Li Ching-wan. / Thesis submitted in: November 2001. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 104-130). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.i / ABSTRACTS (ENGLISH/CHINESE) --- p.iii / LIST OF FIGURES --- p.xi / LIST OF TABLES --- p.xiii / LIST OF ABBREVIATIONS --- p.xiv / Chapter CHAPTER1 --- INTRODUCTION / Chapter 1.1. --- Liver Cancer --- p.1 / Chapter 1.2. --- Hepatocellular Carcinoma --- p.1 / Chapter 1.2.1. --- Types of Liver Cancer --- p.1 / Chapter 1.2.2. --- Epidemiology --- p.4 / Chapter 1.2.2.1. --- Geographical Distribution --- p.4 / Chapter 1.2.2.2. --- Age and Gender Distribution --- p.8 / Chapter 1.2.3. --- Etiologic Factors --- p.9 / Chapter 1.2.3.1. --- Chronic Infection with Hepatitis B (HBV) and C (HCV) Viruses --- p.9 / Chapter 1.2.3.2. --- Aflatoxin B1 --- p.11 / Chapter 1.2.3.3. --- Alcohol --- p.12 / Chapter 1.2.3.4. --- Summary --- p.12 / Chapter 1.3. --- HCC in Hong Kong --- p.14 / Chapter 1.4. --- Role of Viral Hepatitis B in HCC --- p.17 / Chapter 1.4.1. --- HBV Genome --- p.17 / Chapter 1.4.2. --- Consequences of HBV DNA Integration --- p.17 / Chapter 1.4.2.1. --- HBV Integration --- p.17 / Chapter 1.4.2.2. --- Transactivation of Cellular Genes by HBV DNA --- p.19 / Chapter 1.4.2.3. --- Chromosomal DNA Instability --- p.20 / Chapter 1.5. --- Genetic Alterations in HCC --- p.21 / Chapter 1.5.1. --- Tumor Suppressor Gene --- p.21 / Chapter 1.5.2. --- Proto-oncogene --- p.23 / Chapter 1.5.3. --- Genetic Studies in HCC --- p.23 / Chapter 1.5.3.1. --- Loss of Heterozygosity (LOH) --- p.25 / Chapter 1.5.3.2. --- Comparative Genomic Hybridization (CGH) --- p.26 / Chapter 1.5.3.3. --- Array CGH --- p.26 / Chapter 1.5.4. --- Large-Scale Genetic Analysis in HCC --- p.27 / Chapter CHAPTER2 --- RATIONALE IN THIS STUDY --- p.35 / Chapter CHAPTER3 --- MATERIALS AND METHODS / Chapter 3.1. --- Patients and Materials --- p.38 / Chapter 3.1.1. --- DNA Extraction --- p.40 / Chapter 3.2. --- Loss of Heterozygosity Analysis on Chromosome 4q --- p.40 / Chapter 3.2.1. --- Microsatellite Markers --- p.41 / Chapter 3.2.2. --- Amplification of Target Sequences by PCR --- p.42 / Chapter 3.2.2.1. --- 5-end Labeling Primers --- p.42 / Chapter 3.2.2.2. --- Amplification of Target Sequences --- p.42 / Chapter 3.2.3. --- Denaturing Polyacrylamide Gel --- p.44 / Chapter 3.2.3.1. --- Electrophoresis --- p.44 / Chapter 3.2.4. --- Detection of Loss of Heterozygosity (LOH) --- p.45 / Chapter 3.2.5. --- Duplex PCR Analysis of Homozygous Deletion --- p.45 / Chapter 3.3. --- Amplification Analysis by Array-CGH --- p.46 / Chapter 3.3.1. --- Nick-Translation --- p.49 / Chapter 3.3.2. --- Hybridization --- p.49 / Chapter 3.3.3. --- Imaging and Data Analysis --- p.50 / Chapter 3.3.4. --- Determination of Normal Range for All Cases --- p.51 / Chapter 3.3.5. --- Assessment of Data Quality --- p.51 / Chapter 3.4. --- Statistical Analysis --- p.52 / Chapter CHAPTER4 --- RESULTS / Chapter 4.1. --- Loss of Heterozygosity Analysis on Chromosome 4q --- p.53 / Chapter 4.1.1. --- Region I of Smallest Common Deletion Region --- p.54 / Chapter 4.1.2. --- Region II of Smallest Common Deletion Region --- p.54 / Chapter 4.2. --- Amplification Analysis by Array-CGH --- p.62 / Chapter CHAPTER5 --- DISCUSSION / Chapter 5.1. --- LOH Analysis on Chromosome 4q --- p.73 / Chapter 5.1.1. --- LOH of Chromosome 4q in Various Cancers --- p.74 / Chapter 5.1.1.1. --- Hepatocellular Carcinomas --- p.74 / Chapter 5.1.1.2. --- Other Neoplasia --- p.76 / Chapter 5.1.2. --- Functional Studies on Chromosome 4 --- p.76 / Chapter 5.1.3. --- Putative Tumor Suppressors on Chromosome 4q --- p.80 / Chapter 5.1.3.1. --- Region I (4q27-q28.1) --- p.80 / Chapter 5.1.3.1.1. --- MAD2L1 (4q27) --- p.80 / Chapter 5.1.3.2. --- Region II (4q35.2) --- p.81 / Chapter 5.1.3.2.1. --- INGlL(4q35.1) --- p.81 / Chapter 5.1.3.2.2. --- FAT (4q34-q35) --- p.81 / Chapter 5.1.3.2.3. --- Caspase 3 (4q35) --- p.82 / Chapter 5.1.4. --- Limitation of this Study --- p.83 / Chapter 5.1.4.1. --- Markers --- p.83 / Chapter 5.1.4.1.1. --- Limitation of the Markers --- p.83 / Chapter 5.1.4.1.2. --- Location of the Microsatellite Markers --- p.83 / Chapter 5.1.4.2. --- Tissue Samples --- p.84 / Chapter 5.1.4.2.1. --- Normal Reference --- p.84 / Chapter 5.1.4.2.2. --- Pathologic Characterization --- p.85 / Chapter 5.1.5. --- Future Studies --- p.85 / Chapter 5.1.5.1. --- Improvement of the Experiment --- p.85 / Chapter 5.1.5.2. --- Extension of the Present Study --- p.86 / Chapter 5.2. --- Amplification Analysis by Array-CGH --- p.88 / Chapter 5.2.1. --- Amplicons Showing Amplification in HCC --- p.89 / Chapter 5.2.1.1. --- Locus of 17q23 --- p.89 / Chapter 5.2.1.1.1. --- D17S1670 --- p.89 / Chapter 5.2.1.1.2. --- RPS6KB1 --- p.91 / Chapter 5.2.1.2. --- Locus of 1q25-q31 --- p.92 / Chapter 5.2.1.2.1. --- LAMC2 --- p.92 / Chapter 5.2.1.3. --- Locus of 3q26.3 --- p.93 / Chapter 5.2.1.3.1. --- PIK3CA --- p.93 / Chapter 5.2.1.4. --- Locus of 8p22 --- p.94 / Chapter 5.2.1.4.1. --- CTSB --- p.94 / Chapter 5.2.1.5. --- Locus of 6q22 --- p.95 / Chapter 5.2.1.5.1. --- MYB --- p.95 / Chapter 5.2.1.6. --- Locus of 20ql3 --- p.96 / Chapter 5.2.1.6.1. --- CSE1L --- p.96 / Chapter 5.2.1.7. --- Locus of Ip36.2-p35.1 --- p.97 / Chapter 5.2.1.7.1. --- FGR --- p.97 / Chapter 5.2.1.8. --- Locus of 7q21.1 --- p.98 / Chapter 5.2.1.8.1. --- PGY1 --- p.98 / Chapter 5.2.2. --- Amplicons Showing Deletion in HCC --- p.99 / Chapter 5.2.2.1. --- Loss at 11ql3 and 14q32.3 --- p.99 / Chapter 5.2.3. --- Limitation of the Study --- p.100 / Chapter 5.2.3.1. --- Samples and Materials --- p.100 / Chapter 5.2.4. --- Further Study --- p.101 / Chapter 5.2.4.1. --- Confirmation of the Result in Various Levels --- p.101 / Chapter 5.2.4.2. --- Assessment of the Significant Losses on Chromosomes 11ql3 and 14ql3 --- p.101 / Chapter 5.2.5. --- Application of Microarray in Genetic Studies --- p.102 / Chapter 5.2.5.1. --- Deletion Analysis --- p.102 / Chapter 5.2.5.2 --- Tissue Microarray --- p.103 / Chapter 5.2.5.3. --- cDNA Microarray --- p.103 / Chapter chapter6 --- references --- p.104

OBJETIVO: A perda de heterozigosidade (LOH) é capaz de avaliar as alterações genéticas de lesões potencialmente malignas. Este ensaio avalia as regiões cromossômicas polimórficas que estão próximas ou na região dos oncogenes e genes supressores de tumor conhecidos. Os objetivos desta tese foram três principais: 1) Avaliar a frequência de perda de heterozigosidade de leucoplasias bucais com diferentes graus de severidade histopatológico em regiões cromossômicas próximas aos genes supressores de tumores. 2) Comparar e correlacionar o perfil de perda de heterozigosidade entre indivíduos da British Columbia (Canadá) e Rio Grande do Sul (Brasil). 3) Avaliar os danos ao DNA que podem ocorrer durante o processamento e armazenamento das amostras de tecido parafinado. MÉTODOS: Amostras de leucoplasia bucal (com e sem displasias), fixadas em formalina tamponada 10% e parafinadas, obtidas nos laboratório de patologia bucal do Canadá e do Brasil foram selecionadas e microdissectadas. Procedeu-se a extração de DNA, amplificação por PCR das seguintes regiões microssatélites: 4q (D4S243, FABP2), 9p21 (IFNA, D9S171, D9S1748, D9S1751), 17p11.2 (CHRNB1) e 17p13.1 (tp53 e D17S786). Após o produto do PCR foi separado e visualizado em gel de poliacrilamida por autoradiografia. RESULTADOS: Observou-se uma forte correlação entre o perfil de perda de heterozigosidade entre indivíduos com leucoplasia bucal de ambos os países, independentemente da etnicidade. Além disso, pode-se notar que amostras de tecidos parafinados submetidos a mais de 24 horas de fixação em formalina tamponada 10% não serão, em sua maioria, boas amostras para análises de DNA. CONCLUSÃO: As lesões potencialmente malignas, provavelmente não são influenciadas em sua etiopatogênia pelas diferenças étnicas. O modelo de risco genético validado por Zhang e colaboradores (2012) parece ser aplicável em nossa comunidade, sendo necessário a sua validação, respeitando procedimentos técnicos padronizados. Ainda, vale ressaltar, que é imprescindível que a comunidade científica passe a adotar metodologias que preservem o material genético das peças dos bancos de tecidos parafinados, que são de inestimável valor para a pesquisa biomédica. / OBJECTIVE: Loss of heterozygosity (LOH) can evaluate genetic alterations of pre-malignant lesions. This assay evaluates the chromosomal polymorphic regions that are present in tumor suppressor genes and oncogenes. The main objectives of this thesis were: 1) Evaluate the frequency of LOH of oral leukoplakias with different histopathological degrees at chromosomal regions of tumor suppressor genes. 2) Compare the profile of LOH between individuals from British Columbia (Canada) and Rio Grande do Sul (Brazil). 3) Evaluate the DNA damage that may occur with FFPE (formalin-fixed paraffin-embedded) tissues. METHODS: FFPE samples of oral leukoplakia (with and without dysplasia), obtained in Canadian and Brazilian oral pathology laboratories were selected and microdissected. DNA extraction and PCR amplification of the following microsatellite regions were conducted: 4q (D4S243, FABP2), 9p21 (IFNA, D9S171, D9S1748, D9S1751), 17p11.2 (CHRNB1) and 17p13.1 (tp53 and D17S786). PCR products were separated and visualized on polyacrylamide gel by autoradiography. RESULTS: A strong correlation between the LOH profile among individuals with oral leukoplakia from both countries was observed, regardless ethnicity. Furthermore, FFPE tissues subjected to more than 24 hours of fixation in 10% buffered formalin are not, generally, good samples for DNA analysis. CONCLUSION: Pre-malignant lesions etiopathogenesis may not be influenced by ethnicity. The genetic risk model validated by Zhang et al. (2012) seems to be applicable in our community, requiring its own validation, respecting standardized procedures. Still, it is important to emphasize that it is imperative that a scientific community adopts methodologies that preserve the genetic material FFPE tissues that are an invaluable resource for biomedical research.

<p>In diploid yeast cells, mitotic recombination is very important for repairing double-strand breaks (DSB). When repair of a DSB results in crossovers, it may cause loss of heterozygosity (LOH) of markers centromere-distal to the DSB in both daughter cells. Gene conversion events unassociated with crossovers cause LOH for an interstitial section of a chromosome. Alternatively, DSBs can initiate break-induced replication (BIR), causing LOH in only one of the daughter cells. Mapping mitotic LOH contributes to understanding of mechanisms for repairing DSBs and distribution of these recombinogenic lesions. Methods for selecting mitotic crossovers and mapping the positions of crossovers have recently been developed in our lab. Our current approach uses a diploid yeast strain that is heterozygous for about 55,000 SNPs, and employs SNP-Microarrays to map LOH events throughout the genome. These methods allow us to examine selected crossovers on chromosome V and unselected mitotic recombination events (crossovers, gene conversion events unassociated with crossovers, and BIR events) at about 1 kb resolution across the genome.</p><p>Mitotic recombination can be greatly induced by UV radiation. However, prior to my research, the nature of the recombinogenic lesions and the distribution of UV-induced recombination events were relatively uncharacterized. Using SNP microarrays, we constructed maps of UV-induced LOH events in G1-synchronized cells. Mitotic crossovers were stimulated 1500-fold and 8500-fold by UV doses of 1 J/m2 and 15 J/m2, respectively, compared to spontaneous events. Additionally, cells treated with 15 J/m2 have about eight unselected LOH events per pair of sectors, including gene conversions associated and unassociated with crossovers as well as BIR events. These unselected LOH events are distributed randomly throughout the genome with no particular hotspots; however, the rDNA cluster was under-represented for the initiation of crossover and BIR events. Interestingly, we found that a high fraction of recombination events in cells treated with 15 J/m2 reflected repair of two sister chromatids broken at roughly the same position. In cells treated with 1 J/m2, most events reflect repair of a single broken sister chromatid (Chapter 2). </p><p>The primary pathway to remove pyrimidine dimers introduced by UV is the nucleotide excision repair (NER) pathway. In NER, the dimer is excised to generate a 30-nucleotide gap that can be replicated to form DSBs if not filled in before DNA replication. The NER gap can also be expanded by Exo1p to form single stranded gaps greater than one kilobase. Alternatively, in the absence of NER, unexcised dimers could result in blocks of DNA replication forks. Resolving the stalled replication fork could lead to recombinogenic breaks. In Chapter 3 and Chapter 4, we analyzed recombination events in strains defective in various steps of processing of UV-induced DNA damage, including exo1 and rad14 mutants. </p><p>In Chapter 3, I show that Exo1p-expanded NER gaps contribute to UV-induced recombination events. Interestingly, I also found that Exo1p is also required for the hotspot activity of a spontaneous crossover hotspot involving a pair of inverted Ty repeats. In addition to its role of expanding a nick to a long single-stranded gap, Exo1p is also a major player in DSB end resection. Therefore, I examined the gene conversion tract lengths in strains deleted for EXO1. I found that, although crossover-associated gene conversion tracts become shorter in the exo1 mutant as expected, noncrossover tract lengths remained unaffected. As a result, noncrossover tracts are longer than crossover tracts in the exo1 mutant while the opposite result was observed in the wild-type strains. I proposed models to rationalize this observation.</p><p>In Chapter 4, to investigate whether the substantial recombinogenic effect in UV in G1-synchronized cells requires NER, we mapped UV-induced LOH events in NER-deficient rad14 diploids treated with 1 J/m2. Mitotic recombination between homologs was greatly stimulated, which suggests that dimers themselves can also cause recombination without processing by NER. We further show that UV-induced inter-homolog recombination events (noncrossover, crossover and BIR) depend on the resolvase Mus81p, and are suppressed by Mms2p-mediated error-free post-replication repair pathway. </p><p>The research described in Chapters, 2, 3, and 4 are in the publications Yin and Petes (2013), Yin and Petes (2014), and Yin and Petes (2015), respectively.</p> / Dissertation

Epigenetic differences between maternally inherited and paternally inherited chromosomes, such as CpG methylation, render the maternal and paternal genome functionally inequivalent, a phenomenon called genomic imprinting. This functional inequivalence is exemplified with imprinted genes, whose expression is parent-of-origin specific. The dosage of imprinted gene expression is disrupted in cells with uniparental disomy (UPD), which is an unequal parental contribution to the genome. I have derived mouse embryonic stem (ES) cell sub-lines with maternal UPD (mUPD) for mouse chromosome 6 (MMU6) to characterize regulation and maintenance of imprinted gene expression. The main finding from this study is that maintenance of imprinting in mitotic UPD is extremely variable. Imprint maintenance was shown to vary from gene to gene, and to vary between ES cell lines depending on the mechanism of loss of heterozygosity (LOH) in that cell line. Certain genes analyzed, such as Peg10, Sgce, Peg1, and Mit1 showed abnormal expression in ES cell lines for which they were mUPD. These abnormal expression levels are similar to that observed in ES cells with meiotically-derived full genome mUPD (parthenogenetic ES cells). Imprinted CpG methylation at the Peg1 promoter was found to be abnormal in all sub-lines with mUPD for Peg1. Two cell sub-lines which incurred LOH through mitotic recombination showed hypermethylation of Peg1, consistent with the presence of two maternal alleles. Surprisingly, a cell sub-line which incurred LOH through full chromosome duplication/loss showed hypomethylation of Peg1. The levels of methylation observed in these sub-lines correlates with expression, as the first two sub-lines showed a near-consistent reduction of Peg1, while the latter showed Peg1 levels close to wild-type. Altogether these results suggest that certain imprinted genes, like Peg1 and Peg10, have stricter imprinting maintenance, and as a result show abnormal expression in UPD. This strict imprint maintenance is disrupted, however, in UPD incurred through full chromosome duplication/loss, possibly because of the trisomic intermediate stage which occurs in this mechanism.

Epigenetic differences between maternally inherited and paternally inherited chromosomes, such as CpG methylation, render the maternal and paternal genome functionally inequivalent, a phenomenon called genomic imprinting. This functional inequivalence is exemplified with imprinted genes, whose expression is parent-of-origin specific. The dosage of imprinted gene expression is disrupted in cells with uniparental disomy (UPD), which is an unequal parental contribution to the genome. I have derived mouse embryonic stem (ES) cell sub-lines with maternal UPD (mUPD) for mouse chromosome 6 (MMU6) to characterize regulation and maintenance of imprinted gene expression. The main finding from this study is that maintenance of imprinting in mitotic UPD is extremely variable. Imprint maintenance was shown to vary from gene to gene, and to vary between ES cell lines depending on the mechanism of loss of heterozygosity (LOH) in that cell line. Certain genes analyzed, such as Peg10, Sgce, Peg1, and Mit1 showed abnormal expression in ES cell lines for which they were mUPD. These abnormal expression levels are similar to that observed in ES cells with meiotically-derived full genome mUPD (parthenogenetic ES cells). Imprinted CpG methylation at the Peg1 promoter was found to be abnormal in all sub-lines with mUPD for Peg1. Two cell sub-lines which incurred LOH through mitotic recombination showed hypermethylation of Peg1, consistent with the presence of two maternal alleles. Surprisingly, a cell sub-line which incurred LOH through full chromosome duplication/loss showed hypomethylation of Peg1. The levels of methylation observed in these sub-lines correlates with expression, as the first two sub-lines showed a near-consistent reduction of Peg1, while the latter showed Peg1 levels close to wild-type. Altogether these results suggest that certain imprinted genes, like Peg1 and Peg10, have stricter imprinting maintenance, and as a result show abnormal expression in UPD. This strict imprint maintenance is disrupted, however, in UPD incurred through full chromosome duplication/loss, possibly because of the trisomic intermediate stage which occurs in this mechanism.

Malignant hyperthermia is a potentially fatal pharmacological disorder and is triggered by volatile anaesthetics in predisposed individuals. Mutations in the RYR1 gene, encoding the skeletal muscle calcium receptor channel have been linked to MH susceptibility. Over 200 point mutations have been have been found to date in the RYR1 gene linked to MHS worldwide. EBV-immortalization is regularly used worldwide as an effective procedure for inducing long-term growth of human B lymphocytes. In the current study, it was observed that immortalized lymphocytes from MHS patients heterozygous for the missense mutation H4833Y when initially cultured expressed both wild type and mutant allele but after a few weeks of culture they seemed to lose the mutant allele. High resolution melting assays and hybridization probe assays showed the loss of heterozygosity and this was confirmed using DNA sequencing. Genotyping and haplotype analysis using three intragenic RFLPs and two (CA)n repeat microsatellite markers tightly linked to the RYR1 gene showed a definite change in the haplotype, suggesting more widespread changes in the genome upon short-term culture of EBV-immortalized B-lymphocytes

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 6 uppsatser.

OBJETIVO: A perda de heterozigosidade (LOH) é capaz de avaliar as alterações genéticas de lesões potencialmente malignas. Este ensaio avalia as regiões cromossômicas polimórficas que estão próximas ou na região dos oncogenes e genes supressores de tumor conhecidos. Os objetivos desta tese foram três principais: 1) Avaliar a frequência de perda de heterozigosidade de leucoplasias bucais com diferentes graus de severidade histopatológico em regiões cromossômicas próximas aos genes supressores de tumores. 2) Comparar e correlacionar o perfil de perda de heterozigosidade entre indivíduos da British Columbia (Canadá) e Rio Grande do Sul (Brasil). 3) Avaliar os danos ao DNA que podem ocorrer durante o processamento e armazenamento das amostras de tecido parafinado. MÉTODOS: Amostras de leucoplasia bucal (com e sem displasias), fixadas em formalina tamponada 10% e parafinadas, obtidas nos laboratório de patologia bucal do Canadá e do Brasil foram selecionadas e microdissectadas. Procedeu-se a extração de DNA, amplificação por PCR das seguintes regiões microssatélites: 4q (D4S243, FABP2), 9p21 (IFNA, D9S171, D9S1748, D9S1751), 17p11.2 (CHRNB1) e 17p13.1 (tp53 e D17S786). Após o produto do PCR foi separado e visualizado em gel de poliacrilamida por autoradiografia. RESULTADOS: Observou-se uma forte correlação entre o perfil de perda de heterozigosidade entre indivíduos com leucoplasia bucal de ambos os países, independentemente da etnicidade. Além disso, pode-se notar que amostras de tecidos parafinados submetidos a mais de 24 horas de fixação em formalina tamponada 10% não serão, em sua maioria, boas amostras para análises de DNA. CONCLUSÃO: As lesões potencialmente malignas, provavelmente não são influenciadas em sua etiopatogênia pelas diferenças étnicas. O modelo de risco genético validado por Zhang e colaboradores (2012) parece ser aplicável em nossa comunidade, sendo necessário a sua validação, respeitando procedimentos técnicos padronizados. Ainda, vale ressaltar, que é imprescindível que a comunidade científica passe a adotar metodologias que preservem o material genético das peças dos bancos de tecidos parafinados, que são de inestimável valor para a pesquisa biomédica. / OBJECTIVE: Loss of heterozygosity (LOH) can evaluate genetic alterations of pre-malignant lesions. This assay evaluates the chromosomal polymorphic regions that are present in tumor suppressor genes and oncogenes. The main objectives of this thesis were: 1) Evaluate the frequency of LOH of oral leukoplakias with different histopathological degrees at chromosomal regions of tumor suppressor genes. 2) Compare the profile of LOH between individuals from British Columbia (Canada) and Rio Grande do Sul (Brazil). 3) Evaluate the DNA damage that may occur with FFPE (formalin-fixed paraffin-embedded) tissues. METHODS: FFPE samples of oral leukoplakia (with and without dysplasia), obtained in Canadian and Brazilian oral pathology laboratories were selected and microdissected. DNA extraction and PCR amplification of the following microsatellite regions were conducted: 4q (D4S243, FABP2), 9p21 (IFNA, D9S171, D9S1748, D9S1751), 17p11.2 (CHRNB1) and 17p13.1 (tp53 and D17S786). PCR products were separated and visualized on polyacrylamide gel by autoradiography. RESULTS: A strong correlation between the LOH profile among individuals with oral leukoplakia from both countries was observed, regardless ethnicity. Furthermore, FFPE tissues subjected to more than 24 hours of fixation in 10% buffered formalin are not, generally, good samples for DNA analysis. CONCLUSION: Pre-malignant lesions etiopathogenesis may not be influenced by ethnicity. The genetic risk model validated by Zhang et al. (2012) seems to be applicable in our community, requiring its own validation, respecting standardized procedures. Still, it is important to emphasize that it is imperative that a scientific community adopts methodologies that preserve the genetic material FFPE tissues that are an invaluable resource for biomedical research.

OBJETIVO: A perda de heterozigosidade (LOH) é capaz de avaliar as alterações genéticas de lesões potencialmente malignas. Este ensaio avalia as regiões cromossômicas polimórficas que estão próximas ou na região dos oncogenes e genes supressores de tumor conhecidos. Os objetivos desta tese foram três principais: 1) Avaliar a frequência de perda de heterozigosidade de leucoplasias bucais com diferentes graus de severidade histopatológico em regiões cromossômicas próximas aos genes supressores de tumores. 2) Comparar e correlacionar o perfil de perda de heterozigosidade entre indivíduos da British Columbia (Canadá) e Rio Grande do Sul (Brasil). 3) Avaliar os danos ao DNA que podem ocorrer durante o processamento e armazenamento das amostras de tecido parafinado. MÉTODOS: Amostras de leucoplasia bucal (com e sem displasias), fixadas em formalina tamponada 10% e parafinadas, obtidas nos laboratório de patologia bucal do Canadá e do Brasil foram selecionadas e microdissectadas. Procedeu-se a extração de DNA, amplificação por PCR das seguintes regiões microssatélites: 4q (D4S243, FABP2), 9p21 (IFNA, D9S171, D9S1748, D9S1751), 17p11.2 (CHRNB1) e 17p13.1 (tp53 e D17S786). Após o produto do PCR foi separado e visualizado em gel de poliacrilamida por autoradiografia. RESULTADOS: Observou-se uma forte correlação entre o perfil de perda de heterozigosidade entre indivíduos com leucoplasia bucal de ambos os países, independentemente da etnicidade. Além disso, pode-se notar que amostras de tecidos parafinados submetidos a mais de 24 horas de fixação em formalina tamponada 10% não serão, em sua maioria, boas amostras para análises de DNA. CONCLUSÃO: As lesões potencialmente malignas, provavelmente não são influenciadas em sua etiopatogênia pelas diferenças étnicas. O modelo de risco genético validado por Zhang e colaboradores (2012) parece ser aplicável em nossa comunidade, sendo necessário a sua validação, respeitando procedimentos técnicos padronizados. Ainda, vale ressaltar, que é imprescindível que a comunidade científica passe a adotar metodologias que preservem o material genético das peças dos bancos de tecidos parafinados, que são de inestimável valor para a pesquisa biomédica. / OBJECTIVE: Loss of heterozygosity (LOH) can evaluate genetic alterations of pre-malignant lesions. This assay evaluates the chromosomal polymorphic regions that are present in tumor suppressor genes and oncogenes. The main objectives of this thesis were: 1) Evaluate the frequency of LOH of oral leukoplakias with different histopathological degrees at chromosomal regions of tumor suppressor genes. 2) Compare the profile of LOH between individuals from British Columbia (Canada) and Rio Grande do Sul (Brazil). 3) Evaluate the DNA damage that may occur with FFPE (formalin-fixed paraffin-embedded) tissues. METHODS: FFPE samples of oral leukoplakia (with and without dysplasia), obtained in Canadian and Brazilian oral pathology laboratories were selected and microdissected. DNA extraction and PCR amplification of the following microsatellite regions were conducted: 4q (D4S243, FABP2), 9p21 (IFNA, D9S171, D9S1748, D9S1751), 17p11.2 (CHRNB1) and 17p13.1 (tp53 and D17S786). PCR products were separated and visualized on polyacrylamide gel by autoradiography. RESULTS: A strong correlation between the LOH profile among individuals with oral leukoplakia from both countries was observed, regardless ethnicity. Furthermore, FFPE tissues subjected to more than 24 hours of fixation in 10% buffered formalin are not, generally, good samples for DNA analysis. CONCLUSION: Pre-malignant lesions etiopathogenesis may not be influenced by ethnicity. The genetic risk model validated by Zhang et al. (2012) seems to be applicable in our community, requiring its own validation, respecting standardized procedures. Still, it is important to emphasize that it is imperative that a scientific community adopts methodologies that preserve the genetic material FFPE tissues that are an invaluable resource for biomedical research.

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Breast cancer (BC) is one of the most common malignancies in women. Death results from treatment failure and metastatic disease. Thousands of lives might be saved if it was possible to detect and eliminate occult metastatic cells before they become clinically evident. Therefore, there is a critical need to identify new markers to improve treatment options for these patients. Genomic instability is the earliest indication of breast cancer and the use of genomic methodologies is a progress towards early detection and treatment, through the identification of biomarkers that can be translated into novel therapy targets. The interferon regulatory factor-1(IRF-1) gene, localized on chromosome 5q31.1, is believed to act as a tumor suppressor gene in breast cancer. The IRF-1 was found to be inactivated by single nucleotide polymorphism (SNP) in breast cancer suggesting that the loss of its function might be critical to the development of the disease. The phosphatidylinositol 3-kinase (PIK3) signaling pathway mediates key cellular functions and alterations of genes in this pathway, including PIK3CA, serine-threonine protein kinases (AKT1and AKT2), phosphatase and tensin homolog (PTEN), fibroblast growth factor receptor 2 (FGFR2) and ERBB2, whose expression have been demonstrated to be altered in breast cancer patients. In addition, these genes are linked to treatment resistance. vi In this study, we have investigated allelic loss of IRF-1 gene in primary tumors obtained from patients undergoing mastectomy at Groote Schuur hospital (Cape Town, South Africa). These samples were then further analyzed for the DNA copy number changes of specific genes involved in the PIK3/AKT signaling pathway. Statistical analysis has been performed in order to correlate genomic findings with clinical-histopathological and follow up information from the patients and to establish whether these genes can predict prognosis. Our data analysis has indicated that 46 cases (45.5%) out of 101 cases were informative for the IRF-1 dinucleotide marker used for LOH analysis (Figure 3.1). LOH was detected in 23 of these informative cases (23/46; 50%). No statistical significance was found between LOH at the IRF-1 locus and age (≤50 years or >50 years) (P value = 1.0000) and earlier stage (Stages I and II) (P value= 0.4982) based on Fisher’s exact test. Patients presented a high level of DNA copy number changes in genes involved in the PIK3/AKT pathway. The most frequent changes were observed in the PIK3CA and PTEN genes. PIK3CA presented high copy number in 36.8% of the cases. PTEN was observed with low copy number in 47.5% of the cases. This dissertation shows the effectiveness of genomic methodologies as means for the detection of early breast cancer progression in South African women. The PIK 3/AKT genes can validate the usefulness of breast cancer therapies.