digestion. After analysis of the commonly mutated exon regions of VHL for 50 patients, 78% were found to have mutations. 17% of those mutations occurred in multiple patients. DNA from serum and/or urine from these patients was then examined for the identified mutations. 27% of patients had mutations detected in the DNA purified from their serum and/or urine. Contrasting the sensitivity of mutation detection via RFLP of body fluids versus detection via Sequenom indicated that Sequenom was able to identify mutations present in DNA as dilute as 0.05ng, compared to 12.5ng DNA required for RFLP detection.CONCLUSIONS: A panel of the most highly observed VHL polymorphism DNA biomarkers is a potential diagnostic tool for the detection of early stage RCC. High-throughput identification methods of such mutations, such as mass spectrometry, have an increased sensitivity and specificity compared to PCR/RFLP analysis and would therefore better translate into clinical application. VHL mutations identified in DNA purified from patient urine or serum via RFLP and mass spectrometry support the feasibility of developing such a test.
413 Background: Cell cycle dysregulation is prevalent in renal cell carcinoma (RCC). PD-0332991 is an orally active, potent, and selective inhibitor of cyclin-dependent kinases (CDK) 4 and 6, blocking retinoblastoma (Rb) phosphorylation at nanomolar concentrations. Methods: 28 RCC and immortalized kidney cell lines were used to examine the effects of PD-0332991 on proliferation to determine the half maximal inhibitory concentration (IC50). Effects of PD-0332991 on cell-cycle, apoptosis, and Rb phosphorylation were assessed with flow cytometry and western blot analysis for five of the cell lines: RCC-HB and SW 156 (sensitive/malignant), R444 and Hs 891.T (resistant/malignant), and CCD 1103 (resistant/immortalized non-malignant). Molecular markers for response prediction were studied using array CGH and gene expression profiling. Results: Concentration-dependent inhibition of proliferation was identified in response to PD-0332991, with IC50values ranging from 25.0nM up to 700nM; five cell lines were identified as completely resistant at 1000nM. PD-0332991 induced G0/G1 cell cycle arrest, as well as induction of late apoptosis in SW 156, and Rb phosphorylation was blocked in a time-dependent fashion in both sensitive cell lines, while resistant lines were unaffected. Genotype and expression data of CDKN2A and CDKN2B were combined and a consensus was made regarding p16 and p15 status; significant association between loss and sensitivity to PD-0332991 was identified for p16 (p = 0.027). For CCND1, CCNE1, E2F1, Rb, CDK4, and CDK6 no amplifications or homozygous deletions were identified by array CGH; cell lines were then classified as having high or low expression for each of these markers. E2F1 had low expression levels significantly associated with response to PD-0332991 (p = 0.041). Conclusions: PD-0332991 shows anti-proliferative activity in RCC through blockade of the cell cycle. The decreased expression of molecular markers p16 and E2F1 predict for sensitivity to PD-0332991 in RCC.
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