Molecular archaeology of cancer

Författare: Cecilia Williams; Kth; []

Nyckelord: ;

Sammanfattning: 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

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