Biomedical subjects

Weichung Shih

Publications and source records attributed to Weichung Shih.

6 recordsLinked to original sources

PubMed2005

Detection of B-RAF and N-RAS mutations in human melanoma.

BACKGROUND: It is now known that activating point mutations in components of the mitogen-activated protein kinase pathway commonly occur in melanoma. We previously described a method to detect point mutations in heterogenous tissues containing both wild-type and mutant B-RAF and N-RAS genes by using site-directed mutagenesis to introduce new restrictions sites in the cDNA sequence when the specific point mutations are present. We modified this technique to improve sensitivity and used it to determine the incidence of B-RAF and N-RAS mutations in human melanoma. STUDY DESIGN: We screened 115 melanoma samples for the most common B-RAF and N-RAS mutations found in melanoma using a site-directed mutagenesis-based detection technique. Southern blotting was used to increase sensitivity of the basic system. We also tested this method of genetic mutation detection in fine-needle aspiration specimens and paraffin-embedded tissues. RESULTS: Sixty-eight samples (20 of 36 primaries, 18 of 27 regional metastases, 16 of 40 nodal metastases, and 9 of 12 distant metastases) harbored the V599E B-RAF mutation (59%), 17 contained a Q61R N-RAS mutation, and 4 contained a Q61K N-RAS mutation. We were able to detect the V599E mutation in genomic DNA from paraffin-embedded melanoma samples and could routinely detect this mutation in fine-needle aspirations of melanoma tumors. This method of detection was sensitive and specific with no false positives. CONCLUSIONS: Activating mutations of B-RAF and N-RAS were present in approximately 60% and 18%, respectively, of samples tested. The site-directed mutagenesis system of mutation detection was both sensitive and specific in detecting these mutations and will likely prove very clinically useful in future studies.

Biomarkers, Tumor↗

PubMed2005

A genotyping system capable of simultaneously analyzing >1000 single nucleotide polymorphisms in a haploid genome.

A high-throughput genotyping system for scoring single nucleotide polymorphisms (SNPs) has been developed. With this system, >1000 SNPs can be analyzed in a single assay, with a sensitivity that allows the use of single haploid cells as starting material. In the multiplex polymorphic sequence amplification step, instead of attaching universal sequences to the amplicons, primers that are unlikely to have nonspecific and productive interactions are used. Genotypes of SNPs are then determined by using the widely accessible microarray technology and the simple single-base extension assay. Three SNP panels, each consisting of >1000 SNPs, were incorporated into this system. The system was used to analyze 24 human genomic DNA samples. With 5 ng of human genomic DNA, the average detection rate was 98.22% when single probes were used, and 96.71% could be detected by dual probes in different directions. When single sperm cells were used, 91.88% of the SNPs were detectable, which is comparable to the level that was reached when very few genetic markers were used. By using a dual-probe assay, the average genotyping accuracy was 99.96% for 5 ng of human genomic DNA and 99.95% for single sperm. This system may be used to significantly facilitate large-scale genetic analysis even if the amount of DNA template is very limited or even highly degraded as that obtained from paraffin-embedded cancer specimens, and to make many unpractical research projects highly realistic and affordable.

Chromosome Mapping↗

PubMed2003

Detection of mutations in the mitogen-activated protein kinase pathway in human melanoma.

PURPOSE: Recent studies suggest that activating point mutations in B-RAF may commonly occur in melanoma. We devised a method to detect point mutations in heterogeneous tissues containing both wild-type and mutant B-RAF and N-RAS genes by using site-directed mutagenesis to introduce new restrictions sites in the cDNA sequence when the specific point mutations are present. We used this technique to determine the incidence of mitogen-activated protein kinase (MAPK) mutations in human melanoma. EXPERIMENTAL DESIGN: We screened 85 melanoma samples for the most common B-RAF and N-RAS mutations found in melanoma using a site-directed mutagenesis-based detection technique. Western blotting was used to evaluate downstream up-regulation of the mitogen-activated protein kinase pathway in these tissues. RESULTS: Thirty-three samples (7 of 25 primaries, 15 of 25 regional metastases, 5 of 25 nodal metastases, and 6 of 10 distant metastases) harbored the V599E B-RAF mutation (39%), 12 contained a Q61R N-RAS mutation and 5 a Q61K N-RAS mutation. Western blotting with antiphosphorylated extracellular signal-regulated kinase 1/2 antibodies demonstrated up-regulation of the MAPK pathway in samples containing activating B-RAF or N-RAS mutations compared with wild-type samples. This method of detection was sensitive and specific with no false positives. CONCLUSIONS: Activating mutations of the MAPK pathway were present in approximately 60% of samples tested and caused activation of this cellular pathway that appears to be important in the pathogenesis of melanoma.

Base Sequence↗

PubMed2003

A phase I and pharmacologic study of the combination of marimastat and paclitaxel in patients with advanced malignancy.

BACKGROUND: Marimastat is a potent inhibitor of matrix metalloproteinases and in preclinical studies enhances the anti-tumor activity of certain chemotherapeutics. We performed a phase I clinical evaluation of the combination of oral marimastat and intravenous paclitaxel, to determine if these drugs could be co-administered safely, and to determine whether marimastat alters paclitaxel pharmacokinetics. MATERIAL/METHODS: Marimastat was administered twice daily and paclitaxel as a three hour infusion every three weeks. Doses of both marimastat and paclitaxel were escalated in cohorts of patients up to maximal doses of 10 mg for marimastat and 175 mg/m2 for paclitaxel. Paclitaxel plasma pharmacokinetic parameters were assessed in the absence (cycle 1) and presence (cycle 2) of marimastat. Trough marimastat plasma levels were evaluated during cycle 2. RESULTS: A total of 19 patients were treated at three different dose levels. There were no dose-limiting toxicities during the first cycle of therapy, resulting in dose escalation up to the planned maximal dose for each drug. Neutropenia was the most common significant toxicity at the highest dose level, with grade 3 or higher neutropenia occurring in 38% of patients. There were no complete or partial responses. Pharmacokinetic analyses indicate that marimastat does not alter paclitaxel clearance. At the 10 mg dose, the mean trough marimastat level was 14.8 Kg/L. CONCLUSIONS: Marimastat and paclitaxel can be co-administered safely at doses equivalent to those recommended for single-agent administration. Additional studies are necessary to determine whether this combination is more effective in controlling tumor progression than paclitaxel alone.

Adult↗

PubMed2002

Problems in dealing with missing data and informative censoring in clinical trials.

Acommon problem in clinical trials is the missing data that occurs when patients do not complete the study and drop out without further measurements. Missing data cause the usual statistical analysis of complete or all available data to be subject to bias. There are no universally applicable methods for handling missing data. We recommend the following: (1) Report reasons for dropouts and proportions for each treatment group; (2) Conduct sensitivity analyses to encompass different scenarios of assumptions and discuss consistency or discrepancy among them; (3) Pay attention to minimize the chance of dropouts at the design stage and during trial monitoring; (4) Collect post-dropout data on the primary endpoints, if at all possible; and (5) Consider the dropout event itself an important endpoint in studies with many.

Journal Article↗

PubMed2002

A Phase I/pilot study of sequential doxorubicin/vinorelbine: effects on p53 and microtubule-associated protein 4.

PURPOSE: Few molecular determinants of sensitivity to cancer chemotherapy exist. In experimental systems, p53 regulates the sensitivity to antimicrotubule drugs through its effect on microtubule-associated protein 4 (MAP4). MAP4 is the major microtubule-associated protein in nonneuronal tissues and promotes microtubule polymerization. We reported that wild-type p53 induction by doxorubicin in C127 breast cancer cells repressed MAP4, decreased microtubule polymerization, and increased Vinca alkaloid sensitivity. The goals of this Phase I/pilot clinical trial were to determine: (a) the safety of delivering a DNA-damaging agent (doxorubicin) followed in sequence by treatment with an antimicrotubule drug (vinorelbine); and (b) the feasibility of detecting activation of p53 and repression of MAP4 in patients' tissues. EXPERIMENTAL DESIGN: Peripheral blood mononuclear cells (PBMNCs) and tumor were obtained from 16 women with locally advanced (stage IIIb) or metastatic (stage IV) breast cancer before doxorubicin treatment and immediately before treatment with vinorelbine 24 or 48 h later. RESULTS: After doxorubicin treatment, p53 increased in 12 of 14 PBMNC and 4 of 10 tumor samples. Changes in MAP4 were variable; however, in samples in which p53 was induced, MAP4 decreased in 7 of 12 PBMNC and 3 of 4 breast cancer specimens. Immunohistochemistry confirmed lower MAP4 expression in tumor cells after doxorubicin treatment. Seven of 16 patients had a partial response, and treatment was well tolerated. CONCLUSIONS: These data demonstrate the ability to detect the activation of p53 and the repression of MAP4 in normal and malignant tissues in patients treated with a DNA-damaging agent, and that an antimicrotubule drug can be administered safely at a time when cells may be more sensitive to treatment.

Adult↗