinical trials.…”

Relevance of circulating tumor cells, extracellular nucleic acids, and exosomes in breast cancer

“…Studies that investigated the prognostic value of CTCs/DTCs at the time of completion of adjuvant therapy [42, 106,107] showed that the detection of DTCs [42,108] and CTCs [106] after the completion of chemotherapy is associated with an unfavorable clinical outcome [103,105]; further reviewed by Ignatiadis et al [109]. In keeping with this work suggesting diagnostic and prognostic relevance for CK19, more recently full-length CK19 has been detected in bone marrow specimens from breast cancer patients and is associated with the presence of overt metastases and poor survival rates [110].…”