In Western Europe and the United States approximately 1 in 12 women develop breast cancer. A small proportion of breast cancer cases, in particular those arising at a young age, are attributable to a ...highly penetrant, autosomal dominant predisposition to the disease. The breast cancer susceptibility gene, BRCA2, was recently localized to chromosome 13q12-q13. Here we report the identification of a gene in which we have detected six different germline mutations in breast cancer families that are likely to be due to BRCA2. Each mutation causes serious disruption to the open reading frame of the transcriptional unit. The results indicate that this is the BRCA2 gene.
We aimed to investigate the expression pattern of phosphatase and tensin homolog (PTEN), to evaluate the relationship between PTEN expression and clinicopathological characteristics, including fatty ...acid synthase (FAS) expression, and to determine the correlations of PTEN and FAS expression with survival in Chinese patients with hepatocellular carcinoma (HCC). The expression patterns of PTEN and FAS were determined using tissue microarrays and immunohistochemistry. The expression of PTEN was compared with the clinicopathological characteristics of HCC, including FAS expression. Receiver operator characteristic curves were used to calculate the clinical sensitivity and specificity of PTEN expression. Kaplan-Meier survival curves were constructed to evaluate the correlations of PTEN loss and FAS overexpression with overall survival. We found that the loss of PTEN expression occurred predominantly in the cytoplasm, while FAS was mainly localized to the cytoplasm. Cytoplasmic and total PTEN expression levels were significantly decreased in HCC compared with adjacent non-neoplastic tissue (both, p < 0.0001). Decreased cytoplasmic and total PTEN expression showed significant clinical sensitivity and specificity for HCC (both, p < 0.0001). Downregulation of PTEN in HCC relative to non-neoplastic tissue was significantly correlated with histological grade (p = 0.043 for histological grades I-II versus grade III). Loss of total PTEN was significantly correlated with FAS overexpression (p = 0.014). Loss of PTEN was also associated with poor prognosis of patients with poorly differentiated HCC (p = 0.049). Moreover, loss of PTEN combined with FAS overexpression was associated with significantly worse prognosis compared with other HCC cases (p = 0.011). Our data indicate that PTEN may serve as a potential diagnostic and prognostic marker of HCC. Upregulating PTEN expression and inhibiting FAS expression may offer a novel therapeutic approach for HCC.
Changes in DNA copy number are one of the hallmarks of the genetic instability common to most human cancers. Previous microarray-based methods have been used to identify chromosomal gains and losses; ...however, they are unable to genotype alleles at the level of single nucleotide polymorphisms (SNPs). Here we describe a novel algorithm that uses a recently developed high-density oligonucleotide array-based SNP genotyping method, whole genome sampling analysis (WGSA), to identify genome-wide chromosomal gains and losses at high resolution. WGSA simultaneously genotypes over 10,000 SNPs by allele-specific hybridisation to perfect match (PM) and mismatch (MM) probes synthesised on a single array. The copy number algorithm jointly uses PM intensity and discrimination ratios between paired PM and MM intensity values to identify and estimate genetic copy number changes. Values from an experimental sample are compared with SNP-specific distributions derived from a reference set containing over 100 normal individuals to gain statistical power. Genomic regions with statistically significant copy number changes can be identified using both single point analysis and contiguous point analysis of SNP intensities. We identified multiple regions of amplification and deletion using a panel of human breast cancer cell lines. We verified these results using an independent method based on quantitative polymerase chain reaction and found that our approach is both sensitive and specific and can tolerate samples which contain a mixture of both tumour and normal DNA. In addition, by using known allele frequencies from the reference set, statistically significant genomic intervals can be identified containing contiguous stretches of homozygous markers, potentially allowing the detection of regions undergoing loss of heterozygosity (LOH) without the need for a matched normal control sample. The coupling of LOH analysis, via SNP genotyping, with copy number estimations using a single array provides additional insight into the structure of genomic alterations. With mean and median inter-SNP euchromatin distances of 244 kilobases (kb) and 119 kb, respectively, this method affords a resolution that is not easily achievable with non-oligonucleotide-based experimental approaches.
Homozygous deletions of recessive cancer genes and fragile sites are known to occur in human cancers. We identified 281 homozygous deletions in 636 cancer cell lines. Of these deletions, 86 were ...homozygous deletions of known recessive cancer genes, 17 were of sequenced common fragile sites, and 178 were in genomic regions that do not overlap known recessive oncogenes or fragile sites ("unexplained" homozygous deletions). Some cancer cell lines have multiple homozygous deletions whereas others have none, suggesting intrinsic variation in the tendency to develop this type of genetic abnormality (P < 0.001). The 178 unexplained homozygous deletions clustered into 131 genomic regions, 27 of which exhibit homozygous deletions in more than one cancer cell line. This degree of clustering indicates that the genomic positions of the unexplained homozygous deletions are not randomly determined (P < 0.001). Many homozygous deletions, including those that are in multiple clusters, do not overlap known genes and appear to be in intergenic DNA. Therefore, to elucidate further the pathogenesis of homozygous deletions in cancer, we investigated the genome landscape within unexplained homozygous deletions. The gene count within homozygous deletions is low compared with the rest of the genome. There are also fewer short interspersed nuclear elements (SINEs), long interspersed nuclear elements (LINEs), and low-copy-number repeats (LCRs). However, DNA within homozygous deletions has higher flexibility. These features may signal the presence of currently unrecognized zones of susceptibility to DNA rearrangement. They may also reflect a tendency to reduce the adverse effects of homozygous deletions by minimizing the number of genes removed.
A small proportion of breast cancer, in particular those cases arising at a young age, is due to the inheritance of dominant susceptibility genes conferring a high risk of the disease. A genomic ...linkage search was performed with 15 high-risk breast cancer families that were unlinked to the BRCA 1 locus on chromosome 17q21. This analysis localized a second breast cancer susceptibility locus, BRCA2, to a 6-centimorgan interval on chromosome 13q12-13. Preliminary evidence suggests that BRCA2 confers a high risk of breast cancer but, unlike BRCA 1, does not confer a substantially elevated risk of ovarian cancer.
We demonstrate that comparative genomic hybridization (CGH) onto cDNA microarrays may be used to carry out genome-wide screens for regions of genetic loss, including homozygous (complete) deletions ...that may represent the possible location of tumour suppressor genes in human cancer. Screening of the prostate cancer cell lines LNCaP, PC3 and DU145 allowed the mapping of specific regions where genome copy number appeared altered and led to the identification of two novel regions of complete loss at 17q21.31 (500 kb spanning STAT3) and at 10q23.1 (50-350 kb spanning SFTPA2) in the PC3 cell line.
RNAi screening holds the promise of systemizing the search for combination therapeutic strategies. Here we performed a pooled shRNA library screen to look for promising targets to inhibit in ...combination with inhibition of the mitotic regulator polo-like kinase (PLK1). The library contained ~4,500 shRNAs targeting various signaling and cancer-related genes and was screened in four lung cancer cell lines using both high (IC80) and low (IC20) amounts of the PLK1 inhibitor GSK461364. The relative abundance of cells containing individual shRNAs following drug treatment was determined by microarray analysis, using the mock treatment replicates as the normalizing reference. Overall, the inferred influences of individual shRNAs in both high and low drug treatment were remarkably similar in all four cell lines and involved a large percentage of the library. To investigate which functional categories of shRNAs were most prominent in influencing drug response, we used statistical analysis of microarrays (SAM) in combination with a filter for genes that had two or more concordant shRNAs. The most significant functional categories that came out of this analysis included receptor tyrosine kinases and nuclear hormone receptors. Through individual validation experiments, we determined that the two shRNAs from the library targeting the nuclear retinoic acid receptor gene RARA did indeed silence RARA expression and as predicted conferred resistance to GSK461364. This led us to test whether activation of RARA receptor with retinoids could sensitize cells to GSK461364. We found that retinoids did increase the drug sensitivity and enhanced the ability of PLK1 inhibition to induce mitotic arrest and apoptosis. These results suggest that retinoids could be used to enhance the effectiveness of GSK461364 and provide further evidence that RNAi screens can be effective tools to identify combination target strategies.
Breast and ovarian cancer Wooster, Richard; Weber, Barbara L
The New England journal of medicine,
2003-Jun-05, Letnik:
348, Številka:
23
Journal Article
Aurora kinases play critical roles in mitosis and are being evaluated as therapeutic targets in cancer. GSK1070916 is a potent, selective, ATP competitive inhibitor of Aurora kinase B and C. ...Translation of predictive biomarkers to the clinic can benefit patients by identifying the tumors that are more likely to respond to therapies, especially novel inhibitors such as GSK1070916.
59 Hematological cancer-derived cell lines were used as models for response where in vitro sensitivity to GSK1070916 was based on both time and degree of cell death. The response data was analyzed along with karyotype, transcriptomics and somatic mutation profiles to determine predictors of response.
20 cell lines were sensitive and 39 were resistant to treatment with GSK1070916. High chromosome number was more prevalent in resistant cell lines (p-value = 0.0098, Fisher Exact Test). Greater resistance was also found in cell lines harboring polyploid subpopulations (p-value = 0.00014, Unpaired t-test). A review of NOTCH1 mutations in T-ALL cell lines showed an association between NOTCH1 mutation status and chromosome number (p-value = 0.0066, Fisher Exact Test).
High chromosome number associated with resistance to the inhibition of Aurora B and C suggests cells with a mechanism to bypass the high ploidy checkpoint are resistant to GSK1070916. High chromosome number, a hallmark trait of many late stage hematological malignancies, varies in prevalence among hematological malignancy subtypes. The high frequency and relative ease of measurement make high chromosome number a viable negative predictive marker for GSK1070916.
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