Analytical and statistical methods to evaluate microsatellite allelic imbalance in small amounts of DNA.
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Microsatellite analysis is a powerful tool for the assessment of genetic instability and loss of heterozygosity in cancer cells. However, most human tumors harbor significant numbers of normal cells, which may contribute to false-negative results. Recent techniques based on fluorescently labeled primers and semiautomated capillary electrophoresis of polymerase chain reaction (PCR) products allow a reliable quantitative assessment of (PCR) products while requiring very small numbers of cells. We report a highly sensitive protocol for the semiautomated analysis of allelic imbalance based on time-release PCR and capillary electrophoresis. With this protocol, as few as 100 cells can be used to reliably assess allelic imbalance (AI) in DNA samples. Using a panel of seven microsatellite markers, we determined allelic variation in a large set of heterozygous lymphocyte DNA samples and examined the use of different statistical analysis techniques. Using these statistical approaches, we describe a calibration method to evaluate AI from microsatellite results. Using a simple formula, cutoff points at preset confidence levels are used to decide whether allelic imbalance exists in a given sample at the loci under investigation. Our method allows the reliable detection of AI with very small amounts of DNA, and is sufficiently quantitative to assess allelic ratios in nonclonal tissue specimens. (+info)
Gene expression alterations over large chromosomal regions in cancers include multiple genes unrelated to malignant progression.
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In solid tumors, the relationship between DNA copy number and global expression over large chromosomal regions has not been systematically explored. We used a 12,626-gene expression array analysis of head and neck squamous cell carcinoma and normal oral mucosa and annotated gene expression levels to specific chromosomal loci. Expression alterations correlated with reported data using comparative genomic hybridization. When genes with significant differences in expression between normal and malignant lesions, as defined by significance analysis of microarrays (SAM), were compared to nonsignificant genes, similar chromosomal patterns of alteration in expression were noted. Individual tumors underwent microsatellite analysis and chi(2) analysis of expression at 3p and 22q. Significant 3p underexpression and 22q overexpression were found in all primary tumors with 3p and 22q allelic imbalance, respectively, whereas no tumor without allelic imbalance on these chromosomal arms demonstrated expression differences. Loss and gain of chromosomal material in solid cancers can alter gene expression over large chromosomal regions, including multiple genes unrelated to malignant progression. (+info)
Allelic imbalance studies of chromosome 9 suggest major differences in chromosomal instability among nonmelanoma skin carcinomas.
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CONTEXT: Loss of heterozygosity in the 9p21-p22 region, has been frequently described in a wide range of human malignancies, including familial melanomas. Also, losses and gains in other regions of chromosome 9 have frequently been observed and may indicate additional mechanisms for basal cell tumorigenesis. OBJECTIVE: To investigate allelic imbalance in the 9p21-p22 region, among basal cell carcinomas. TYPE OF STUDY: Microsatellite analysis. SETTING: Two dermatology services of public universities in Sao Paulo and the Laboratory of Cancer Molecular Genetics of Universidade Estadual de Campinas (Unicamp). PARTICIPANTS: 13 patients with benign skin lesions consecutively referred to the outpatient dermatology clinics of Unicamp and Universidade Estadual de Sao Paulo (Unesp) and 58 with malignant skin tumours. MEAN MEASUREMENTS: We examined 13 benign cases including four of solar keratosis, three keratoachanthomas, three melanocytic nevi, two of Bowen's disease and one of neurofibroma, and 58 malignant skin tumors: 14 of squamous cell, 40 basal cell carcinomas and four melanomas. Participating patients had the main tumor and a normal portion of non-adjacent skin surgically removed. DNA was extracted from the tumor and matching normal tissue. We used four sets of primers to amplify polymorphic microsatellite repeats on chromosome 9, two of them targeting the 9p21-p22 region. RESULTS: We identified eight cases (20%) of allelic imbalance among basal cell carcinomas, two cases of loss of heterozygosity and six cases of microsatellite instability in the 9p21-p22 region. Additional markers were also involved in three of these tumors. No events were detected among the benign or the other malignant cases. CONCLUSION: This phenotype dependency suggests that there is a major distinction between the two most important forms of nonmelanoma skin cancers in their tendency to present microsatellite instability in chromosome 9. Since the CDKN2a/p16INK4a, p19ARF and p15INK4b tumor suppressor genes do not appear to be responsible for the observed abnormalities, other genes at 9p21-p22 may be involved in the pathogenesis and progression pathway of basal cell carcinomas. (+info)
Modulation of patched-associated susceptibility to radiation induced tumorigenesis by genetic background.
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We described previously a basal cell carcinoma (BCC) and medulloblastoma (MB) phenotype for CD1Ptch1(neo67/+) mice exposed to ionizing radiation. Ptch1 heterozygous mice mimic the predisposition to BCC and MB development of patients affected by nevoid BCC syndrome that inherit a mutant Patched (Ptch1) allele. To examine the impact of genetic background on development of BCCs and other tumors we used two outbred mouse lines characterized by extremely high, carcinogenesis-susceptible (Car-S), and low, carcinogenesis-resistant (Car-R), susceptibility to skin carcinogenesis. Crosses between Ptch1(neo67/+) mice and Car-S (F1S) or Car-R mice (F1R) were exposed to ionizing radiation. F1SPtch1(neo67/+) mice were highly susceptible to radiation-induced BCCs, whereas F1RPtch1(neo67/+) mice were completely resistant, indicating that tumor penetrance can be modulated by genetic background. Development of microscopic and macroscopic BCC lesions was influenced by Car-S and Car-R genotypes, suggesting a genetic-background effect on both initiation and progression of BCC. Susceptibility was additionally increased in N2 backcross mice (Car-S x F1SPtch1(neo67/+)), showing a contribution from recessive-acting Car-S modifiers. The modifying effects of Car-S-derived susceptibility alleles were tissue specific. In fact, despite higher susceptibility to BCC induction, Car-S-derived lines had lower MB incidence compared with CD1Ptch1(neo67/+) mice. BCC-associated somatic events were not influenced by genetic background, as shown by similar rate of wild-type Ptch1 loss in BCCs from F1SPtch1(neo67/+) (93%) and CD1Ptch1(neo67/+) mice (100%). Finally, microsatellite analysis of BCCs showed Ptch1 loss through interstitial deletion. These results are relevant to humans, in which BCC is the commonest malignancy, because this model system may be used to study genes modifying BCC development. (+info)
Allelic imbalance on 12q22-23 in serum circulating DNA of melanoma patients predicts disease outcome.
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Allelic imbalance (AI) encompassing the apoptotic protease-activating factor 1 (APAF-1) locus (12q22-23) is found frequently in metastatic melanoma. Circulating DNA with AI on 12q22-23 in serum was evaluated as a surrogate marker to predict biochemotherapy (BC) treatment response in melanoma patients. Sera were collected from 49 American Joint Committee on Cancer stage IV melanoma patients treated with BC. Serum AI of the 12q22-23 region was demonstrated to be present before and/or after BC. BC responders showed a significantly lower frequency of AI (5 of 24, 21%) compared with nonresponders (11 of 20, 55%; Fisher's exact test, P < 0.029). Serum AI on 12q22-23 was associated with worse prognosis (log-rank test, P < 0.046). These findings indicate that serial serum genetic analysis of tumor-related AI on 12q22-23 may have clinical use in predicting tumor response to therapy. (+info)
Allelic imbalance of 8p indicates poor survival in gastric cancer.
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Gastric cancer is a common tumor worldwide and a tremendous health burden. However, the underlying mechanisms of tumorigenesis in this cancer's development are primarily undefined. Allelic imbalance (AI) of 8p has been reported in many cancers, yet, the target(s) of alteration and the importance of allelic imbalance on this chromosomal arm in gastric carcinoma development remained to be characterized. Our findings confirmed a high rate of AI on 8p in gastric cancers. Moreover, we demonstrated that AI on 8p, either overall or at marker D8S560, was associated with poorer survival in patients with gastric cancer. Finally, gastric cancers with a high rate of microsatellite instability were significantly associated with noncardia tumors and with female gender. (+info)
Allelic imbalance at 1p36 may predict prognosis of chemoradiation therapy for bladder preservation in patients with invasive bladder cancer.
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Invasive bladder cancers have been treated by irradiation combined with cis-platinum (CDDP) as a bladder preservative option. The aim of this study was to find a marker for predicting patient outcome as well as clinical response after chemoradiation therapy (CRT) by investigating allelic loss of apoptosis-related genes. A total of 67 transitional cell carcinomas of the bladder treated by CRT (median dose: 32.4 Gy of radiation and 232 mg of CDDP) were studied. We investigated allelic imbalances at 14 loci on chromosomes 17p13 and 1p36 including the p53 and p73 gene regions by fluorescent multiplex PCR based on DNA from paraffin-embedded tumour specimens and peripheral blood. The response to CRT was clinical response (CR) in 21 patients (31%), partial response (PR) in 31 (46%), and no change(NC) in 15 (22%). There was no statistical correlation between treatment response and clinical parameters, such as tumour grade, stage, radiation dose, or CDDP dose. The frequencies of allelic imbalance for TP53 and TP73 were 21 and 56%, respectively; neither was correlated with clinical treatment response and tumour stage or grade. There was no statistical correlation between treatment response and allelic imbalance at the other 12 loci. We found a significant correlation between cancer-specific survival and an imbalance of D1S243 (P=0.0482) or TP73 (P=0.0013) using a Log-rank test, although other loci including TP53 did not correlate with survival (P=0.4529 Multivariate analysis showed performance status (P=0.0047), recurrence (P=0.0017), and radiation doses (P=0.0468) were independent predictive factors for cancer-specific survival. However, an allelic imbalance of TP73 was the most remarkable independent predictive factor of poor patient survival (P=0.0002, risk ratio: 3382). Our results suggest that the allelic loss of the p73 gene predicts a clinical outcome of locally advanced bladder cancer when treated by CRT. (+info)
Influence of template DNA degradation on the genotyping of SNPs and STR polymorphisms from forensic materials by PCR.
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Detection of single nucleotide polymorphisms (SNPs) and short tandem repeat (STR) polymorphisms by PCR is widely used to analyze degraded DNAs in forensic science. The success of DNA analysis from human remains largely depends on the quality of the template DNA. We examined two SNPs (HLA-DQA1 and ABO) and two STR polymorphisms (VWA and CD4) by SSCP gel or denaturing gel electrophoresis, using two kinds of degraded DNA samples (165 teeth and blood stains contaminated with saliva) derived from the same person and investigated the influence of template DNA degradation on genotyping. As the degradation of DNA proceeds, unbalanced amplification of alleles occurred in the analysis of both SNPs and STRs, followed by allele drop, and further by loss of amplification. Non-target allelic products of STRs were amplified from highly degraded DNA samples; however, false allelic products of SNPs were not amplified from them. Amplification efficiency increased in proportion to the decrease of PCR target size, but reduction of the PCR target sizes also increased the chances of amplifying contaminating DNA, especially in highly degraded DNA specimens. The present results will help investigators to evaluate the genotyping of highly degraded DNA samples in forensic casework. (+info)