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Biomedical subjects

O Fodstad

Publications and source records attributed to O Fodstad.

At least 73 records · Page 4Linked to original sources
PubMed1995

Semiquantitative polymerase chain reaction for t(14;18) in follicular lymphomas: a colorimetric approach.

BACKGROUND: Autologous bone marrow transplantation is increasingly being used in the management of several types of cancer, and to avoid reintroduction of malignant cells, bone marrow purging is often performed. In such cases, sensitive quantitation methods are needed both to assess the efficacy of the purging and for surveillance of patients in remission. Polymerase chain reaction (PCR) has the necessary sensitivity for this application, but it requires that the cancer cells can be recognized by a defined genetic abnormality. In addition, PCR is in principle a qualitative technique and must be modified for quantitative purposes. In follicular non-Hodgkin's lymphomas, the translocation t(14;18) (q32;q21) is common and is used here for model experiments. EXPERIMENTAL DESIGN: A PCR-based method for the quantitation of translocation-positive cells was developed on the basis of coamplification of cancer-specific target molecules with competitor molecules of known concentration. Gel electrophoresis was substituted by a colorimetric quantitation system to cope with patient PCR products of the same size as the competitor product and ease automation of the method at a later stage. Cell line Karpas 422, which contains the t(14;18) (q32;21) translocation, was used to validate the method. The method was used to assess the efficacy of a patient bone marrow purging where the initial infiltration levels were too low for traditional detection systems. RESULTS: A reproducible and near linear response was obtained between 70 pg and 200 ng K422 DNA, equivalent to 10 K422 cells and 30,000 K422 cells, respectively. Bone marrow infiltration in one patient was 0.6 to 0.7% before malignant cell removal and 3 to 7 x 10(-4) after removal. The corresponding figures for the other patient were 2% and 3 to 7 x 10(-4), respectively. CONCLUSIONS: The method presented has a sufficient dynamic range for applications like evaluation of bone marrow purging or monitoring of minimal residual disease. Adaptation of this method to other translocations is discussed.

Base Sequence↗
PubMed1994

MDM2 gene amplification and transcript levels in human sarcomas: relationship to TP53 gene status.

BACKGROUND: Alterations of the TP53 tumor suppressor gene appear to be implicated in the tumorigenesis and progression of several types of human cancer, including different histologic subtypes of sarcomas. The MDM2 (murine double minute-2) gene encodes a nuclear phosphoprotein that may interact with both mutant and wild-type p53 proteins, thereby inhibiting p53-mediated transactivation in a dose-dependent manner. Recently it has been suggested that mdm2 and p53 proteins are components of an autoregulatory loop in which the MDM2 gene is transactivated by p53. PURPOSE: Our purpose was to examine the frequency of MDM2 amplifications in larger panels of sarcomas, determine if the mRNA level could be elevated in tumors without concomitant gene amplification, and relate MDM2 findings to the TP53 status of the tumors. METHODS: Sarcoma tissue of different histologic subtypes was obtained from 68 patients at the time of surgery and from 26 human xenografts in nude mice. In addition, two human sarcoma cell lines (OSA and U2OS) were studied. Genomic DNA from tumor tissue, in vitro cell lines, and peripheral blood cells were isolated by Southern-blot analysis methods to determine MDM2 gene amplification. Tumor DNA was analyzed for possible TP53 gene mutations in exons 5, 7, and 8 by constant denaturing gel electrophoresis. To determine the MDM2 and TP53 mRNA levels, Northern-blot analysis was performed. RESULTS: Amplification of the MDM2 gene was detected in 10 tumors (10.3%). Whereas MDM2 amplification and/or over-expression were found only in two (U2OS and OSA cell lines) of 18 osteosarcomas, one of 20 malignant fibrous histiocytomas (MFHs), and in none of 14 leiomyosarcomas, such alterations were observed in two of two fibrosarcomas, three of six malignant schwannomas, three of 19 liposarcomas, and in the one hemangiopericytoma examined. MDM2 overexpression was found in all nine examined cases with and in three tumors without amplification. TP53 mutations were detected in 12 cases (five osteosarcomas, four MFHs, and three leiomyosarcomas), of which none showed amplification, but one had increased levels of MDM2 mRNA. None of the fibrosarcomas, malignant schwannomas, and liposarcomas examined had mutated TP53. The six sarcomas that showed high TP53 mRNA expression in the absence of gene mutation also had elevated levels of MDM2 mRNA. CONCLUSIONS: The present data provide further indications that increased MDM2 expression level, caused by gene amplification or altered regulation of transcription, is involved in tumor progression of some, but not all, sarcoma subtypes.

Animals↗
PubMed1994

Targeted therapy with immunotoxins in a nude rat model for leptomeningeal growth of human small cell lung cancer.

Metastasis to the central nervous system in patients with small cell lung cancer is not uncommon, and a fraction of the cases have leptomeningeal disease for which no effective therapy is available. To establish an experimental model for evaluation of new therapeutic approaches for such tumor lesions, 1 x 10(6) human H-146 cells were injected directly into the cerebrospinal fluid in the cisterna magna of nude rats. Small, superficial leptomeningeal tumors developed, consistently resulting in symptoms of central nervous system involvement after a mean latency of 20 days. The model was used to study the efficacy of intrathecal targeted therapy with immunotoxins. The monoclonal anti-carcinoma antibodies MOC-31 and NrLu10 and the growth factor transferrin were conjugated to Pseudomonas exotoxin A (PE), and 1 day after tumor cell inoculation instilled in the cisterna magna as a single bolus dose of 1.5 micrograms. The antibody conjugates, which were highly cytotoxic to target cells in a protein synthesis inhibition assay in vitro, increased the symptom-free latency by 35-46%. PE had no effect, reflecting a lower in vitro cytotoxicity and possibly also a down-regulation of transferrin-receptor expression in the meningeal H-146 tumors. Delayed or repeated treatment with MOC-31-PE was less effective than day 1 administration, whereas the addition of 10% glycerol to the injection solution increased the symptom-free period to 72%. The efficacy of MOC-31-PE is superior to reported effects obtained in similar models with other therapies, and the results support the development of this immunotoxin towards clinical evaluation in small cell lung cancer patients with leptomeningeal carcinomatosis.

ADP Ribose Transferases↗
PubMed1994

Validity and usefulness of human tumor models established by intratibial cell inoculation in nude rats.

Intratibial injection in nude rats of 1 x 10(6) OHS, MHMX, and LOX human tumor cells resulted in each case in progressively growing bone tumors. When the diameter of the affected leg had increased by 2-3 mm, the animals were examined for uptake of 99mTc-methylenediphosphonate. The OHS osteosarcoma tumors caused sclerotic lesions with high and uniform isotope uptake, and the MHMX unclassified sarcoma showed a mixed pattern with both sclerotic and lytic areas, whereas the LOX melanoma caused lytic bone lesions with low uptake of the radionuclide. These findings were compared with the results of analogous investigations previously performed in the patients from whom the tumor lines originated. Striking similarities in both the morphology and the scintigraphic images were observed between corresponding tumors in rats and humans, with results supporting the clinical relevance of the model systems. When the LOX model was used for therapy experiments, doxorubicin had no effect on the growth of the tibial tumors, which in the control group appeared after a latency of 13.5 days. The alkylating agent mitozolomide increased the median tumor-free latency to 47 days in 7 rats, and 5 animals did not develop tumors within the observation period of 60 days. Doxorubicin was ineffective also against the OHS tumor, whereas ifosfamide and the radionuclide 89Sr-chloride showed significant antitumor activity. The disease-free latency increased from 20 days, in the control animals, to 45 and 28.5 days, respectively, in the 2 treated groups, in which 2 of 7 and 2 of 10 rats were without tumors at 60 days. The data demonstrate that the tibial models discriminated between the action of the different therapeutic agents, and suggest that they may be useful in selecting compounds with clinical activity against skeletal tumors.

Animals↗
PubMed1994

Suppression of the neoplastic phenotype in vivo by an anti-ras ribozyme.

In this study, the efficacy of an anti-ras ribozyme in reversing the neoplastic phenotype was investigated. Murine NIH3T3 cells were transfected with cellular DNA from the FEMX-I human melanoma cell line expressing the activated H-ras gene. The transformed cells displayed the neoplastic phenotype in vitro and were tumorigenic in nude mice in vivo. When the transformants were transfected by a ribozyme designed to cleave only activated H-ras RNA, the transformed phenotype was abrogated. In contrast, expression of a mutant ribozyme, essentially acting only as antisense, into the transformed cells resulted in less dramatic changes in cell growth and tumorigenicity. These results reinforce the potential role of anti-oncogene ribozymes as suppressors of neoplastic growth, with possible implications for gene therapy.

3T3 Cells↗
PubMed1994

Expression of the neuroectodermal intermediate filament nestin in human melanomas.

Nestin is a newly identified intermediate filament expressed in proliferating neuronal progenitor cells, but not in the adult brain. Nestin expression reappears in many tumors of the central nervous system and has in human glioblastomas been associated with a high degree of malignancy. Because melanocytes are of neuroectodermal origin, we studied nestin expression in benign and malignant cells of the melanocytic lineage using Northern blot and immunohistochemical analyses. Nestin mRNA was detected in 24 of 34 metastatic melanomas and in 1 of 4 benign nevi, whereas the protein was expressed in 10 of 15 primary melanomas, in 29 of 34 metastatic tumors, and in 3 of 4 nevi. Neither normal melanocytes nor any of 4 basal cell carcinomas showed detectable levels of the protein. The high fraction of melanocytic tumors which express nestin, particularly the metastatic melanomas, suggests that nestin may be a useful marker for such malignancies. Furthermore, although no significant correlation between nestin expression and tumor malignancy was observed, the protein was most abundantly expressed in the infiltrating part of the tumors, indicating a possible involvement of nestin in tumor invasion.

Biomarkers, Tumor↗
PubMed1994

Comparison of two antibody-based methods for elimination of breast cancer cells from human bone marrow.

Three monoclonal antibodies reactive with antigens abundantly expressed on human carcinoma cells were used to develop and compare the efficacy of immunotoxins (ITs) and immunobeads for purging breast cancer cells from bone marrow. ITs constructed as conjugates of the monoclonal antibodies and Pseudomonas exotoxin A showed high specific cytotoxicity against three breast cancer cell lines, inhibiting protein synthesis by 50% at concentrations of 4 x 10(-13) M to 1 x 10(-10) M. Tested in a reproducible clonogenic assay, two of the ITs used at a concentration of 0.1 microgram/ml killed > 5 log units of MCF7 cells, the maximal sensitivity for assessing cytotoxic effects, and 1.5 log of T-47D tumor cells. At 1 microgram/ml, each of the three ITs eliminated > 5 log of both cell lines. The immunobead procedure removed 2.0-4.1 log of tumor cells with one purging cycle and up to 6.0 log with two cycles. The mixture of the three ITs or immunobeads was not clearly superior in efficacy, compared to the use of individual molecules, probably reflecting an overlap in expression of the respective antigens in these cell lines. For both methods, the purging efficacy was not reduced when the tumor cells were admixed with normal bone marrow cells at a ratio of 1:10. The survival of colony-forming units, granulocyte/macrophage, was 49-86% with the immunobeads and 44-75% even at high concentrations (up to 2.5 micrograms/ml x 3) of the ITs. The results indicate that each of the two immunological methods can be safely used for effective elimination of tumor cells from the graft of breast cancer patients undergoing autologous bone marrow transplantation.

Antibodies, Monoclonal↗
PubMed1994

Microenvironment revisited: time for reappraisal of some prevailing concepts of cancer metastasis.

The development and biological characteristics of tumor metastasis are influenced by multiple cell and host-associated factors. To study these factors experimentally, the necessity of choosing adequate in vivo model systems for human tumor metastasis is emphasized. Recent research has provided results that highlight the role of the microenvironment in determining important characteristics of the metastatic cells, including their degree of differentiation and sensitivity to drugs. Furthermore, evidence is presented as background for discussing the general validity of the notion of clonal selection of metastatic cells, and whether the metastatic phenotype is acquired through the last of a series of mutational events occurring during tumor progression.

Animals↗
PubMed1994

Nude rat models for human tumor metastasis to CNS. Procedures for intracarotid delivery of cancer cells and drugs.

Models for hematogenous spread of human cancer to the central nervous system (CNS) were established by injecting human tumor cells into the internal carotid artery of nude rats. With 4 out of 10 cell lines, belonging to four different tumor types, metastases developed in all injected animals. Tumor growth manifested clinically as neurological symptoms which appeared after a median latency ranging from 19-87 days for the different tumors. The H-146 and DMS-273 small cell lung cancers and the LOX melanoma almost exclusively gave meningeal tumors, whereas with FEMX-I melanoma cells bone metastases in the skull dominated. For these tumor types a correlation was found between the capacity for experimental metastasis formation and the s.c. tumorigenicity. In agreement with clinical experience, none of the 2 sarcoma and 2 glioblastoma lines gave CNS metastases. With a modified microsurgical technique, allowing for repeated ipsilateral intracarotid injections, we analyzed the drug concentrations obtained in tumor and surrounding brain tissue after i.v. treatment with doxorubicin. The concentration in the LOX tumor reached therapeutic levels and was approximately 100 x higher than in normal brain tissue, both with and without intraarterial pretreatment with arabinose. In the same model, the tissue concentrations of 9.2.27-abrin immunotoxin 10 min after intracarotid injection were examined. Although the levels were low, a tumor to brain concentration ratio of up to 9 was achieved. The data demonstrate that clinically relevant tumor models can be established with the techniques described, and these models may successfully be used to evaluate the pharmacokinetics and effect of intravenous or intraarterial therapy.

Animals↗
PubMed1994

The protooncogene CHOP/GADD153, involved in growth arrest and DNA damage response, is amplified in a subset of human sarcomas.

The C/EBP-homologous transcription factor CHOP (GADD153) is inducible by growth inhibition or DNA damage, and has been shown to be oncogenically activated by the specific (12;16) translocation in human myxoid liposarcoma. We have now found CHOP amplification in two sarcoma cell lines with previously reported amplification of the nearby GLI gene. Among 98 other human sarcomas of various types, CHOP was amplified in a hemangiopericytoma, a liposarcoma, and two osteosarcoma. High constitutive expression levels of CHOP were observed in tumors with gene amplification, but also in some other samples. The nearby MDM2 gene, which codes for a protein that may inactivate wild-type p53, has previously been reported to be frequently amplified in sarcoma. In our sarcoma panel, MDM2 was amplified in 9 cases. MDM2 and CHOP were co-amplified in two of these, whereas the two osteosarcomas had amplified CHOP but not MDM2. CHOP was amplified in both cell lines with GLI amplification, and MDM2 only in one. No mutations in the TP53 gene have been found in samples with amplification of MDM2. In contrast, the cell line in which CHOP but not MDM2 was amplified had mutated TP53, suggesting that selection of this amplicon was not mediated through p53 inactivation.

CCAAT-Enhancer-Binding Proteins↗
PubMed1994

Immunoscintigraphy of bone sarcomas--results in 5 patients.

The feasibility of using the murine monoclonal antibody, TP-1, for clinical immunoscintigraphy was examined in a pilot study involving 5 patients with bone sarcomas. 131I-labelled F(ab')2 antibody fragments were injected in doses of 0.8-1.0 mg (90-130 MBq), and the accumulation of radioactivity was examined by scintigraphy, and assessed by direct measurements on biopsied tumour and normal tissue. One osteosarcoma patient had a primary tumour in the femur, whereas the other 4 had single lung metastases detected by other diagnostic methods. Immunoscintigraphy of the femoral primary was optimally visualised after 22 h. In 2 patients, the method failed to detect lung metastasis, in 1 of the cases possibly related to less than optimal methodological conditions. In 2 other patients, increased accumulation of radioactivity indicated one and three lung tumours, in addition to the single metastasis observed by X-ray and CT scanning, tumours that were later confirmed and removed surgically. The concentration of radioactivity in tumour and normal tissues 44-72 h after antibody injection could be measured in 4 patients. The tumour to blood ratios were in the range of 1.2-4.2, compared to 0.1-0.8 for various normal tissues. The results indicate that immunoscintigraphy with TP-1 antibody fragments have a potential for early detection of lung metastases in patients with bone sarcoma.

Adolescent↗
PubMed1994

Helix pomatia agglutinin binding in human tumour cell lines: correlation with pulmonary metastases in nude mice.

The extent of lectin binding by three human melanoma (LOX, FEMX-1 and SESX) and two sarcoma lines (MHMX and OHSX) was related to their potential for experimental metastasis formation in athymic nude mice. The Helix pomatia agglutinin (HPA), which recognises the N-acetyl-D-galactosamine ligand, showed differential binding to the cell lines in a manner that correlated with their ability to give lung colonies after i.v. injection in the mice (P < 0.005). The degree of HPA binding and lung colony formation of the cell lines studied was ranked in the following order, LOX > MHMX > OHSX > SESX > FEMX-I. Similar patterns were not observed with the other lectins used in this study (WGA, Con A, PNA and UEA-I). The high HPA reacting LOX melanoma line shows extensive pulmonary metastatic formation with no extrapulmonary colonies, whereas the low HPA reacting FEMX-I cells give only extrapulmonary metastases with no detectable colonies in the lungs. Precoating of tumour cells with HPA prior to injection did not reduce the ability of cells to give pulmonary metastases, suggesting that the HPA epitope was not functionally associated with the pulmonary metastatic potential observed in nude mice. These findings support recent human studies of a correlation between HPA binding and incidence of metastasis, however, our data indicate that there is no causal relationship. Further analyses are required to identify the specific HPA-binding glycoconjugates that may be involved.

Animals↗
PubMed1994

TP53 allele loss, mutations and expression in malignant melanoma.

p53 alterations at the DNA, mRNA and protein levels were studied in tumour metastases sampled from 30 patients with malignant melanoma. Paraffin-embedded sections from these and an additional 12 patients were examined for the presence of p53 protein. TP53 gene aberrations were found in 7 of 30 (23%) of the patients, six of which showed loss of heterozygosity (LOH). Point mutations were detected in only two cases, one of which had LOH whereas the other was non-informative. Increased levels of p53 mRNA were present in only one tumour with, but in six cases without, detectable DNA abnormalities. Four of the latter and six tumours with normal transcript levels had immunohistochemically detectable levels of p53 protein. In 25 cases in which corresponding primary and metastatic lesions could be compared, closely similar immunoreactivity patterns were observed. Increased expression of the MDM2 gene was found in only one tumour in parallel with overexpression of p53. Altogether, the data indicate that inactivation of the p53 regulatory pathway is not of major significance in the tumorigenesis of malignant melanoma. However, a significant association was found between p53 immunoreactivity and the relapse-free period in patients with superficial spreading melanoma. That increased protein expression was predominantly found in tumours without DNA alterations might suggest a role for the wild-type p53 protein in restricting malignant cell proliferation in these cases.

Alleles↗
PubMed1994

Preclinical phase II studies in human tumor xenografts: a European multicenter follow-up study.

BACKGROUND: The EORTC New Drug Development Office has initiated a multicenter collaborative program to evaluate the use of human tumor xenografts to predict phase II clinical activity. A first study confirmed the efficacy of doxorubicin and inactivity of amsacrine against human tumor xenografts (Boven et al., Cancer Res: 52, 5940, 1992). In the follow-up study reported here, the activities of cisplatin, AZQ (diaziquone), pazelliptine and retelliptine have been evaluated against a panel of 40 established tumor lines grown subcutaneously in nude mice. DESIGN: The xenografts used represent carcinomas of the breast, colon, head+neck, ovary, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC) and melanoma. Drugs were administered intravenously on days 0 and 7. Doses were for cisplatin 5 mg/kg, AZQ 3-7 mg/kg, pazelliptine 20-80 mg/kg and retelliptine 6-12.5 mg/kg and were selected to give a median loss of about 10%-15% body weight. RESULTS: When activity was defined as a specific growth delay > 1 and a tumor growth inhibition > 50%, then cisplatin demonstrated activity in 15 of 40 xenografts tested (3 of 5 breast, 1 of 6 colon, 0 of 5 head+neck, 2 of 6 NSCLC, 4 of 7 SCLC, 1 of 5 melanoma and 4 of 6 ovarian cancers); AZQ was active in 23 of 38 xenografts (2 of 3 breast, 2 of 7 colon, 4 of 5 head+neck, 3 of 6 NSCLC, 6 of 6 SCLC, 2 of 5 melanoma, 4 of 6 ovarian cancers); pazelliptine was active in 2 of 38 xenografts (1 of 5 breast cancers, 1 of 5 melanoma) while retelliptine was active in 1 of 39 xenografts (a breast cancer xenograft) tested. CONCLUSIONS: These results are reasonably consistent with the clinical activity of cisplatin, but overpredict the clinical efficacy of AZQ. Since pazelliptine and retelliptine are investigational compounds, the clinical phase II studies will provide a prospective test for this model. The results of the present study and the previous one indicate that the human tumor xenograft model could be suitable for predicting the activity of novel compounds to be developed for treatment of cancer patients.

Animals↗
PubMed1994

In vivo efficacy of intrathecal transferrin-Pseudomonas exotoxin A immunotoxin against LOX melanoma.

Neoplastic meningitis due to the dissemination of systemic cancer or primary central nervous system tumors through the cerebrospinal fluid carries a very poor prognosis. Current treatments for this disease are ineffective, and new therapeutic modalities such as immunotoxins may be beneficial. We created an animal model of human carcinomatous meningitis with LOX melanoma-derived tissue-culture cells in athymic rats for testing the efficacy of intrathecal therapy with transferrin-Pseudomonas exotoxin A (Tfn-PE) immunotoxin. An injection of 5 x 10(5) LOX cells into the intrathecal space through an indwelling catheter resulted in the reproducible development of lower-extremity paraplegia at 9.24 +/- 1.77 days because of focal deposits of tumor growth adjacent to the thoracic and lumbar spinal cord. A dose of 2.5 or 5 micrograms of intrathecal Tfn-PE immunotoxin was neurotoxic and resulted in the deaths of 8 of 10 animals within 24 hours. Histological evidence of central nervous system damage was seen as hemorrhagic degeneration around the central canal or a pathological cleft at the level of the cervical spinal cord. Because no neurotoxicity was seen with 1 microgram of intrathecal Tfn-PE immunotoxin, this dose was administered in treatment experiments. Twenty-four hours after the intrathecal instillation of LOX cells, 10 animals received intrathecally either 1 microgram of Tfn-PE or phosphate-buffered saline with 0.1% human serum albumin (control group).(ABSTRACT TRUNCATED AT 250 WORDS)

ADP Ribose Transferases↗
PubMed1993

Eradication of small cell lung cancer cells from human bone marrow with immunotoxins.

The potential of autologous bone marrow transplantation to improve the treatment results for patients with small cell lung cancer (SCLC) may be limited by the presence of tumor cells in the graft. We constructed immunotoxins (ITs) involving 4 monoclonal antibodies and Pseudomonas exotoxin A and investigated the cytotoxicity of the ITs to H-146 SCLC cells in the presence and absence of normal human bone marrow (BM) cells. The Pseudomonas exotoxin A conjugate with the MOC-1 antibody, which recognizes an NCAM antigen, was inactive, as tested in a reproducible soft agar assay. Conjugates involving the monoclonal antibodies MOC-31, NrLu10, and MLuC1 killed about 3.5 log tumor cells at 0.1 microgram/ml and > 5.0 log at 1 microgram/ml. In the absence of BM cells, the combination of the 3 ITs was not superior to each IT used individually. However, when H-146 cells were admixed to nucleated BM cells at the ratio of 1:10, > 5 log tumor cell kill was obtained at a concentration as low as 0.1 microgram/ml of each IT. Survival of normal BM progenitor cells was only moderately reduced by the IT treatment, even in experiments in which the 3 IT3s were used at 2.5 micrograms/ml each. Freezing and thawing of the BM, as required in a clinical setting, reduced the colony-forming unit, granulocyte-macrophage, and colony-forming unit, granulocyte-erythroid-macrophage-megakaryocyte, by 30-60% in both treated and untreated cultures. We conclude that the use of a mixture of the 3 ITs provides a safe, rapid, and effective method for eradicating SCLC cells from BM used for autologous bone marrow transplantation following high-dose chemotherapy.

Bone Marrow Purging↗