The Results of Stricter Inclusion Criteria in an Immunomagnetic Detection Study of Micrometastatic Cells in Bone Marrow of Uveal Melanoma Patients - Relevance for Dormancy.

Approximately 50% of uveal melanoma patients develop metastases. We want to evaluate the effect of stricter criteria on our data from our previous study correlating survival and bone marrow (BM) micrometastasis results using our immunomagnetic separation (IMS) method. Mononuclear cell fractions (MNC) isolated from BM were examined for tumour cells and the patients were classified as BM positive (BM+) or BM negative (BM-). The study originally included 328 consecutive patients with uveal melanoma from 1997 to 2006. The cohort was limited to 217 patients when we introduced cyto- or histopathological verification of melanoma cells in the patient as a main new criterion for inclusion. Tumour cells were found in BM-samples in 38.7% (95% CI, 32-45) at enrolment. Until the latest work-up 43.8% (95% CI, 38-50) of patients had developed melanoma metastases. After a minimum follow-up time of 8.5 years, 60.4% (95% CI, 54-66) of patients had died. The causes were: melanoma metastases 69.5%, another type of cancer 5.4% and non-cancerous causes 19.5%. Overall median survival was shorter for the BM- patients (11.3 years) (95% CI, 10-12) compared to the BM+ (16.5 years) (95% CI, 12-14), p = 0.04, log rank test. All-cause mortality and specific melanoma mortality estimated after 12 year follow-up showed a highly significant difference comparing BM- and BM+, p = 0.010 and p = 0,017, respectively. IMS yields a high fraction of BM+ samples due to micrometastasis at diagnosis and these cells appear to have a positive prognostic impact strengthening our previous report. The late recurrences support the concept of tumour dormancy.

Immunomagnetic detection of micrometastatic cells in bone marrow of uveal melanoma patients: a paradox.

PURPOSE: Our objective was to study survival rates with the bone marrow (BM) results in a cohort of uveal melanoma patients with long follow-up. METHODS: Mononuclear cell fractions isolated from BM were examined for tumour cells using our immunomagnetic separation (IMS) method. The patients were classified as BM positive or BM negative. Clinical follow-up, histopathological findings, vital status and cause of death were registered. RESULTS: The study included 328 consecutive patients with uveal melanoma from 1997 to 2006. Tumour cells were found in BM samples in 29% (95% CI, 25-34) at enrolment (96 cases). After a minimum follow-up time of 6 years, 156 (48%) (95% CI, 42-53) melanoma patients had died. The causes were as follows: melanoma metastases 92 (59%), another cancer 20 (13%) and non-cancer 44 (28%). Nine patients were still living with melanoma metastases. Until the latest work-up, 101(31%) (95% CI, 26-36) patients had developed melanoma metastases. Cyto- or histopathological verification of the metastatic lesions was obtained in 85 cases (84%). In the group with melanoma metastases, 28 tested BM positive at study entry (28%) (95% CI, 19-38). In total, 39 of 101 with metastases tested positive at least once after a maximum of three tests (39%) (95% CI, 29-49). The overall median survival from the first BM test was shorter for the BM negative patients (9.5 years) compared with the BM positive (14.4 years), p = 0.02, log rank test. CONCLUSION: Ocular melanoma cells detected in BM seem to have a positive prognostic impact on survival in contrast to our original hypothesis.

In vitro and in vivo properties of CD133 expressing cells from human lung cancer cell lines.

BACKGROUND: Tumor development is recently hypothesized to depend on a rare cell population with stem cell properties, such cells are called cancer stem cells (CSCs) or tumor-initiating cells (TICs). From various cancer tissues or cancer cell lines, CD133 expressing cells were found to define a unique CSC/TIC phenotype. To study whether that also could be the case in lung cancer, we examined different lung cancer cell lines for CD133 expression. RESULTS: Among the 4 cell lines studied, only the cell line LC-42 expressed CD133. Therefore, LC-42 was further characterized and studied with special emphasis on identifying the presence of CD133+ CSCs/TICs. FACS sorted CD133high and CD133dim subpopulations from LC-42 showed no differences in soft agar colony-forming capacity and spheres-forming capacity in serum-free cultures. LC-42 cells contained Side Population (SP), and only SP cells were able to form spheres. Furthermore, Nanog expression was significantly higher in SP than in non-SP. However, no difference was observed of CD133 expression in SP and non-SP. When CD133high and CD133dim cells were serially xeno-transplanted in NOD/SCID mice, both formed tumours similar to their parental LC-42 cells. There were no expression differences for NANOG, OCT4 and SOX2 examined immunohistochemically in the xenografts from both cell fractions. CONCLUSION: Our data do not show a difference in tumorigenic potential of CD133high and CD133dim cells with respect to any of the parameters analyzed in vitro and in vivo, suggesting that CD133 expression is not restricted to cancer-initiating cells in the human lung cancer cell line LC-42.

Disseminated tumour cells in bone marrow of patients with uveal melanoma.

PURPOSE: Approximately 50% of patients with uveal melanomas develop metastases. Thus, it is important to improve our understanding of how melanoma metastases develop. METHODS: As part of a uveal melanoma micrometastasis study, we compared the detection rates of immunomagnetically selected (IMS) tumour cells in bone marrow (BM) with positively stained tumour cells using immunocytochemistry (ICC). Bone marrow mononuclear cells were isolated. Immunocytochemistry cytospin preparations were immunocytochemically stained in parallel with two different melanoma antibodies, 9.2.27 and HMB45. Using IMS, melanoma cells were selected from BM mononuclear cell fractions using immunomagnetic beads coated with the 9.2.27 antibody and identified by light microscopy. RESULTS: In cytospin preparations from 226 patients, melanoma cells were detected in 24 (10.6%), 10 with 9.2.27 and 17 with the HMB45 antibody. In three cases, we found positive cells with both antibodies. Six of the 226 (2.6%) patients that stained positively with ICC died with metastatic disease, all also positive with IMS. Sixty-six (29.2%) patients had positive BM samples with IMS at the first examination. Immunomagnetic selection (IMS) was positive in 36.8% of the 57 patients who later developed clinical metastases. Twenty-one IMS-positive patients and 31 IMS-negative patients died of metastases, in total 52 of 226 patients (23.0%). The mortality rate of melanoma metastasis was 24% (6/24) after at least 4 ½ years in ICC-positive patients compared to 38.5% (20/52) in IMS-positive patients. CONCLUSION: The presence of melanoma cells in BM of patients with uveal melanoma is documented in our study with IMS and ICC. Immunomagnetically selected is more sensitive than ICC in detecting tumour cells in BM. However, statistically, we did not find any prognostic impact of the presence of melanoma cells in BM.

Osteoblast-induced EGFR/ERBB2 signaling in androgen-sensitive prostate carcinoma cells characterized by multiplex kinase activity profiling.

Bone metastases in prostate cancer are predominantly osteoblastic. To study regulatory mechanisms underlying the establishment of prostate cancer within an osteoblastic microenvironment, human androgen-sensitive prostate carcinoma cells (LNCaP) were treated with culture medium conditioned by human osteoblast-derived sarcoma cells (OHS), and activated signalling pathways in the carcinoma cells were analyzed using microarrays with tyrosine kinase substrates. Network interaction analysis of substrates with significantly increased phosphorylation levels revealed that signalling pathways mediated by EGFR and ERBB2 were activated in LNCaP cells under OHS influence but also by androgen treatment. Activation of EGFR/ERBB2 signalling was also found in LNCaP cells in cocultures with OHS cells or osteoblastic cells that had been differentiated from human mesenchymal stem cells. Our experimental data suggests osteoblast-directed induction of signalling activity via EGFR and ERBB2 in prostate carcinoma cells and may provide a rationale for the use of EGFR or ERBB2 inhibition in systemic prevention or treatment of metastatic prostate cancer in the androgen-sensitive stage of the disease.

Immunomagnetic detection of micrometastatic cells in bone marrow in uveal melanoma patients.

PURPOSE: Our objective was to introduce immunomagnetic separation (IMS) in ocular research by evaluating the possibility of detecting tumour cells in bone marrow (BM) and peripheral blood (PB) samples and validating the captured cells as melanocytic cells. METHODS: Mononuclear cell (MNC) fractions isolated from BM and PB in uveal melanoma patients were examined for tumour cells using our IMS method. Sheep-anti-mouse IgG antibody-coated super paramagnetic particles were conjugated to an anti-melanoma antibody. Microscopy of the magnetic fraction isolated from MNCs was performed to identify and count the number of bead-rosetted cells. The finding of at least two rosettes with coated beads in a 20-microl fraction of a sample was registered as a positive test. The melanocytic nature of the tumour cells was ascertained with a double labelling procedure using fluorescent microparticles. RESULTS: Using IMS in a study of 328 patients, tumour cells were at initial diagnosis found in BM and PB in 29.9% and 1.6% of cases, respectively. In positive samples, a median of 56 tumour cells (range 2-500) were identified. The captured cells were documented to be of melanocytic origin by the simultaneous binding of fluorescent beads coated with another melanoma-associated antibody. CONCLUSIONS: The IMS method was sensitive and efficient in the detection of occult melanoma tumour cells in BM. The validity of the immunomagnetic technique was strengthened by verifying the melanocytic characteristics of the isolated cells. The IMS procedure identifies intact, vital tumour cells, permitting further molecular characterization, an advantage which makes this method attractive for extended use. The clinical relevance of the findings will be further investigated in follow-up studies with repeated sampling and characterization of the isolated tumour cells.

S100B in bone marrow aspirates in healthy individuals and malignant melanoma patients.

The aim of this study was to evaluate S100B in bone marrow (BM) plasma from malignant melanoma patients. BM aspirates and peripheral blood (PB) plasma from 56 patients and BM aspirates from 29 healthy volunteers were collected. S100B was measured using an immune radiometric assay, which is a two-site sandwich assay based on monoclonal antibodies recognizing the beta-subunit. In the control population, the median S100B level in BM plasma was 9.0 microg/l (26 women and three men), an unexpectedly high value compared with the median S100B level in PB<0.05 microg/l. S100B levels in BM seems to be sex dependent. Median S100B levels in samples taken from male melanoma patients was 26.7 microg/l in contrast to 9.3 microg/l in female patients (Mann-Whitney P<0.002). The elevated BM S100B in melanoma patients could not be explained by presence of melanoma cells in the BM, as the values also were increased to the same extent in patients with no detectable BM metastases. In attempts to identify the source of S100B in BM, cytospins from five patients with high S100B values were stained, but none of the BM cells stained positive. S100B levels in PB were dependent on the stage of melanoma disease and there was a significant shorter survival time in the group of patients with elevated S100B compared with the group with normal S100B values, (log rank test: P=0.04). In BM taken from melanoma patients, however, there were no association between S100B levels and survival. The median S100B level in BM aspirates from healthy female volunteers and BM samples from female melanoma patients were 8.1 and 9.3 microg/l both manifold higher than the cut-off value for S100B in PB (0.2 microg/l). The median S100B in the samples taken from male melanoma patients was nearly three times higher than in the female patients. Unlike S100B in PB, S100B in BM demonstrated no prognostic value. The explanation for the unexpected high S100B in BM remains elusive.

Specific isolation of disseminated cancer cells: a new method permitting sensitive detection of target molecules of diagnostic and therapeutic value.

Molecular studies of rare cells, such as circulating cancer cells, require efficient pre-enrichment steps to obtain a pure population of target cells for further characterization. We have developed a two-step approach, starting with immunomagnetic enrichment, followed by specific isolation of individual, easily identifiable bead-rosetted target cells using a new semi-automated CellPick system. With this procedure, 1-50 live target cells can now be isolated. As a model system, we spiked a small number of tumor cells into millions of normal mononuclear cells (MNCs). Efficient isolation of pure target cells was obtained by use of the CellPick system, and the nature of isolated, bead-rosetted cells was verified by use of FISH. Single breast cancer cells were picked directly into an RNA preserving lysis buffer, reverse transcribed, and PCR amplified with two cDNA specific primer sets. With the isolated cells we consistently obtained both ubiquitously expressed and tumor cell specific PCR products. We also performed a successful mutation analysis of single cells using PCR and cycling temperature capillary electrophoresis (CTCE). This may have significant clinical implications in cancer and in other diseases, e.g. in characterizing micrometastatic cancer cells in blood and lymph nodes to help identifying patients who most likely will respond to therapies like tyrosine kinase inhibitors and compounds targeting specific mutations. By use of the CellPick system it is possible to specifically isolate bead-rosetted or otherwise labelled target cells from a heterogeneous cell population for further molecular characterization.

Immunomagnetic detection and clinical significance of micrometastatic tumor cells in malignant melanoma patients.

PURPOSE: Positive associations between the presence of micrometastatic tumor cells and disease aggressiveness have been reported in several tumor types, but the clinical implications are still not established. We wanted to test a new, sensitive immunomagnetic detection method on bone marrow (BM) and peripheral blood (PB) samples from patients with malignant melanoma and relate the findings to clinical outcome. EXPERIMENTAL DESIGN: Samples from 210 patients admitted for relapse of cutaneous melanoma were examined. Mononuclear cell fractions isolated from BM and PB were incubated with superparamagnetic particles coated with antimelanoma antibodies. Live tumor cells with bound beads were isolated with a magnet and identified in a microscope as cell-bead rosettes. Beads without antibody or with an irrelevant antibody were used as controls. The whole procedure was completed within 2-3 h. The identity of the cells was confirmed with a new double labeling procedure with fluorescent microparticles. RESULTS: Rosetted melanoma cells were found in BM aspirates of 35 of 186 (19%) patients, but in only 2 of 208 (1%) PB samples. The controls were all negative. After a median observation time of 1.1 year (range, 0-6.8 years), patients with tumor cells in BM showed a significantly shorter overall survival from time of BM aspiration (P = 0.009). In multiple regression analysis, a positive BM test was a strong indicator of overall survival (P = 0.021), associated with disease stage (American Joint Committee on Cancer) and with the number of metastatic sites, but not with the primary (Breslow) tumor depth and morphology. CONCLUSIONS: The results demonstrate the prognostic significance of detecting BM micrometastasis in melanoma patients. The results strengthen the validity of the immunobead technique. In contrast to other techniques, the method identifies intact, live tumor cells that can be further characterized, making the assay attractive for extended use.