Detection of mutations in exon 8 of TP53 by temperature gradient 96-capillary array electrophoresis.

Various capillary electrophoresis applications have increasingly been utilized in mutation detection. Separation of two species is either based on secondary structure or differences in melting of DNA due to the mutation. Detection of the mutant is based on its mobility difference in the sieving matrix. We have adapted a regular 96-capillary sequencing instrument, the MegaBACE 1000, for mutation detection based on thermodynamic stability and mobility shift during electrophoresis. Denaturation of the lower melting domain of the DNA was achieved with a gradually decreasing temperature gradient in combination with a chemical denaturant. Samples were analyzed for mutants in exon 8 of the TP53 genefrom tumor samples and controls. Genomic DNA was PCR-amplified with one fluorescein labeled primer and one GC-clamped primer, diluted in water, and analyzed by temperature gradient 96-capillary array electrophoresis. Tumor samples and PCR reconstruction experiment samples were resolved by capillary gel electrophoresis under appropriate temperature gradient denaturing conditions. Ninety-six samples were analyzed in one run, with an analysis time of 30 min and a sensitivity to detect mutated alleles in wild-type background down to 0.4%. The technique proved to be robust, in that the gradient compensatesfor temperature differences within the capillary chamber; thus, each capillary will pass through the optimal separating conditions around the theoretical melting temperature for TP53 exon 8, separating homoduplexes and heteroduplexes. This technique is applicable to any sequence previously analyzed by DNA melting gel techniques or sequences harboring iso-melting domains of 100-120 bp.

Mutation analysis of TP53 exons 5-8 by automated constant denaturant capillary electrophoresis.

DNA fragments melt characteristically according to their nucleotide sequence and length, when exposed to denaturants such as temperature, urea or formamide. Small differences within a defined sequence, like a base mutation, will result in a slightly different melting behavior of the aberrant DNA fragment compared to that of the wild type sequence. This feature has previously been exploited for mutation detection by constant denaturant capillary electrophoresis (CDCE). In this report, we describe an automated approach (ACDCE) using a commercially available apparatus (ABI 310 Genetic Analyzer) to analyze mutations in exons 5-8 of TP53. The running conditions were determined by temperature titration of the fragments on the apparatus, and an operating sensitivity showed that 0.1% mutated alleles could be detected against a background of wild-type alleles. Up to 48 samples can be analyzed by ACDCE without any need for operator intervention. The apparatus is commercially available, and there is no need for instrument modification. To our knowledge this is the first report on the analysis of TP53 exons 5-8 by ACDCE.

Automated constant denaturant capillary electrophoresis applied for detection of KRAS exon 1 mutations.

In this study, we have applied automated constant denaturant capillary electrophoresis (ACDCE) for the detection of KRAS exon 1 mutations. Samples from 191 sporadic colon carcinomas previously analyzed for KRAS mutations with allele-specific PCR (ASPCR), temporal temperature gradient electrophoresis (TTGE), and constant denaturant capillary electrophoresis (CDCE) were analyzed. In ACDCE, an unmodified ABI Prism 310 genetic analyzer with constant denaturant conditions separated fluorescein-labeled PCR products. Temperature in combination with a chemical denaturant was used for separation. The optimal separation conditions for PCR-amplified KRAS exon 1 fragments were determined by adjusting the temperature before electrophoresis. In the ACDCE analysis, the sequence of a mutant was determined by comparing the electropherogram of the fragment to that of known mutations followed by mixing the sample with control mutations before reanalysis. In a titration experiment mixing mutant and wild-type alleles, the sensitivity for mutation detection was shown to be 0.6% in this automated CDCE technique. The automation of CDCE allowed rapid analysis of a large number of test samples over as short period of time and with a commercially available apparatus.

Two-point fluorescence detection and automated fraction collection applied to constant denaturant capillary electrophoresis.

Constant denaturant capillary electrophoresis (CDCE) has been shown to be a sensitive method to detect point mutations in DNA sequences of 100-bp lengths. Here, we report a significant modifications for the instrumental setup that allows a highly accurate prediction of the elution time of DNA fragments from the capillary and an efficient collection of separated fractions. Fluorescently labeled DNA fragments of TP53 exon 8 wild-type and two mutants (base pair number 14480 and 14525) are detected at two separate points of the same capillary. This permits the precise calculation of the fragment velocity after separation in the heated zone because, at room temperature, all DNA fragments of the same length have the same velocity. Such precision permits the selective collection of separated fragments using an automated fraction collector for additional CDCE analysis or sequencing. Also, the two-point detection allows one to rapidly distinguish between double-stranded and single-stranded DNA fragments of the same length, a process that cannot be achieved with a one-point detection system alone. Both modifications greatly improve the procedure to detect novel mutations by means of CDCE.

Detection of low-frequency mutations in exon 8 of the TP53 gene by constant denaturant capillary electrophoresis (CDCE).

We extended our development of the means to measure point mutations at the DNA level to the problem of detecting TP53 gene variants in the area around tumors where mutant fractions are expected to be as low as one mutant per 1000 wild-type (WT) sequences. We met this criterion by using the following methods. The TP53 exon 8 sequence was amplified from genomic DNA samples under conditions of high polymerase fidelity using a fluorescein-labeled primer. This mixture of WT and mutant sequences was converted to heteroduplexes of mutant and WT sequences by melting and re-annealing. The mixture was resolved by capillary gel electrophoresis under appropriate constant denaturing conditions. Using laser-induced fluorescence, we found that fractions as low as 1/1000 could be detected without any prior enrichment for mutant sequences, and that the melting properties of the amplified DNA will leave "fingerprints" in the electropherogram that can be used to identify the specific mutation. This method is fast and robust and should be applicable to clinical analyses in which rapid scanning for any mutation in an exonic sequence is important.

Alterations in methotrexate pharmacokinetics by naproxen in the rat as measured by microdialysis.

Reports of a potentially life-threatening interaction between the antifolate methotrexate (MTX) and drugs belonging to the NSAID class instigated a study of MTX pharmacokinetics by a microdialysis technique in the presence and absence of the NSAID naproxen in anesthetized rats. After pretreatment with naproxen, the animals received either 750 or 1,000 mg/kg MTX as a 6 h continuous intravenous infusion. During infusions, microdialysis effluents were obtained from probes situated intravenously, intrahepatically and intrarenally. In all three compartments, time-concentration AUCs for both MTX and its major extracellular metabolite, 7-hydroxymethotrexate (7-OH-MTX), increased about two-fold in the presence of naproxen. The mechanisms responsible for the MTX-NSAID interaction are briefly discussed. The study demonstrate that the microdialysis technique offers a means to investigate pharmacokinetic drug-drug interactions.

Continuous intratumoral microdialysis during high-dose methotrexate therapy in a patient with malignant fibrous histiocytoma of the femur: a case report.

We used a microdialysis technique to assay intratumoral methotrexate (MTX) levels during high-dose (12 g/m2 given as a 4-h infusion) therapy in a 43-year-old man with a malignant fibrous histiocytoma in the medial femoral condyle. Additional microdialysis probes were implanted in muscle tissue contralateral to the tumor and in an antecubital vein. Microdialysis was attempted during the initial two high-dose courses, but the two latter probes were removed at the start of the second treatment cycle due to leakage. No attempt to correct for microdialysis recovery was made. The intratumorally localized probe gave reproducible data on tumor MTX exposure of 9.3-14% of unbound systemic MTX. There was a close correlation between tumor and systemic levels for both MTX and its major extracellular metabolite 7-hydroxymethotrexate. Although limited to the study of MTX pharmacokinetics in a single subject, the experiment demonstrates that intratumoral microdialysis may provide data on tumor drug exposure, although of an indirect nature and dependent on the probe characteristics, the flow rate, and, possibly, the time after probe implantation. We propose that the application of microdialysis may prove useful for elucidation of the relationship between local drug exposure and the therapeutic response in normally inaccessible compartments during cancer pharmacotherapy.

Intratumoral differences in methotrexate levels within human osteosarcoma xenografts studied by microdialysis.

A central tenet in oncology is the assumed relationship between drug concentration and cytotoxicity. Determinations of drug levels in tumor tissues are, however, generally not undertaken. Microdialysis is a method where continuous drug monitoring may be achieved by sampling of low molecular weight substances from the extracellular space. By employing this technique it is possible to observe variable drug levels within tissues, including tumors, over time. Herein, we present results from a nude rat model where subcutaneous human osteosarcoma xenografts were established prior to the administration of the antifolate methotrexate as an intravenous infusion. Significant differences in drug exposure within single tumors were evident. Generally, peak drug concentrations were lower and drug efflux slower from the center of the tumors as compared to the periphery. The use of microdialysis could be an important tool for optimizing current strategies in anticancer chemotherapy.

Determination of extracellular methotrexate tissue levels by microdialysis in a rat model.

We used a microdialysis technique to determine tissue methotrexate (MTX) levels during steady state in a rodent model. Two different approaches were employed to measure the actual extracellular MTX concentrations in muscle, liver, and kidney tissues of anesthetized Wistar rats. With the reduced-perfusion-rate technique, the flow in the microdialysis perfusate was gradually decreased toward zero to permit calculation of zero-flow intercepts. Using the net change technique, microdialysis probes were perfused with different MTX concentrations to allow an assessment of equilibrium drug levels. For these two methods to be used, drug concentrations in the matrix to be analyzed must remain unchanged during the experimental procedure. In the animal model, steady state was attained after 1.5 h and maintained throughout the rest of the experiments by the administration of MTX as continuous infusions through a venous catheter. In vitro and in vivo, both the reduced-perfusion-rate and net change techniques gave reproducible data that permitted the estimation of extracellular drug concentrations in the dialyzed tissue compartments.The data suggest that the level of unbound MTX in the circulation is fairly similar to the extracellular concentrations in the muscle and liver. In the kidney, MTX levels were measured to be 3-8 times higher than those of unbound, circulating MTX, and a considerable discrepancy between the two methods used for estimations was apparent. These results demonstrate that both the net change and reduced-flow microdialysis techniques can produce reproducible and precise data. The results may constitute a basis for determining recoveries and, thus, true extracellular drug levels during in vivo microdialysis of MTX. This may be of importance in delineation of the relationship between tissue MTX levels and outcome in a variety of normally inaccessible compartments during cancer pharmacotherapy.

Pharmacokinetics of different doses of methotrexate at steady state by in situ microdialysis in a rat model.

We used a microdialysis technique to monitor extracellular methotrexate (MTX) levels during the steady state in a rodent model. Microdialysis probes were implanted in the muscle, liver, and kidney of anesthetized male Wistar rats. MTX (18.75-500 mg/kg) was given as a continuous infusion through a venous catheter, and blood samples were obtained through a second venous catheter. Heparinized plasma, ultrafiltered plasma, microdialysis effluent from tissues, and tissue samples (obtained at the end of experiments) were analyzed for MTX content by high-performance liquid chromatography (HPLC). Steady state was demonstrated in the blood and tissues from 2 h until the end of the experiments (6 h). Extracellular drug levels in muscle and liver displayed a linear correlation with doses, whereas kidney levels reached a plateau at an MTX dose of 150 mg/kg per 6 h. Microdialysis-fluid endpoint levels for muscle, liver, and kidney were positively correlated to the endpoint total tissue levels (r2 = 0.80, 0.85, and 0.68, respectively). In the kidneys, the maximal relative tissue MTX accumulation was measured at a total dose of 75 mg/kg per 6 h. At higher doses, the relative drug sequestration declined to less than half of the values observed at this dose. This study demonstrates that the microdialysis technique can provide reproducible data on MTX tissue exposure in an animal model and that it offers a means of serial and reproducible monitoring of extracellular-tissue MTX levels at steady state and over a wide dose range. Pending additional studies, microdialysis may be a helpful technique for elucidating the kinetics of drug delivery to both targeted and toxicity-prone tissues during chemotherapy.