| 1. |
- Ahmadian, Afshin, et al.
(författare)
-
Genetic instability in the 9q22.3 region is a late event in the development of squamous cell carcinoma.
- 1998
-
Ingår i: Oncogene. - : Springer Science and Business Media LLC. - 0950-9232 .- 1476-5594. ; 17:14
-
Tidskriftsartikel (refereegranskat)abstract
- Squamous cell carcinoma (SCC) of the skin represents a group of neoplasms which is associated with exposure to UV light. Recently, we obtained data suggesting that invasive skin cancer and its precursors derive from one original neoplastic clone. Here, the analysis were extended by loss of heterozygosity (LOH) analysis in the chromosome 9q22.3 region. A total of 85 samples, taken from twenty-two sections of sun-exposed sites, corresponding to normal epidermis, morphological normal cells with positive immuno-staining for the p53 protein (p53 patches), dysplasias, cancer in situ (CIS) and squamous cell carcinomas (SCC) of the skin were analysed. Overall, about 70% of p53 patches had mutations in the p53 gene but not LOH in the p53 gene or 9q22.3 region. Approximately 70% of the dysplasias showed p53 mutations of which about 40% had LOH in the p53 region but not in the 9q22.3 region. In contrast, about 65% of SCC and CIS displayed LOH in the 9q22.3 region, as well as frequent (80%) mutations and/or LOH in the p53 gene. These findings strongly suggest that alterations in the p53 gene is an early event in the progression towards SCC, whereas malignant development involves LOH and alterations in at least one (or several) tumor suppressor genes located in chromosome 9q22.3.
|
|
| 2. |
- Odeberg, J, et al.
(författare)
-
Context-dependent Taq-polymerase-mediated nucleotide alterations, as revealed by direct sequencing of the ZNF189 gene : implications for mutation detection.
- 1999
-
Ingår i: Gene. - 0378-1119 .- 1879-0038. ; 235:1-2
-
Tidskriftsartikel (refereegranskat)abstract
- We have recently reported on the genetic organisation of a novel Krüppel-like zinc finger, ZNF189, located to 9q22-q31. In that study we found no mutations in the coding sequence when using ZNF189 as a candidate gene for sporadic basal cell cancer and squamous cell cancer. Here, by direct sequencing of the proximal promotor of ZNF189, mutations were found to appear in a small hot-spot region in over 50% of analysed tumour samples, the majority being G to A substitutions. The hot-spot region spans a 24bp G-rich region. Repeated analyses of the original sample lysates fail to confirm each of these mutations; and frequently new mutations appear at neighbouring positions. Subsequent analysis with serial dilutions of genomic DNA and a cosmid harbouring the wild-type ZNF189 gene demonstrate that these sequence-specific alterations arise in the outer PCR-amplification when 50 copies or less of template are used. Although the mechanism of how these context-specific alterations arise is not proven, the results demonstrate a previously unreported type of PCR-mediated sequence-specific alteration that easily could have been interpreted as being of clinical relevance. The phenomena observed show that mutations detected by direct sequencing can be caused by PCR-introduced alterations. Consequently, this should be of general caution in mutation analysis of disease gene candidates when using small amounts of template, such as microdissected biopsies.
|
|
| 3. |
- Pontén, F, et al.
(författare)
-
Genomic analysis of single cells from human basal cell cancer using laser-assisted capture microscopy.
- 1997
-
Ingår i: Mutation research. - 0027-5107 .- 1873-135X. ; 382:1-2
-
Tidskriftsartikel (refereegranskat)abstract
- In this study, we show that direct mutational analysis of genomic DNA can be performed on single somatic cells extracted from a frozen, immunohistochemically stained tissue section using laser-assisted capture microscopy. Eighty-nine single tumor cells were separately dissected from one case of human basal cell cancer (BCC) and p53 mutations were analyzed by direct semi-automated sequencing of PCR fragments. Amplification was obtained for at least one of the two analyzed exons from approximately 50% of the single tumor cells. Identical p53 mutations were found in widely spread areas of the tumor, suggesting a clonal proliferation originating from one cell. Interestingly, comparison between results of immunohistochemistry and genetic analysis of the single cells revealed the same p53 mutations irrespective of the p53 immunoreactivity. We propose that this approach has a great potential to allow investigation of genotypic differences in single cells and more specifically to resolve important and fundamental questions determining cancer heterogeneity.
|
|
| 4. |
- Williams, Cecilia, 1969-, et al.
(författare)
-
A high frequency of sequence alterations is due to formalin fixation of archival specimens.
- 1999
-
Ingår i: American Journal of Pathology. - 0002-9440 .- 1525-2191. ; 155:5
-
Tidskriftsartikel (refereegranskat)abstract
- Genomic analysis of archival tissues fixed in formalin is of fundamental importance in biomedical research, and numerous studies have used such material. Although the possibility of polymerase chain reaction (PCR)-introduced artifacts is known, the use of direct sequencing has been thought to overcome such problems. Here we report the results from a controlled study, performed in parallel on frozen and formalin-fixed material, where a high frequency of nonreproducible sequence alterations was detected with the use of formalin-fixed tissues. Defined numbers of well-characterized tumor cells were amplified and analyzed by direct DNA sequencing. No nonreproducible sequence alterations were found in frozen tissues. In formalin-fixed material up to one mutation artifact per 500 bases was recorded. The chance of such artificial mutations in formalin-fixed material was inversely correlated with the number of cells used in the PCR-the fewer cells, the more artifacts. A total of 28 artificial mutations were recorded, of which 27 were C-T or G-A transitions. Through confirmational sequencing of independent amplification products artifacts can be distinguished from true mutations. However, because this problem was not acknowledged earlier, the presence of artifacts may have profoundly influenced previously reported mutations in formalin-fixed material, including those inserted into mutation databases.
|
|
| 5. |
- Williams, Cecilia, 1969-, et al.
(författare)
-
Assessment of sequence-based p53 gene analysis in human breast cancer : messenger RNA in comparison with genomic DNA targets.
- 1998
-
Ingår i: Clinical Chemistry. - : Oxford University Press (OUP). - 0009-9147 .- 1530-8561. ; 44:3
-
Tidskriftsartikel (refereegranskat)abstract
- The high prevalence of p53 mutations in human cancers and the suggestion from several groups that the presence or absence of p53 mutations might have both prognostic and therapeutic consequences point to the importance of optimal methods for p53 determination. Several strategies exploring this have been described, based either on mRNA or genomic DNA as a template. However, no comparative study on the reliability of the two templates has been performed. The principal aim of this study was to study the concordance of RNA- and DNA-based direct sequencing methods in detecting p53 mutations in breast tumors. In 100 tumors, 22 mutations were detected by both methods. Furthermore, one stop mutation, two splice-site mutations, and one intron alteration were found only by genomic sequencing. In addition, the comparative study suggests that cells with missense mutations have increased steady-state concentrations of p53-specific mRNA, in contrast to cells with a gene encoding a truncated protein.
|
|
| 6. |
|
|
| 7. |
- Williams, Cecilia, 1969-, et al.
(författare)
-
Clones of normal keratinocytes and a variety of simultaneously present epidermal neoplastic lesions contain a multitude of p53 gene mutations in a xeroderma pigmentosum patient.
- 1998
-
Ingår i: Cancer Research. - 0008-5472 .- 1538-7445. ; 58:11
-
Tidskriftsartikel (refereegranskat)abstract
- A patient with xeroderma pigmentosum group C was extensively examined for mutations in the p53 gene in normal skin exposed to varying degrees of sunlight and in excisional biopsies of basal cell cancer, squamous cell cancer, and squamous cell dysplasia. Seventy-three samples were analyzed by microdissection of small cell clusters, followed by PCR and direct DNA sequencing. In skin taken from areas that most likely had never been exposed to the sun, no mutations were found. However, in skin exposed to the sun, we observed a multitude of mutations in the p53 gene. UV light-induced mutations were found in all types of lesions, as well as in clusters of morphologically normal epidermal cells. Twenty-nine distinct mutations were found in exons 5-8, all missense or nonsense, of which 27 (93%) were UV-specific C --> T or CC --> TT transitions at dipyrimidine sites of the nontranscribed strand. Two types of normal skin areas containing p53 mutations were observed: areas that stain strongly with p53 antibody (p53 patches) and those that do not stain. Because no silent or intron mutations were found in these cell clusters, the alterations in the p53 gene of morphologically normal cells are likely to have resulted in a selective growth advantage. The poor correlation between mutations and morphological phenotypes demonstrates that p53 mutations alone do not determine the phenotypes observed.
|
|
| 8. |
- Williams, Cecilia
(författare)
-
Molecular archaeology of cancer
- 1999
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Alterations of the p53 tumor suppressor gene plays animportant role in the development of many types of cancers.This and the suggestion that the presence or absence of p53mutations might have both prognostic and therapeuticconsequences, imply the importance of reliable techniques forp53 sequence determination. This thesis describes thedevelopment, evaluation and applications of such methods.Initially, a method for multiplex amplification of the p53gene, was designed and applied on microdissected tumorbiopsies. This approach reduced the required number of cellsfor analysis and minimized any variation in the amplificationsof individual p53 exons. Amplified fragments were analyzed bydirect DNA sequencing in a semi-automated mode. Analysis of theHLA locus was included for control of sample identity. Using amodified version of this method in combination with amicroscope equipped with a laser to assist microdissectionenabled the analysis of single somatic cells from histologictissue sections. The concordance and benefits of basing theanalysis on p53 gene transcripts (mRNA) or its genomiccounterpart (DNA) as templates were evaluated in a double blindanalysis of 100 breast tumors. The results showed that somestop and splice site mutations found in the analysis of genomicDNA were non-detectable in the analysis of mRNA. Further, astudy comparing the analysis of frozen material with formalinfixed archival specimens was performed, concluding that a highfrequency of artifact sequence alterations occur when formalinfixed tissues are used. A related type of artifact mutationsappeared in the analysis of a novel skin cancer candidate gene(ZNF189). We could demonstrate that these artifacts arise inthe PCR-amplification when very few and/or damaged copies oftemplate are used.This method was subsequently applied in the analysis of skincancer. The frequency of p53 mutations, clonality of andgenetic progression in human basal cell cancer (BCC) wereinvestigated. By following a strategy of "molecular archeology"we showed that several BCCs were heterogeneous, containedmultiple p53 mutations and exhibited genetic progression. Next,the correlation between mutations of the p53 gene andmorphological phenotypes of lesions was studied in a patientwith the xeroderma pigmentosum DNA repair disorder. Here, inskin exposed to the sun, we observed a multitude of ultravioletlight-induced mutations in all types of lesions as well as inclusters of morphologically normal epidermal cells. We coulddemonstrate that the alterations in the p53 gene ofmorphologically normal cells were likely to result in aselective growth advantage, and that the poor correlationbetween mutations and morphological phenotypes shows that p53mutations alone do not determine the phenotypes observed.Keywords:p53, DNA sequencing, mutation, PCR, mRNA,cancer, BCC, genetic progression, formalin fixation, artifact,single cell.© Cecilia Williams, 1999
|
|
| 9. |
- Yngveson, A, et al.
(författare)
-
p53 Mutations in lung cancer associated with residential radon exposure.
- 1999
-
Ingår i: Cancer Epidemiology, Biomarkers and Prevention. - 1055-9965 .- 1538-7755. ; 8:5
-
Tidskriftsartikel (refereegranskat)abstract
- Unusual mutation patterns in lung tumors among underground miners have been indicated, suggesting radon-specific alterations in the genome, but the data are not consistent. To investigate the association between residential radon exposure and p53 mutations in lung tumors, we performed a study on cases from a nation-wide population-based investigation in Sweden. Our study included 83 nonsmoking lung cancer cases and 250 smoking lung cancer cases, diagnosed 1980-1984, with a time-weighted average radon exposure over 140 Bq/m3 or up to 50 Bq/m3. Radon was measured in dwellings occupied by the study subjects at some time since 1947. Information on smoking habits and other risk factors was obtained from questionnaires. After exclusions because of the initiation of treatment or insufficient material, the p53-status of 243 tumors was determined using PCR-single-stranded conformation polymorphism analysis and sequencing determination of exons 5-8. The overall mutation prevalence was 23.9%. An increased mutation prevalence was suggested among those with high exposure to residential radon [odds ratio (OR), 1.4; 95% CI, 0.7-2.6], especially among nonsmokers (OR, 3.2; 95% CI, 0.7-15.5), but no specific mutational pattern was indicated. Furthermore, the mutation prevalence seemed to be higher among smoking lung cancer cases than among nonsmoking cases (OR, 2.1; 95% CI, 0.9-5.0), and particularly among those smoking less than 10 cigarettes per day. It may be concluded that residential exposure to radon seems to contribute to a higher mutation prevalence of the p53 gene in lung tumors.
|
|
