The rationale for using small molecule inhibitors of oncogenic proteins as cancer therapies depends, at least in part, on the assumption that metastatic tumors are primarily clonal with respect to mutant oncogene. With the emergence of BRAFV600E as a therapeutic target, we investigated intra- and inter-tumor heterogeneity in melanoma using detection of the BRAFV600E mutation as a marker of clonality. BRAF mutant-specific PCR (MS-PCR) and conventional sequencing were performed on 112 tumors from 73 patients, including patients with matched primary and metastatic specimens (n = 18). Nineteen patients had tissues available from multiple metastatic sites. Mutations were detected in 36/112 (32%) melanomas using conventional sequencing, and 85/112 (76%) using MS-PCR. The better sensitivity of the MS-PCR to detect the mutant BRAFV600E allele was not due to the presence of contaminating normal tissue, suggesting that the tumor was comprised of subclones of differing BRAF genotypes. To determine if tumor subclones were present in individual primary melanomas, we performed laser microdissection and mutation detection via sequencing and BRAFV600E-specific SNaPshot analysis in 9 cases. Six of these cases demonstrated differing proportions of BRAFV600Eand BRAFwild-type cells in distinct microdissected regions within individual tumors. Additional analyses of multiple metastatic samples from individual patients using the highly sensitive MS-PCR without microdissection revealed that 5/19 (26%) patients had metastases that were discordant for the BRAFV600E mutation. In conclusion, we used highly sensitive BRAF mutation detection methods and observed substantial evidence for heterogeneity of the BRAFV600E mutation within individual melanoma tumor specimens, and among multiple specimens from individual patients. Given the varied clinical responses of patients to BRAF inhibitor therapy, these data suggest that additional studies to determine possible associations between clinical outcomes and intra- and inter-tumor heterogeneity could prove fruitful.

BackgroundSorafenib monotherapy in patients with metastatic melanoma was explored in this multi-institutional phase II study. In correlative studies the impact of sorafenib on cyclin D1 and Ki67 was assessed.Methodology/Principal FindingsThirty-six patients treatment-naïve advanced melanoma patients received sorafenib 400 mg p.o. twice daily continuously. Tumor BRAFV600E mutational status was determined by routine DNA sequencing and mutation-specific PCR (MSPCR). Immunohistochemistry (IHC) staining for cyclin D1 and Ki67 was performed on available pre- and post treatment tumor samples. The main toxicities included diarrhea, alopecia, rash, mucositis, nausea, hand-foot syndrome, and intestinal perforation. One patient had a RECIST partial response (PR) lasting 175 days. Three patients experienced stable disease (SD) with a mean duration of 37 weeks. Routine BRAFV600E sequencing yielded 27 wild-type (wt) and 6 mutant tumors, whereas MSPCR identified 12 wt and 18 mutant tumors. No correlation was seen between BRAFV600E mutational status and clinical activity. No significant changes in expression of cyclin D1 or Ki67 with sorafenib treatment were demonstrable in the 15 patients with pre-and post-treatment tumor samples.Conclusions/SignificanceSorafenib monotherapy has limited activity in advanced melanoma patients. BRAFV600E mutational status of the tumor was not associated with clinical activity and no significant effect of sorafenib on cyclin D1 or Ki67 was seen, suggesting that sorafenib is not an effective BRAF inhibitor or that additional signaling pathways are equally important in the patients who benefit from sorafenib.Trial registrationClinical Trials.gov NCT00119249

Background: Treatment options for patients with recurrent superficial bladder cancer are limited, necessitating aggressive exploration of new treatment strategies that effectively prevent recurrence and progression to invasive disease. We assessed the effects of belinostat (previously PXD101), a novel histone deacetylase inhibitor, on a panel of human bladder cancer cell lines representing superficial and invasive disease, and on a transgenic mouse model of superficial bladder cancer.

Mutations in the BRAF oncogene at amino acid 600 have been reported in 40 to 70% of human metastatic melanoma tissues, and the critical role of BRAF in the biology of melanoma has been established. Sampling the blood compartment to detect the mutational status of a solid tumor represents a highly innovative advance in cancer medicine, and such an approach could have advantages over tissue-based techniques. We report the development of a fluorescence-based polymerase chain reaction (PCR) assay to detect mutant BRAF alleles in plasma. A mutant-specific PCR assay was optimized to specifically amplify the mutant BRAF allele without amplifying the wild-type allele. Experiments mixing DNA from a BRAF mutant melanoma cell line with wild-type human placental DNA in varying proportions were performed to determine the threshold of this assay and to compare it with routine DNA sequencing. The assay was then applied to tissue and plasma specimens from patients with metastatic melanoma. The assay detected 0.1 ng of mutant DNA mixed in 100 ng of wild-type DNA and was 500-fold more sensitive than DNA sequencing. The assay detected mutant BRAF alleles in plasma samples from 14 of 26 (54%) metastatic melanoma patients. These data demonstrate the feasibility of blood-based testing for BRAF mutations in metastatic melanoma patients. (J Mol Diagn 2007, 9:178 -183;