Optimisation and Application of Comparative Genomic Hybridisation (CGH) in Cancer Cytogenetics

Kim, Mee Hye (2000) Optimisation and Application of Comparative Genomic Hybridisation (CGH) in Cancer Cytogenetics. PhD thesis, University of Glasgow.

Abstract

The aim of this study was to assess to the applicability of the CGH technique to analysis of various kinds of malignancies in particular hematological malignancies and solid tumours, and to optimise a CGH technique for use on very small amounts of DNA from paraffin embedded tissue. During the initial part of the study several FISH experiments were also performed. First, interphase FISH with selected chromosome specific alpha satellite probes was applied to 22 cases with acute lymphoblastic leukemia, to investigate whether this could contribute to the detection of hidden hyperdiploidy or detect aneuploidy in cases where cytogenetic analysis had failed. Then CGH and interphase FISH were both performed in 19 cases with acute lymphoblastic leukemia to compare the efficiency of the two approaches. CGH showed potential as a comprehensive screening method for detection of hyperdiploid cases and additionally unbalanced DNA copy number changes including one amplification on 6p23-pter were detected. Interphase FISH also might be a complementary method to confirm CGH results, especially in hyperdiploid or hypotriploid cases which failed by conventional karyotyping. CGH analysis in twenty-two cases of AML, MDS or MDS in transformation, revealed that net gains and losses of chromosomal material could be detected more simply and accurately by CGH than by conventional karyotyping in cases with a complex karyotype. CGH followed by FISH using specific probes which were chosen according to discordance between CGH and conventional karyotyping could identify the origin of marker chromosomes, and CGH also could detect amplification sites where candidate genes related to pathogenesis of cancer might be harbored. A further fourteen cases of other hematological malignancies were analysed by CGH and another hidden abnormality which was missed by conventional karyotyping was detected in 1 case. Next, CGH experiments moved to fresh frozen solid tumours. Five cases of well differentiated liposarcoma and seventeen cases of malignant melanoma were analysed. In well differentiated liposarcoma CGH could detect additional amplifications including one amplification (3ql2-ql3.3) which was hitherto unreported and helped to evaluate the composition of marker chromosomes. In cases with malignant melanoma, CGH could identify frequently involved regions of gains and losses, the inter-relationship between abnormalities, and found three new sites of amplification (1 pl 1.1-p12, 3p24, and 22) and amplification on 17q24-qter in malignant melanoma. The CGH technique was next applied to very small amounts of DNA from paraffin-embedded tissue of solid tumours. For success of this experiment, several steps from the extraction of DNA to CGH itself had to be optimised. Although it would have been ideal to move to the next step after completing the optimisation of one step, most experiments involving the extraction of DNA went on to attempt CGH because these test samples were precious material and the final goal of this experiment was to obtain successful CGH result. For obtaining an adequate quantity of high quality of DNA, fifteen different parameters were investigated to discover the optimal method for extraction of DNA from paraffin-embedded tissue. According to this optimisation process, in a very small sample (1 x 7mum) a single step cell lysis method (without phenol/chloroform extraction) and higher dosage of proteinase K (1-2 mg/ml) with shorter duration (≤24 hrs) showed better results. For whole genome amplification, two methods - primer extension preamplification (PEP) and degenerate oligonucleotide primed-PCR (DOP-PCR) were used. DOP-PCR was performed with 8 different minor modifications. The DOP-PCR method without disruption from start to finish was the better method for avoidance of contamination and cases using thermosequenase showed better results than those using combination of topoisomerase and T7 sequenase. PEP was performed with several modifications to assess if whole genome amplification (WGA) using PEP could produce analysable CGH images and if so what volume of starting template DNA could produce CGH images by this method. Another aim was to evaluate the difference of results in comparison with DOP-PCR. Although PEP produced some successful CGH images, it showed some limitation to amplify a sufficient amount of DNA to produce reliable CGH from very small samples. By comparing results between both methods, there are some discrepancies of amplification pattern. After the optimisation process was almost completed, this technique was applied to three kinds of serially diluted samples (DNA from normal female blood, abnormal bone marrow with AML/MDS, and thick-sectioned paraffin-embedded ovarian cancer tissue). These experiments showed that the smallest amount of template DNA which could produce reliable CGH images was above 10 pg of DNA from normal female blood, above 50 pg of DNA from abnormal bone marrow with complex chromosome aberrations, and above 100 pg of DNA from thick sectioned paraffin-embedded ovarian cancer tissue. This study also revealed that the application of the same amplification and labelling method to both test and control DNA was the ideal method for decreasing bias developed during the amplification and labelling process.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: J M Connor
• Keywords: Genetics, Biomedical engineering
• Date of Award: 2000
• Depositing User: Enlighten Team
• Unique ID: glathesis:2000-75521
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 19 Nov 2019 19:34
• Last Modified: 19 Nov 2019 19:34
• URI: https://theses.gla.ac.uk/id/eprint/75521