Microsatellite instability (MSI) is a characteristic feature of hereditary nonpolyposis colorectal cancer and is also observed in sporadic colorectal and endometrial cancers. Alterations in the mismatch repair genes hMLH1 and hMSH2 are important for the development of MSI. It has recently been demonstrated that hypermethylation of the hMLH1 promoter region is associated with MSI and appears to be a common mechanism for gene inactivation. For endometrial carcinoma, however, previous studies have been relatively small and have not been population based. We therefore wanted to assess the frequency and prognostic significance of hypermethylation of the hMLH1 and hMSH2 genes in conjunction with hMLH1 protein expression in a prospective and population-based series of endometrial carcinoma patients with known MSI status and complete follow-up. A total of 138 patients were studied, and methylation of hMLH1 was found in 23% of tumors with conclusive results, whereas methylation of hMSH2 was seen in only 1% of tumors. Methylation of hMLH1 was significantly correlated with MSI (P < 0.001). Loss of nuclear staining of hMLH1 protein was seen in 14% of the cases and was significantly correlated with hMLH1 methylation and MSI (P < 0.001). Normal expression of hMLH1 was seen in all of the unmethylated tumors (100%). Of the 14 MSI-positive tumors that were also methylated, all but 1 (93%) showed a loss of nuclear expression of hMLH1. None of the tumors with loss of hMLH1 expression or hMLH1 methylation were aneuploid (P for both < or = 0.05), and loss of hMLH1 expression and hMLH1 methylation was significantly correlated with lack of p53 overexpression (P for both < or = 0.05). Nuclear hMLH1 staining and hMLH1 methylation did not significantly influence survival. In conclusion, hMLH1 methylation was common and was significantly correlated with loss of hMLH1 protein expression, MSI, diploid tumors, and lack of p53 overexpression. In contrast, hMSH2 methylation was infrequent in this prospective and population-based series of endometrial carcinomas.
Abstract Identifying patients prior to treatment that are less likely to benefit from or most likely to experience adverse events from chemotherapeutic agents is essential. Even though humans are the most relevant system, pharmacogenomic discovery in the field of oncology is plagued by difficulties in executing large clinical trials and confounding factors such as co-morbidities, dosage, and concomitant medications. Therefore, we developed a genome-wide cell-based approach evaluating single nucleotide polymorphisms (SNPs) and gene expression to predict chemotherapy-induced response and toxicity and then validated our findings in clinically relevant patient cohorts. Our model utilizes the International HapMap lymphoblastoid cell lines (LCLs). Genome-wide association studies were performed to identify SNPs significantly associated with carboplatin sensitivity through their effects on mRNA expression. The significant findings were evaluated in an independent LCL replication set and in patient samples obtained from the Australian Ovarian Cancer Study (AOCS) and two Phase II head and neck clinical trials (UC12019 and UC13881) conducted at the University of Chicago. Four hundred and nine ovarian cancer patients receiving carboplatin and paclitaxel were analyzed in AOCS; while 60 and 32 head and neck cancer patients were evaluated in UC12019 and UC13881 trials. Carboplatin was used as induction and concomitant chemo-radiation therapy in UC12019 and UC13881 trials, respectively. Using LCLs, our genome-wide model identified 65 SNPs that are associated with at least one carboplatin sensitivity phenotype through the expression of 61 genes. Five of them were replicated in a separate set of LCLs. In AOCS, SNP (rs1649942) was significantly associated with progression-free survival (Plog-rank=0.009) and overall survival (Plog-rank=0.03). In the head and neck cancer trials, 2 other SNP-phenotype associations were identified and replicated in both trials, including the association between SNP (rs4946514) and overall response to carboplatin, and the association between SNP (rs7134205) and post treatment platelet changes. Given the obstacles to performing large, replicable pharmacogenomic studies in patients, the cell-based model is proven to be an effective alternative in novel pharmaco-SNP discovery. We demonstrate germline SNPs identified through the cell-based genome-wide approach are clinically important predictors of chemotherapy response and toxicity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2761.
Abstract H‐Ras oncogene plays a critical role in the transformation of normal cells to a malignant phenotype through constitutive activation of the GTP bound protein leading to uncontrolled cell proliferation in several human cancers. Thus, H‐Ras oncoprotein serves as an excellent target for anticancer drug discovery. To identify novel H‐Ras inhibitors, we performed structure‐based virtual screening of the Maybridge HitFinder™ library using Schrodinger suite. Thirty ligands from the chemical library were identified as they showed preferential in silico binding initially to H‐Ras proteins with Gly12Val, Gly13Asp, and Gly12Val‐Gly13Asp mutations. Absorption, distribution, metabolism, excretion, and toxicity profile confirmed drug‐like properties of the compounds. Three representative molecules were tested for antiproliferative effect on T24 urinary bladder carcinoma cell line, MCF‐7 breast cancer cell line and HDF‐7 normal dermal fibroblast cells using 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay. Two compounds (Cmpds) showed antiproliferative activity exclusively in the cancer cell lines with minimal effect on the control HDF‐7 cells. The effect of compound treatment on cell cycle progression, assessed by propidium iodide (PI) staining, depicted increased arrest of T24 cell line in the sub G1 phase. Further, Annexin‐V PI dual staining and pan caspase inhibitor Z‐VAD‐fmk indicated caspase‐dependent apoptotic activity of Cmpds 1 and 3. Our findings demonstrate caspase‐dependent apoptotic activity of Cmpds 1 and 3 selectively against Gly12Val mutated T24 cancer cell line implicating a potential for treatment of bladder cancer. We envisage that these molecules may be promising candidates with potential therapeutic value in H‐Ras mutation‐associated cancers.
Rett syndrome is a dominant X-linked disorder caused by point mutations (approximately 80%) or by deletions or insertions (approximately 15% to 18%) in the MECP2 gene. It is most common in females but lethal in males, with a distinctly different phenotype. Rett syndrome patients have severe neurological and behavioral problems. Clinical genetic testing laboratories commonly use characterized genomic DNA reference materials to assure the quality of the testing process; however, none are commercially available for MECP2 genetic testing. The Centers for Disease Control and Prevention's Genetic Testing Reference Material Coordination Program, in collaboration with the genetic testing community and the Coriell Cell Repositories, established 27 new cell lines and characterized the MECP2 mutations in these and in 8 previously available cell lines. DNA samples from the 35 cell lines were tested by eight clinical genetic testing laboratories using DNA sequence analysis and methods to assess copy number (multiplex ligation-dependent probe amplification, semiquantitative PCR, or array-based comparative genomic hybridization). The eight common point mutations known to cause approximately 60% of Rett syndrome cases were identified, as were other MECP2 variants, including deletions, duplications, and frame shift and splice-site mutations. Two of the 35 samples were from males with MECP2 duplications. These MECP2 and other characterized genomic DNA samples are publicly available from the NIGMS Repository at the Coriell Cell Repositories.
The fragile X syndrome is predominantly caused by a large expansion of a CGG trinucleotide repeat in the promoter region of the FMR1 gene, which is associated with methylation and downregulation of transcription. The molecular diagnosis of this disorder is based on repeat size and methylation analysis of the FMR1 gene usually by Southern blot analysis. We describe a PCR-based method for the analysis of methylation of the FMR1 gene, which involves bisulfite treatment of DNA prior to amplification. Fifty-two normal and 48 affected, premutation, or mosaic males were analyzed in a blinded study by this method. A prospective study of 30 males suspected of fragile X was also performed. Amplification specific for the methylated FMR1 sequence was readily observed in all individuals with a full mutation, whereas all normal and premutation individuals showed only amplification-specific for the unmethylated sequence, thus, allowing affected and unaffected males to be distinguished. A full mutation in the presence of mosaicism was also detectable by this method. Methylation-specific PCR appears to be a rapid and reliable tool for the diagnosis of fragile X males.
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