Heterogeneous proliferative potential of occult metastatic cells in bone marrow of patients with solid epithelial tumors

Abstract

Bone marrow is a major homing site for circulating epithelial tumor cells. The present study was aimed to assess the proliferative capacity of occult metastatic cells in bone marrow of patients with operable solid tumors especially with regard to their clinical outcome. We obtained bone marrow aspirates from 153 patients with carcinomas of the prostate (n = 46), breast (n = 45), colon (n = 33), and kidney (n = 29). Most of the patients (87%) had primary disease with no clinical signs of overt metastases [tumor-node-metastasis (TNM)-stage UICC (Union Internationale Contre le Cancer) I-III]. After bone marrow was cultured for 21-102 days under special cell culture conditions, viable epithelial cells were detected by cytokeratin staining in 124 patients (81%). The cultured epithelial cells harbored Ki-ras2 mutations and numerical chromosomal aberrations. The highest median number of expanded tumor cells was observed in prostate cancer (2,619 per flask). There was a significant positive correlation between the number of expanded tumor cells and the UICC-stage of the patients (P = 0.03) or the presence of overt metastases (P = 0.04). Moreover, a strong expansion of tumor cells was correlated to an increased rate of cancer-related deaths (P = 0.007) and a reduced survival of the patients (P = 0.006). In conclusion, the majority of cancer patients have viable tumor cells in their bone marrow at primary tumor diagnosis, and the proliferative potential of these cells determines the clinical outcome.

Figure 1

Correlation between the tumor type and the extent of in vitro expansion of CK-positive tumor cells. The y axis shows the total number of cultured CK-positive cells per flask, which was calculated by multiplying the concentration of epithelial cells per cytospin with the total number of all cultured cells. The bars represent the median values in each subgroup of patients.

Figure 2

Correlation between the UICC-stage of the patients and the extent of in vitro expansion of CK-positive cells. The data from all patients with different tumor types and available staging information (n = 146) were pooled. The y axis shows the total number of cultured CK-positive cells per cytospin, which was calculated by multiplying the concentration of epithelial cells per flask with the total number of all cultured cells. The bars represent the median values in each subgroup of patients.

Figure 3

Overall survival of all patients depending on the detection (primary screening) and in vitro growth (culture screening) of CK-positive cells. According to the results of the CART analysis, patients were divided into two subgroups: category 1, weak expansion (n CK⁺ cells per flask < 2,105]; category 2, strong expansion (n CK⁺ cells per flask ≥ 2,105]

Figure 4

Representative cell nuclei after hybridization of the five-color probe mix. Centromere 7 is depicted in red, centromere 8 in green, centromere 11 in blue, centromere 17 in yellow, and centromere 18 in purple. (a and b) Interphase cells of patient E.E.: the cell shown in a is in the tetraploid range with loss of one signal for centromere 8 and loss of three centromere 17 signals; the diploid cell in b has three centromere 7 signals. (c–e) Interphase cells of patient M.P.: (c) diploid cell with two copies of each probe; (d and e) interphase cells with loss of one centromere 18 signal (d), and loss of signals for centromeres 11 and 18 (e). (f) Interphase cell of patient K.H. demonstrating a large variety of different signal numbers.