Showing papers by "Ross C. Donehower published in 2009"
TL;DR: To resolve the discordance in the mutation status of KRAS, DNA from this patient’s plasma was tested using a highly sensitive technique termed “BEAMing,” which was named after components of this method (Beads, Emulsifi cation, Amplifi cations, and Magnetics), as previously described.
Abstract: KRAS mutations have clearly emerged as a pharmacogenomic marker that can predict which metastatic colorectal cancers will be resistant to treatment with antibodies that inhibit the epidermal growth factor receptor (EGFR) ( 1 , 2 ). The evaluation of patients for mutations in KRAS is rapidly becoming part of routine practice in clinical oncology and so far has relied mostly on formalinfixed paraffin-embedded (FFPE) tumor tissue. Accurate KRAS testing is critical because it determines which patients may benefit from anti-EGFR therapy. However, the selection of specimens with a sufficient number of tumor cells, possible genetic heterogeneity between different tumor sites (eg, between primary tumor and metastases), the quality of extracted DNA, and different detection methods for KRAS mutations can interfere with accurate analysis. In addition, formalin fixation often indiscriminately and irreversibly damages DNA, increasing sample-to-sample variability and decreasing the amount of DNA available for molecular analysis. A recent article by Tol et al. ( 3 ), on the effects of KRAS mutations on first-line therapy of colorectal cancer patients with anti-EGFR therapies, highlights this issue. Eight patients had to be excluded from the study because of the discordance in the mutation status of KRAS as assessed by two independent sequencing methods, both performed on FFPE sections of tumor tissue. As an additional example, we would like to report the case of a 58-year-old man with metastatic colorectal cancer whose tumor was being evaluated for mutations in KRAS . Inadvertently, testing was performed by two independent laboratories and revealed two different results. In both laboratories, tissue sections were reviewed by a pathologist, DNA was purifi ed from the malignant areas of microdissected tumor specimens, a region of exon 2 from the KRAS [GenBank accession No. NM_004985.3] gene was amplifi ed by polymerase chain reaction (PCR) and analyzed for the presence of mutations at codons 12 and 13. The fi rst laboratory reported the presence of only wild-type KRAS by melting curve analysis. The second laboratory detected a 35G>T mutation, which causes a glycine to valine substitution at codon 12 of KRAS (G12V), using single-nucleotide primer extension analysis. To resolve these discordant fi ndings, we tested DNA from this patient’s plasma for KRAS mutations using a highly sensitive technique termed “BEAMing,” which was named after components of this method (Beads, Emulsifi cation, Amplifi cation, and Magnetics), as previously described ( 4 ). This method uses standard laboratory tools and reagents to create a water-in-oil emulsion wherein each aqueous microdroplet houses an individual fragment of DNA bound to a bead. This setting allows billions of compartmentalized PCRs to be performed in parallel in a single test tube. The products of these reactions coat each bead with thousands of copies of DNA fragments that are identical to the single DNA molecule originally present. In this case, the result is millions of beads coated entirely with either KRAS mutant or KRAS wild-type DNA. To distinguish mutant from wild-type coated beads, allele-specifi c fl uorescent probes complementary to the known wild-type or mutant sequences of KRAS are simultaneously added to the beads for hybridization. The beads are then assessed via fl ow cytometry to detect rare mutant DNA molecules among a much larger number of normal DNA molecules ( 5 ). BEAMing is a digital assay that is able to count the frequency of
85 citations
TL;DR: The combination of 5-AC and phenylbutyrate across three dose schedules was generally well tolerated and safe, yet lacked any real evidence for clinical benefit.
Abstract: Purpose: This was a phase I trial to determine the minimal effective dose and optimal dose schedule for 5-azacytidine (5-AC) in combination with sodium phenylbutyrate in patients with refractory solid tumors. The pharmacokinetics, pharmacodynamics, and antineoplastic effects were also studied. Experimental Design: Three dosing regimens were studied in 27 patients with advanced solid tumors, and toxicity was recorded. The pharmacokinetics of the combination of drugs was evaluated. Repeat tumor biopsies and peripheral blood mononuclear cells (PBMC) were analyzed to evaluate epigenetic changes in response to therapy. EBV titers were evaluated as a surrogate measure for gene re-expression of epigenetic modulation in PBMC. Results: The three dose regimens of 5-AC and phenylbutyrate were generally well tolerated and safe. A total of 48 cycles was administrated to 27 patients. The most common toxicities were bone marrow suppression–related neutropenia and anemia, which were minor. The clinical response rate was disappointing for the combination of agents. One patient showed stable disease for 5 months whereas 26 patients showed progressive disease as the best tumor response. The administration of phenylbutyrate and 5-AC did not seem to alter the pharmacokinetics of either drug. Although there were individual cases of targeted DNA methyltransferase activity and histone H3/4 acetylation changes from paired biopsy or PBMC, no conclusive statement can be made based on these limited correlative studies. Conclusion: The combination of 5-AC and phenylbutyrate across three dose schedules was generally well tolerated and safe, yet lacked any real evidence for clinical benefit. (Clin Cancer Res 2009;15(19):6241–9)
82 citations
TL;DR: Adjuvant chemoradiation following curative resection for ampullary adenocarcinoma did not lead to a statistically significant benefit in overall survival, and a significant proportion of patients still developed distant metastatic disease suggesting a need for more effective systemic adjuvant therapy.
46 citations
TL;DR: A rapid assay was rationally developed that not only identified cases sensitive to ON 01910.Na, but also anticipated the pharmacodynamic events occurring after in vivo exposure, and showed a correlation between the ex vivo assay and sensitivity to the tested agent.
Abstract: The pupose of this study was to evaluate the activity of ON 01910.Na, a mitotic inhibitor, in in vitro and in vivo models of pancreatic cancer and to discover biomarkers predictive of efficacy. Successive in vitro and in vivo models were used; these included cell line-derived and patient-derived tumors from our PancXenoBank, a live collection of freshly generated pancreatic cancer xenografts. ON 01910.Na showed equivalent activity to gemcitabine against pancreatic cancer cell lines in vitro. The activity of the agent correlated with suppression of phospho-CDC25C and cyclin B1. These markers were optimized for a fine-needle aspirate ex vivo rapid assay. Cyclin B1 mRNA evaluation yielded the most optimal combination of accuracy and reproducibility. Next, nine patient-derived tumors from the PancXenoBank were profiled using the assay developed in cell lines and treated with ON01910.Na for 28 days. Two cases were cataloged as potential responders and seven as resistants. There was a correlation between the ex vivo assay and sensitivity to the tested agent, as the two cases prospectively identified as sensitive met prespecified criteria for response. Of the seven tumors of predictive resistant, only one was found to be sensitive to ON 01910.Na. In addition, there was a good correlation between cyclin B1 downregulation ex vivo and changes in cyclin B1 protein post-treatment. The novel mitotic inhibitor, ON 01910.Na, showed activity in preclinical model of pancreatic cancer. A rapid assay was rationally developed that not only identified cases sensitive to ON 01910.Na, but also anticipated the pharmacodynamic events occurring after in vivo exposure.
31 citations