Hanne K. Høifødt

Hematogenous Micrometastases in Osteosarcoma Patients

Bone marrow and peripheral blood samples from 60 patients with suspected bone sarcoma were examined for the presence and number of micrometastatic osteosarcoma cells by a sensitive immunomagnetic detection assay, using in parallel two osteosarcoma-associated antibodies. Forty-nine of the patients had osteosarcoma, and of these, as many as 31 (63%) had tumor cells in bone marrow, in many cases with a high number of cells. Only four (8%) were positive also in blood. None of 38 control bone marrow samples were positive, including 11 from patients with suspected bone sarcoma at time of sampling who later were found not to have osteosarcoma. Fifteen of 28 patients without overt metastases at primary diagnosis (54%) were positive, 12 of whom had localized high-grade primary tumors in the extremity. Four of these have relapsed compared with none of 10 negative patients. In the group of 22 patients with extremity localized nonmetastatic osteosarcoma, information was available on the histologic response to preoperative chemotherapy in 15 patients. None of the three patients in the bone marrow–negative group who had a poor response to chemotherapy have relapsed, whereas two of the four poor responders in the bone marrow–positive cohort are dead of disease. Among 12 patients with overt metastasis at primary diagnosis, 11 (92%) were positive in bone marrow with a very high number of osteosarcoma cells. The immunomagnetically isolated cells were further characterized by the use of fluorescent latex microparticles with surface-bound antibodies targeting different membrane markers. Moreover, in cases with numerous osteosarcoma cells in bone marrow attempts to grow the selected cells in vitro were successful in two of eight attempts, and in two of five cases, isolated cells produced tumors with osteosarcoma characteristics in nude mice. In conclusion, already at primary diagnosis, a very high fraction of osteosarcoma patients had malignant cells in bone marrow, and a correlation between the presence of tumor cells, clinical stage, and disease progression was found. The data show the clinical potential of this immunomagnetic method. Attempts to subgroup osteosarcoma patients for more individualized treatment based on the presence of micrometastatic cells should be studied in a larger cohort of patients.

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.

Sensitive fluorescent in situ hybridisation method for the characterisation of breast cancer cells in bone marrow aspirates.

AIM: The presence of malignant cells in the blood and bone marrow of patients with cancer at the time of surgery may be indicative of early relapse. In addition to their numbers, the phenotypes of the micrometastatic cells might be essential in determining whether overt metastases will develop. This study aimed to establish a sensitive method for the detection and characterisation of malignant cells present in bone marrow. METHODS: In spiking experiments, SKBR3 cells were mixed with mononuclear cells in known proportions to mimic bone marrow samples with micrometastatic cells. Tumour cells were extracted using SAM-M450 Dynabeads coupled to the MOC-31 anti-epithelial antibody, and were further analysed for amplification of erbB2 and int2 by fluorescent in situ hybridisation (FISH). erbB2 and int2 copy numbers were also determined in 15 primary breast cancers, and bone marrow samples from patients with amplification were analysed for micrometastatic cells by immunomagnetic enrichment and FISH. RESULTS: In model experiments, cells with amplification could be detected in bead selected fractions when ratios of tumour cells (SKBR3) to mononuclear cells were as low as 10:10(7). Among the tumour samples, eight showed increased copy numbers of erbB2 and/or int2, and three of these patients had detectable numbers of tumour cells in their bone marrow: 4000, 540, and 26 tumour cells/10(7) mononuclear cells, respectively. The patient with 540 tumour cells/10(7) mononuclear cells showed high level amplification of erbB2 and suffered from a particularly aggressive disease, whereas the patient with 4000 tumour cells/10(7) mononuclear cells had favourable disease progression. CONCLUSION: These results demonstrate the feasibility and advantage of combining immunomagnetic selection and FISH characterisation of cancer cells in bone marrow samples. It is possible that molecular characterisation of such cells could provide prognostically valuable information.

Immunobead-based detection and characterization of circulating tumor cells in melanoma patients.

The presence of circulating tumor cells in bone marrow and peripheral blood of cancer patients may reflect the aggressiveness of the disease. This also applies to cancers that rarely give rise to overt bone marrow metastases. The clinical validity of micrometastasis detection for staging and prognostication depends on the sensitivity and reliability of the detection method. In malignant melanoma, most studies have used reverse transcriptase polymerase chain reaction (RT-PCR) techniques, commonly with tyrosinase mRNA as the target molecule. Unfortunately, highly inconsistent results have been reported, raising doubts about this approach. In a study of 81 melanoma patients with metastatic disease, we used an immunobead rosetting method in which live melanoma cells are selected and identified by binding of paramagnetic beads coated with the 9.2.27 antibody against the high molecular weight melanoma-associated antigen. In bone marrow samples obtained from 60 patients, 14 (23.3%) were positive, compared to only two of 81 in blood. A highly significant correlation (p = 0.0001, log rank test) was found between micrometastasis positivity and overall survival from time of removal of the primary tumor. Moreover, in regression analysis it was found that the presence of micrometastatic cells was an independent and the most important indicator of poor prognosis, with a relative risk of 5.38. The immunomagnetic method is simple, rapid, and highly sensitive and will be used in further prospective clinical studies.

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.

Human malignant melanoma harbours a large fraction of highly clonogenic cells that do not express markers associated with cancer stem cells

Department of Tumour Biology, Oslo University Hospital Radiumhospital, Oslo, Norway Department of Pathology, Oslo University Hospital Radiumhospital, Oslo, Norway Faculty Division, The Norwegian Radium Hospital, University of Oslo, Oslo, Norway Department of Surgery, Oslo University Hospital Radiumhospital, Oslo, Norway Department of Clinical Cancer Research, Oslo University Hospital Radiumhospital, Oslo, Norway Correspondence Lina Prasmickaite, e-mail: linap@rr-research.no

Bone marrow micrometastases in advanced stage non-small cell lung carcinoma patients

BACKGROUND The clinical relevance of bone marrow micrometastases in non-small cell lung cancer (NSCLC) is undetermined, and the value of such analyses in advanced stage patients has not been assessed previously. METHODS Immunomagnetic selection with the MOC31 (anti-EpCam) antibody was performed to isolate and detect tumor cells in bone marrow aspirates obtained from 196 patients with NSCLC, and the patients were subjected to follow-up for the assessment of survival. Repeated bone marrow samples, 2-7 samples per patient, were obtained from 13 long-term survivors. RESULTS MOC31 positive tumor cells were detected in 107 of 196 (55%) samples, a frequency similar to results reported in low-stage patients prior to curative surgical resection for NSCLC. No association was found between the presence of bone marrow micrometastases and disease stage or histological subgroup, and survival was similar in patients with and without detectable tumor cells in bone marrow. Repeated bone marrow analysis revealed, for the first time in this group of patients, the continued presence of tumor cells regardless of the given therapy and treatment response. CONCLUSION The immunomagnetic method utilized is a feasible strategy for the detection of bone marrow micrometastases in NSCLC. In advanced stage patients, the presence of MOC31 positive cells in bone marrow does not predict survival. Repeated analyses of bone marrow samples from long-term survivors revealed tumor cells in bone marrow which may represent dormant cells of particular interest for further characterization and follow-up.

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.

Comparison of in vitro Cloning Assays for Drug Sensitivity Testing of Human Brain Tumours

Three in vitro clonogenic assays were used to determine the sensitivity of an established human glioblastoma cell line (U251-MG) to five chemotherapeutic agents. The colony-forming efficiency of untreated culture was 0.695 +/- 0.170 in a monolayer assay with irradiated feeder cells, 0.018 +/- 0.006 in a low-O2 agar assay, and 0.049 +/- 0.021 in a two-layer agar system with nutrient-enriched medium (p less than 0.001). Comparison of the slope of the regression line for the dose-response curve and the interpolated ID90 for each drug showed that U251-MG was equally sensitive to aziridinylbenzoquinone and dianhydrogalactitol in all three assays. The sensitivity of this cell line to 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU), cis-dichlorodiammineplatinum (II) (CDDP) and 9-hydroxy-2-N-methylellipticine (HME), however, varied depending on the assay used. In no instance did U251-MG show greater sensitivity (lower ID90 or steeper slope) in the low-O2 agar assay than in the other assays. BCNU and CDDP were least active in the monolayer assay, whereas HME showed both the lowest ID90 and steepest slope using this technique. We conclude that different in vitro tumour clonogenic assays show different colony-forming efficiencies for the same cell line and may show different responses to certain drugs. Identification of accurate predictive models of drug sensitivity will require correlative in vivo and in vitro studies.

Bone marrow micrometastases studied by an immunomagnetic isolation procedure in extremity localized non-metastatic osteosarcoma patients

Hematogenous spread of tumor cells is an early event in osteosarcoma and present in the majority of patients at primary diagnosis. Eradication of such micrometastases by adjuvant combination chemotherapy is crucial for survival. However, a survival plateau of 60-70% was reached over two decades ago, above which it seems difficult to further advance with the currently available therapies. In this study we have, by an immunomagnetic isolation procedure, examined the presence and prognostic impact of disseminated tumor cells in bone marrow aspirates taken at primary diagnosis in a cohort of 41 non-metastatic patients with extremity localized, high-grade osteosarcoma.