Correlation between molecular metastases in sentinel lymph nodes of breast cancer patients and St Gallen risk category.

AIMS: To evaluate the clinical significance of tumour metastases detected using real-time reverse transcription-PCR (RT-PCR) in sentinel lymph nodes (SLN) of breast cancer patients. METHODS: Sixty-seven patients with T1-T2 primary breast cancer were included in a prospective study. SLN were analysed for the presence of metastatic tumour cells using standard histopathology staining, immunochemistry (IHC) and multimarker real-time RT-PCR assay for mammaglobin (MMG), carcinoembryonic antigen (CEA) and cytokeratin-19 (CK19) mRNA expression. Correlations between molecular metastases and traditional clinicopathological prognostic factors, including St Gallen risk categories were studied. RESULTS: Of the 67 patients, 15 (22.3%) had one or more pathology-positive SLN. Five (9.6%) pathology-negative SLN were positive by IHC and 19 (36.5%) by RT-PCR. Of note, RT-PCR analysis was also positive in all cases with pathology- or IHC-positive SLN. MMG was the most informative tumour marker in the panel. Molecularly detected metastases were significantly associated with intermediate St Gallen risk category (p=0.023). CONCLUSION: Molecular staging of SLN using real-time RT-PCR for early breast cancer could serve as a useful complement to standard clinicopathological risk factors. Studies with long-term follow-up are necessary to define the impact of molecular metastases on disease free survival and overall survival.

Interleukin-6 overexpression as a marker of malignancy in human gliomas.

OBJECT: Glioblastomas multiforme (GBMs) grow rapidly and are highly resistant to treatment compared with other glioma types and grades. Consequently, it is of major interest to identify markers of aggressiveness in these tumors that could represent new therapeutic targets. Interleukin (IL)-6 is frequently produced in gliomas and, given its manifold properties, could be considered as a candidate marker. Expression of IL-6 may be involved in cell growth, resistance to chemotherapy and radiotherapy (via an antiapoptotic pathway), and angiogenesis. This study was conducted to test this hypotheses and to evaluate the suitability of IL-6 as a target in the treatment of GBMs. METHODS: The authors studied the relationship between the level of IL-6 gene expression as assessed using semiquantitative reverse transcription-polymerase chain reaction and by determining various histological types and grades in a series of 59 gliomas. It was found that GBMs displayed a significantly higher level of IL-6 expression than other types of glioma (p < 0.001). Immunohistochemical analysis revealed that IL-6 was produced mainly by malignant cells and a few vascular endothelial cells. CONCLUSIONS: It can be inferred from these findings that IL-6 gene expression is related to glioma aggressiveness and that IL-6 may play a central role in GBM behavior. Interleukin-6, therefore, could be considered as a new potential target in the treatment of GBMs.

Effects of MDR reversing agent combinations on the 3H-daunomycin accumulation in drug-sensitive and drug-resistant human cancer cells.

BACKGROUND: As multidrug resistant (MDR) tumour cells generally exhibit a drug accumulation deficit, the effects of three prototype modulators and their combinations were investigated by studying the modulation of 3H-dounomycin cellular accumulation. MATERIALS AND METHODS: Two cell lines derived from a rhino-pharingeal human carcinoma, either sensitive (KB-3-1) or selected as MDR (KB-A1) were used. Verapamil (10mumol.L-1), PSC 833 (lmumol.L-1) and S9788 (5mumol.L-1) were tested alone or in association two by two. The cells were characterized by reverse transcriptase polymerase chain reaction (RT-PCR) in terms of pleiotropic resistance gene expression. RESULTS: A strong mdr1 and a light LRP gene expression were found in KB-A1 resistant cells compared to KB-3-1, whereas MRP expression was found to a similar extent. Relative to the KB-3-1, cells, accumulation of 3H-daunomycin was reduced to 31 +/- 5% in the KB-A1 cells. In these KB-A1 cells, the three agents tested significantly increased the 3H-daunomycin intracellular concentration, S9788 being the most active (311 +/- 37%) and inducing a near complete reversion to the basal level of the sensitive cells. Verapamil and PSC 833 demonstrated an additive effect (252 +/- 69% compared to 188 +/- 33% and 126 +/- 27%, respectively). On KB-3-1 sensitive cells, S9788 had no effect, while verapamil or PSC 833 moderately increased the 3H-daunomycin accumulation, without additive effect. CONCLUSION: These results show a strong MDR reversing effect of S9788, which appears specific to P-glycoprotein (Pgp) and an additive effect between verapamil and PSC 833, suggesting a better therapeutic efficiency if used in well defined combinations.

O(6)-methylguanine-DNA methyltransferase gene (MGMT) expression in human glioblastomas in relation to patient characteristics and p53 accumulation.

Repair of cytotoxic DNA damage by O(6)-methylguanine-DNA methyltransferase (MGMT) is a potentially important factor of chemoresistance to chloroethylnitrosoureas (CENUs), commonly used in the treatment of glioblastoma multiforme (GBM). The value of p53 as a prognostic factor in GBMs remains unclear, but a possible relationship between MGMT gene expression and p53 has been suggested. To further examine these GBM characteristics in vivo, we assessed MGMT gene expression using semi-quantitative RT-PCR and p53 alteration by immuno-histochemistry on a series of 39 GBMs. MGMT gene expression was inversely correlated with age (p < 0.03), consistent with the results of others. Interestingly, tumors from male patients had higher MGMT mRNA amounts than tumors from female patients (p < 0.03). No prognostic implication was observed either for MGMT gene expression or for p53 accumulation. However, MGMT gene expression was significantly lower in p53-altered GBM, regardless of the percentage of positive cells (p < 0.01). Our observation suggests that in human glial tumors, a low level of MGMT gene expression might promote p53 alteration, probably via mutation of its gene. Int. J. Cancer (Pred. Oncol.) 84:416-420, 1999.

Comparative 99mTc-sestamibi and 3H-daunomycin uptake in human carcinoma cells: relation to the MDR phenotype and effects of reversing agents.

UNLABELLED: Because 99mTc-sestamibi (MIBI) appears to be a potent candidate for multidrug resistance (MDR) evaluation in tumors, its cellular uptake should be similar to that of 3H-daunomycin in a variety of conditions of expression and inhibition of MDR activity. METHODS: We used a human rhinopharyngeal carcinoma cell line (KB-3-1) and its MDR variant (KB-A1). Cells were incubated 2 h with 99mTc-MIBI and 3H-daunomycin under control conditions or in the presence of a reversing agent such as verapamil (10 pmol/L), PSC833 (1 micromol/L) or S9788 (5 micromol/L). RESULTS: Relative to the KB-3-1-sensitive cells, accumulations of 99mTc-MIBI and 3H-daunomycin were reduced to 31% +/- 5% and 36% +/- 11% (P < 0.001 for both) in KB-A1-resistant cells. In sensitive cells, accumulation of both agents was increased by verapamil and PSC833 (range 115%-140%; P < 0.05) but not by S9788. In KB-A1 cells, only S9788 significantly increased the cellular uptake of 99mTc-MIBI (138% +/- 25%; P < 0.01), whereas the intracellular uptake of 3H-daunomycin was markedly increased with the three reversing agents (up to 311% +/- 37% with S9788; P < 0.001). With this last treatment, uptake of 3H-daunomycin in KB-A1 cells nearly returned to its basal level in sensitive cells. CONCLUSION: 99mTc-MIBI monitors the MDR phenotype of tumor cells effectively but responds to reversing agents differently than 3H-daunomycin.

In vivo tissue extracellular volume fraction measurement by dynamic spin-lattice MRI relaxometry: application to the characterization of muscle fiber types.

RATIONALE AND OBJECTIVES: The extracellular volume fraction (v) was estimated in leg rabbit muscles by MRI dynamic longitudinal relaxation rate (R1) relaxometry to distinguish between slow- and fast-twitch muscle fiber types. METHOD: The extracellular volume fraction was calculated from the dynamic increase of the longitudinal relaxation rate after intravenous administration of a gadolinium (Gd-DTPA) contrast bolus, assuming a biexponential plasma concentration model. RESULTS: It has been shown that the extracellular volume fraction increases with the slow fiber content (oxidative type I); the maximal value (v = 0.186+/-0,018) was obtained in pure slow-twitch muscle fiber (100% type I). CONCLUSION: NMR extracellular volume estimates closely agree with those obtained using the more classic invasive isotopic method (99mTc-DTPA) carried out on the same rabbit strain and with data reported in the literature. The method has potential applications to characterize the pathophysiologic status of tissues. It is also applicable to a wide range of tissues and pathologies, in particular for the characterization of malignant tissues and their response to therapies.

Comparison between technetium-99m-sestamibi and hydrogen-3-daunomycin myocardial cellular retention in vitro.

UNLABELLED: This study was undertaken to verify whether 99mTc-sestamibi uptake parallels that of 3H-daunomycin in cells treated with multidrug resistance (MDR) reversing agents. Since we have detected in a previous work a moderate typical MDR phenotype in rat cardiac cells, a model of cultured myocardial cells was used. METHODS: Newborn-rat cultured myocardial cells were incubated 120 min with the MDR-reversing agent verapamil 50 microM, PSC833 1 microM or S9788 10 microM alone or in combination, and the cellular retention of 3H-daunomycin and 99mTc-sestamibi was counted. RESULTS: Hydrogen-3-daunomycin cellular accumulation was never modified by more than 15% when compared to control values, while 99mTc-sestamibi decreased to 75% +/- 32% (m +/- s.d.) of controls in the presence of S9788 and to 44% +/- 19% when S9788 was associated with verapamil. CONCLUSION: The variations of 99mTc-sestamibi and 3H-daunomycin cellular accumulation induced by MDR-reversing agents in cultured myocardial cells can be dramatically different. While some MDR-reversing agents can significantly increase the 3H-daunomycin retention in cardiac cells, they have unexpected effects on that of 99mTc-sestamibi.

French multicentric evaluation of mdr1 gene expression by RT-PCR in leukemia and solid tumours. Standardization of RT-PCR and preliminary comparisons between RT-PCR and immunohistochemistry in solid tumours. French Network of the Drug Resistance Intergroup, and Drug Resistance Network of Assistance Publique-Hôpitaux de Paris.

Since there is no consensus on the techniques for multidrug resistance (MDR) phenotype evaluation, many discrepancies concerning the importance and frequency of mdr1 gene expression in leukemias and solid tumors are observed in the literature. In order to establish an inter-laboratory consensus in France, a multicenter study was carried out to propose further guidelines for MDR phenotype evaluation. The techniques used by the 38 laboratories participating in the trial were: immunodetection (immunohisto and/or cytochemistry, flow cytometry), functional tests, reverse transcription-polymerase chain reaction (RT-PCR) or Northern blot. We present the results obtained by 19 laboratories concerning the measurement of mdr1 gene expression assessed by RT-PCR or Northern blot in: (1)19 samples of tumor cells obtained from leukemic patients; (2) six solid tumor samples obtained at surgery; (3) eight cell lines exhibiting variable levels of resistance, and; (4)10 preparations of RNA and of cDNA obtained from solid tumors. Standardization of the RT-PCR technique and preliminary results comparing RT-PCR with immunohistochemistry in solid tumors are also reported.

In vitro detection of the MDR phenotype in rat myocardium: use of PCR, [3H]daunomycin and MDR reversing agents.

A decrease in the intracellular drug concentration in resistant cells as compared to sensitive cells is one of the characteristics of the MDR phenotype. P-glycoprotein (Pgp) is thought to be responsible for an active efflux of some lipophilic drugs such as anthracyclines. Anthracyclines such as daunomycin are highly effective anticancer agents but induce a well-described, while incompletely explained, cardiac toxicity. In this study, we investigated the MDR phenotype in rat myocardium in terms of gene expression, detection of Pgp and indirect evaluation of Pgp function. A clear mdr1a gene specific expression in rat cultured myocardial cells and cardiac tissue was detected by RT-PCR. The incorporation of [3H]daunomycin in myocardial cell cultures was studied with and without reversing agents. Daunomycin was found to have a high accumulation in cardiac cells illustrated by a Ci/Ce ratio of 2890. This high accumulation was moderately but significantly (p < 0.05) increased in the presence of a MDR reversing agent such as verapamil, PSC 833 or S9788. These results suggest that blockade of the Pgp in humans may result in an increased toxicity of several Pgp substrates in normal tissues like the myocardium.

Study of cell changes induced by radiolabelling of human lymphocytes with 99m-technetium.

With the aim of evaluating cell changes possibly induced by labelling lymphocytes with 99m-technetium (99mTc), several rosette tests and stimulation assays with mitogens have been carried out before and after radiolabelling. The results presented here show that (i) as the rosette tests are not modified statistically, the original labelling method does not seem to induce short term membrane structure modification and (ii) as stimulation by mitogens decreases after labelling with 99mTc, medium term radiation damage, linked to autoirradiation of the cells, can be evoked. This study indicates that there is reason to limit the time taken to perform scintigraphic explorations with labelled lymphocytes in man.

Labeling of human lymphocytes with 99mTc by means of stannous pyrophosphate. Scintigraphic applications.

An original 99mTc-labeling method applied to human lymphocytes is described. This technique is based on the use of stannous pyrophosphate to reduce sodium pertechnetate. The proposed procedure has two main advantages: firstly, the labeling process takes place in a neutral and buffered medium which prevents any cellular aggregation; secondly, the labeling yield obtained is compatible with scintigraphic studies in man. The influence of each parameter on the labeling quality had been studied as well as the binding stability of the technetium fixed on cells. A systematic study of lymphocyte surface markers, before and after labeling, has led to the suggestion that the labeling process seems to affect cellular membranes, probably because of the technetium binding. Finally, scintigraphic results are presented, the study being performed on eight healthy volunteers. These results are compared with those published by other authors who used either a different radioisotope or a different labeling method.