INTRODUÇÃO: O aprimoramento dos métodos de rastreamento e a conscientização da população geral contribuíram para o diagnóstico cada vez mais precoce do câncer de mama e proporcionou, juntamente com o avanço na terapêutica, altas taxas de sobrevida. O estado do acometimento axilar por metástase é um dos principais fatores prognósticos em pacientes com câncer de mama, particularmente naquelas com diagnóstico em estádio inicial. Na última década, esforços científicos foram direcionados para simplificar a amostragem dos linfonodos axilares, diminuindo a morbidade, mas respeitando os princípios oncológicos. Nesse sentindo, a biópsia do linfonodo sentinela foi considerada o avanço mais importante. Ao se obter um método preditor do estado axilar, que apresente os benefícios da abordagem padrão dissecção axilar e biópsia de linfonodo sentinela porém sem seus efeitos colaterais e que seja facilmente reproduzível, realizaremos um grande avanço na avaliação e terapêutica do câncer de mama inicial. MÉTODOS: Foi realizado estudo transversal retrospectivo com base nos prontuários de pacientes com câncer de mama invasivo, não metastático, com qualquer idade, atendidas entre 1999 e 2007 no Setor de Mastologia da Disciplina de Ginecologia do Departamento de Obstetrícia e Ginecologia da Faculdade de Medicina da Universidade de São Paulo e Clínica Professor José Aristodemo Pinotti, cujo estudo histopatológico e imunoistoquímico foi supervisionado por um único médico patologista. Realizamos uma subdivisão imunoistoquímica dos tumores, sendo considerado luminal A os tumores com receptores hormonais positivos e HER 2 negativo; luminal B os com receptores hormonais positivos e HER 2 positivo; HER 2 as pacientes com receptores hormonais negativos e HER 2 positivo e triplo negativo aquelas com receptores hormonais e HER 2 negativos. Correlacionamos esses subtipos com as variáveis clínicas idade e tamanho tumoral para predizer a probabilidade de acometimento linfonodal axilar. RESULTADOS: Duzentos e trinta e nove casos foram analisados. No subtipo luminal A, a possibilidade de metástase foi maior quanto menor a idade da paciente e maior o tamanho do tumor. Essa foi a única associação que apresentou diferença estatisticamente significante. As pacientes que possuíam tumores triplo negativo tiveram, aproximadamente, 90% menos chance de metástase linfonodal que as pacientes com tumor luminal A. CONCLUSÕES: As pacientes com tumor luminal A apresentaram, significativamente, maior probabilidade de metástase linfonodal axilar. As pacientes com de tumores triplo negativo, com idade superior a 55 anos ou tumores menores que 2 cm, revelaram menor probabilidade de metástase axilar / INTRODUTION: The improvement of screening methods and awareness of general population contributed to the increasingly early diagnosis of breast cancer and provided, together with advances in therapy, high survival rates. The status of axillary involvement is a major prognostic factor in patients with breast cancer, particularly those with early stage. In the last decade, research efforts were directed to simplify the sampling of axillary lymph nodes, decreasing the morbidity, but respecting the oncological principles. In this sense, the sentinel lymph node biopsy was considered the most important advance. If we obtain a method to predict the axillary status, with the benefits of the standard approach - axillary dissection and sentinel lymph node biopsy - without its side effects and easily reproducible, we will hold a major advance in the assessment and treatment of early breast cancer. METHODS: We conducted a retrospective cross-sectional study based on records of patients with invasive breast cancer, non metastatic, with any age, treated between 1999 and 2007 in the breast cancer Sector of the Gynecology Discilpine of the Department of Obstetrics and Gynecology, Faculty of Medicine, University of São Paulo and Private Clinic of Professor José Aristodemo Pinotti, whose histopathological studies were supervised by a single pathologist. We performed an immunohistochemical subdivision of the tumors, and considered luminal A tumors with positive hormonal receptors and negative HER 2, luminal B with positive hormonal receptors and positive HER 2, HER 2 patients with negative hormonal receptors and positive HER 2 and the triple-negative those with negative hormonal receptors and HER 2. Those subtypes were correlated with the clinical variables age and tumor size in predicting the likelihood of axillary lymph node involvement. RESULTS: Two hundred and nine cases were analyzed. In the luminal A, the possibility of axillary metastasis was higher in the younger patients and larger tumors. That was the only combination that showed statistically significant difference. The patients who had triplenegative tumors had approximately 90% less chance of lymph node metastasis than patients with tumors luminal A. CONCLUSIONS: The patients with luminal A tumors showed a significantly association with greater likelihood of axillary lymph node metastasis. The patients with triple negative tumors, age over 55 years or tumors smaller than 2 cm showed a lower likelihood of axillary lymph node metastasis

This thesis is about the use of massive DNA sequencing to investigate the transcriptome. During recent decades, several studies have made it clear that the transcriptome comprises a more complex set of biochemical machinery than was previously believed. The majority of the genome can be expressed as transcripts; and overlapping and antisense transcription is widespread. New technologies for the interroga- tion of nucleic acids have made it possible to investigate such cellular phenomena in much greater detail than ever before. For each application, special requirements need to be met. The work presented in this thesis focuses on the transcrip- tome and the development of technology for its analysis. In paper I, we report our development of an automated approach for sample preparation. The procedure was benchmarked against a publicly available reference data set, and we note that our approach outperformed similar manual procedures in terms of reproducibility. In the work reported in papers II-IV, we used different massive sequencing technologies to investigate the transcriptome. In paper II we describe a concatemerization approach that increased throughput by 65% using 454 sequencing,and we identify classes of transcripts not previously described in Populus. Papers III and IV both report studies based on SOLiD sequencing. In the former, we investigated transcripts and proteins for 13% of the human gene and detected a massive overlap for the upper 50% transcriptional levels. In the work described in paper IV, we investigated transcription in non-genic regions of the genome and detected expression from a high number of previ- ously unknown loci. / QC 20100723

1. Despite extensive research on oral squamous cell carcinoma (OSCC), its five-year survival rate has not improved for the last two decades. Effective treatment of OSCC requires the identification of molecular targets to design appropriate therapeutic strategies. To this end, DDRT-PCR analysis was used to identify molecular markers, which could be used as therapeutic targets. 2. DDRT-PCR in combination with reverse Northern analysis identified 25 differentially expressed genes in oral tumors. Fourteen genes did not show homology to any known gene in the database and therefore may represent non-specific genomic DNA sequences or novel genes that have not yet been identified. The remaining 11 genes showed homology to known genes such as DIAPH1, NJMU-R1, RBM28, PCNA, GLTP, MTATP6, ZKSCAN1, TNKS2, PAM, TUBB2C and C14orf154. TNKS2, PAM, TUBB2C and C14orf154 showed downregulation and the remaining seven genes were upregulated in oral tumor samples. 3. To reconfirm the results of DDRT-PCR and reverse Northern blot analyses, Northern blot analysis was carried out on matched normal and tumor samples for a few genes. As expected, PCNA, NJMU-R1 and ZKSCAN1 showed upregulation, whereas TUBB2C showed downregulation in the tumor sample. PCNA was also found to be upregulated in tumor samples at the protein level. 4. The expression of eight differentially expressed genes (viz., DIAPH1, NJMU-R1, RBM28, PCNA, GLTP, TNKS2, PAM and TUBB2C) was also validated in a panel of 16 matched normal and tumor samples. The mean mRNA expression levels of GLTP, PCNA, RBM28, NJMU-R1 and DIAPH1 were significantly greater in tumor samples than in normal samples. The mean expression levels of TNKS2, PAM and TUBB2C were significantly lower in tumor samples than in normal samples. 5. As some of the genes like NJMU-R1, RBM28, GLTP and PAM are found to differentially regulated in a majority of the tumors, they could be used as potential markers in oral cancer. 6. Tuberin and hamartin have been placed as a complex in the insulin signaling pathway and are known to negatively regulate this pathway. Since overexpression of TSC2 has been previously shown to exert antitumor effect on two oral cancer cell lines, and some components of the insulin signaling pathway have already been implicated in head and neck cancers, we reasoned that both TSC genes and other key players of this pathway might be differentially regulated in oral tumors. Northern blot analysis showed downregulation of the TSC2 gene in an oral tumor sample. In order to further validate the expression pattern of the TSC2 gene, a semiquantative RT-PCR analysis was carried out in a panel of 16 matched normal and tumor samples. The mean expression level of TSC2 was significantly lower in tumor samples than in normal tissue samples. The mean expression level of its interacting partner TSC1 was also significantly lower in tumor samples than in normal tissue samples, suggesting the involvement of these genes in the etiology of oral cancer. TSC1 and TSC2 were also downregulated in eight matched normal and tumor samples at the protein level. We wanted further to determine the expression of both TSC genes in cell lines. Interestingly, TSC2 did not show a detectable level of expression in an oral cancer cell line SCC 131, whereas it was expressed in two other oral cancer cell lines KB and SCC 104 as well as in four non-oral cell lines: A549, HEK-293T, HeLa and HepG2 at the protein level. The TSC2 expression in KB was, however, lower than in other cell lines. TSC1 was expressed in all the cell lines, albeit at different levels. The TSC1 expression was lower in SCC 131 as compared to two other cell lines KB and SCC 104. 7. Given the fact that both are tumor suppressors, it was hypothesized that LOH, inactivating somatic mutations and/or promoter methylation might be playing a role for their downregulation in oral tumors. Mutation analysis of all the coding regions of both the TSC genes failed to detect any mutation in a panel of 25 tumor samples. However, seven normal population variants were identified in different patients. Our analysis of the matched peripheral blood and tumor DNA samples from 52 patients showed LOH at both the TSC loci. At the TSC1 locus, 17/48 (35.42%) tumors showed an allelic loss for one or more markers. At the TSC2 locus, LOH was found in 18/48 (37.5%) informative cases. Nine patients (9/48, 18.75%) had LOH at both the TSC loci. Since PTEN is another tumor suppressor in the insulin signaling pathway, we then sought to determine if LOH is also present in the PTEN candidate region in a panel of 50 matched samples. Microsatellite analysis using three markers showed a low LOH rate of 13% in tumor samples. 8. As the OSCC cell line SCC 131 did not show a detectable level of TSC2 expression, we treated this cell line with methylation inhibition drug 5-azacytidine. The treatment restored the expression of TSC2 and increased the expression of TSC1, suggesting that the promoter methylation and LOH are the important mechanisms for their downregulation. In order to see if the downregulation of the TSC genes is due to their promoters being methylated in tumors from the patients, we examined the methylation status of their promoters in 16 oral tumors, three normal oral tissues, two peripheral blood DNA samples from normal individuals and two cell lines HeLa and SCC 131 by COBRA. Our repeated efforts to amplify the TSC1 promoter using different DNA polymerases failed. However, we were able to successfully amplify the 571 bp long TSC2 promoter. Our analysis showed methylation of the TSC2 promoter in all tumors and two cell lines. As expected, the TSC2 promoter was not methylated in normal oral tissues and control blood DNA samples. Our bisulfite sequencing data suggested a low level and a considerable heterogeneity of methylation. 9. Using Fisher’s exact test, no correlation was found between LOH at the TSC loci and different clinical parameters such as age, sex, T classification, stage, grade, histology, tobacco habits and lymph node metastasis. 10. Using Fisher’s exact test, no correlation was found between the TSC2 promoter methylation and its downregulation in 16 tumor samples. We believe that this could be due to small sample size. 11. Since TSC1 and TSC2 are important regulators of the insulin pathway, it was hypothesized that other key players of this pathway might also be dysregulated in oral cancer. To this end, the expression pattern of some of the major regulators of the insulin pathway (viz., PI3K, AKT, PDK1, RHEB, mTOR, S6K1, S6, eIF4E, 4E-BP1, PTEN, 14-3-3ﾟ and IRS1) was investigated using semiquantative RT-PCR in a panel of 16 matched normal and tumor samples. The mean expression levels of the following genes showed significant upregulation in tumor samples: AKT, PI3K, PDK1, RHEB, mTOR, S6K1, S6 and eIF4E. On the other hand, 4E-BP1 and PTEN showed significant downregulation in tumor tissues. No significant difference in the expression was found for 14-3-3ﾟ and IRS1 between tumor and normal tissues. The expression pattern of some of these genes was also analyzed at the protein level using Western blot analysis and eight matched normal and tumor tissues. The level of total AKT was upregulated in 2/8 tumor samples only. However, phosphorylated-AKT (Thr308) showed upregulation in 6/8 samples. p70S6K1 and phosphorylated-p70S6K1 (Thr389) were upregulated in 8/8 and 6/8 tumor samples, respectively. Increase in the phosphorylated forms of both AKT and its downstream effector p70S6K1 suggested an increase in their kinase activity, indicating a constitutive activation of this pathway in oral cancer. 12. Based on our findings of mutation analysis, LOH study, 5-azacytidine treatment of an oral cancer cell line and COBRA analysis, we suggest that LOH at the TSC gene loci and promoter methylation are important mechanisms for the downregulation of the TSC genes. Loss of function of these genes may thus contribute to the constitutive activation of the insulin signaling pathway in oral cancer, leading to overall cell growth and proliferation. Our studies have shown that several key members of this pathway show aberrant expression in a subset of cancers of the oral cavity and can provide useful therapeutic targets. Several inhibitors of the insulin signaling pathway, such as rapamycin and its derivatives which inhibit mTOR and the PI3K inhibitor wortmannin, are now being actively evaluated for clinical trials for other cancers. We suggest that these inhibitors could also be evaluated for the treatment of oral cancer in future. Our differential display analysis has served to identify several genes that may be important for the onset and progression of oral cancer. Further analysis of these genes is warranted.

Microarray expression analysis was carried out to identify genes with a role in epithelial ovarian cancer (EOC). The U133A Affymetrix GeneChipRTM was used to determine the expression patterns of the 3p25.3-ptel genes represented on the microarray in 14 primary cultures of normal ovarian surface epithelial (NOSE) samples, 25 frozen malignant ovarian tumor samples and four EOC cell lines. Seven genes with differential expression patterns in the tumor samples compared to the NOSE samples were identified as candidates for further analysis, starting with ARPC4, SRGAP3 and ATP2B2. Although none of the candidates had been previously studied in ovarian cancer, several had either family or pathway members that had. Expression patterns seemed unaffected by either tumor histopathological subtype or the allelic imbalances observed with loss of heterozygosity (LOH) analysis. The absence of association with genomic context suggested that differential expression was the result of transcriptional regulation rather than direct targeting.

DNA is the code for all life. Via intermediary RNA the information encoded by the genome is relayed to proteins executing the various functions in a cell. Together, this repertoire of inherently linked biological macromolecules determines all characteristics and features of a cell. Technological advancements during the last decades have enabled the pursuit of novel types of studies and the investigation of the cell and its constituents at a progressively higher level of detail. This has shed light on numerous cellular processes and on the underpinnings of several diseases. For the majority of studies focusing on nucleic acids, an amplification step has to be implemented before an analysis, scoring or interrogation method translates the amplified material into relevant biological information. This information can, for instance, be the genotype of particular SNPs or STRs, or the abundance level of a set of interesting transcripts. As such, amplification plays a significant role in nucleic acid assays. Over the years, a number of techniques – most notably PCR – has been devised to meet this amplification need, specifically or randomly multiplying desired regions. However, many of the approaches do not scale up easily rendering comprehensive studies cumbersome, time-consuming and necessitating large quantities of material.Trinucleotide threading (TnT) – forming the red thread throughout this thesis – is a multiplex amplification method, enabling simultaneous targeted amplification of several nucleic acid regions in a specific manner. TnT begins with a controlled linear DNA thread formation, each type of thread corresponding to a segment of interest, by a gap-fill reaction using a restricted trinucleotide set. The whole collection of created threads is subsequently subjected to an exponential PCR amplification employing a single primer pair. The generated material can thereafter be analyzed with a multitude of readout and detection platforms depending on the issue or characteristic under consideration.TnT offers a high level of specificity by harnessing the inherent specificities of a polymerase and a ligase acting on a nucleotide set encompassing three out of the four nucleotide types. Accordingly, several erroneous events have to occur in order to produce artifacts. This necessitates override of a number of control points.The studies constituting this thesis demonstrate integration of the TnT amplification strategy in assays for analysis of various aspects of DNA and RNA. TnT was adapted for expression profiling of intermediately-sized gene sets using both conventional DNA microarrays and massively parallel second generation 454 sequencing for readout. TnT, in conjunction with 454 sequencing, was also employed for allelotyping, defined as determination of allele frequencies in a cohort. In this study, 147 SNPs were simultaneously assayed in a pool comprising genomic DNA of 462 individuals. Finally, TnT was recruited for parallel amplification of STR loci with detection relying on capillary gel electrophoresis. In all investigations, the material generated with TnT was of sufficient quality and quantity to produce reliable and accurate biological information.Taken together, TnT represents a viable multiplex amplification technique permitting parallel amplification of genomic segments, for instance harboring polymorphisms, or of expressed genes. In addition to these, this versatile amplification module can be implemented in assays targeting a range of other features of genomes and transcriptomes. / QC 20100819

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2007. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 6 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 5 uppsatser.

Colorectal cancer (CRC) is a common malignancy which develops due to old age and lifestyle factors, low percent of patients afflicted by a genetic disorders. Half of all colorectal cancer patients are diagnosed after metastasis. The high rate of the late detection, emphasizes on the requirement of convenient and inexpensive diagnostic methods for comprehensive screening programs. The aim of this study was to discover proto-oncogenes mutation and assessment of tumor suppressor genes expression. Formalin fixed paraffin embedded (FFPE) histologically verified colorectal cancer samples were used. APC, KRAS and BRAF mutations were investigated using polymerase chain reaction (PCR) fragments and direct sequencing. Gene expression assessment of MLH1, MGMT and CDKN2A were achieved via quantitative polymerase chain reaction (qPCR). In the present study we could detect a novel transversion heterozygous mutation in APC gene codon 1365 in three patients. BRAF codon 600 mutation were detected in one patient. KRAS codon 12 mutation was discovered in one sample and also a novel transition mutation in codon 15 was detected in 6 patients. In 80% of cases, MLH1 and MGMT expression were undetectable, in remaining 20%, MLH1 expression were reduced, but MGMT showed both reduced and increased expression compared to control. In 100% of patients CDKN2A expression was undetectable. The rate of mutations in predetermined hotspot codons and amount of uncommon mutations into APC, BRAF and KRAS in Nepalese patients indicates the requirement of further investigation in CRC patients from that part of the world. Also, the expression rate of MLH1, MGMT, CDKN2A and deficiency of an information source emphasizes the necessity of whole genome CRC expression profiling data to comparison and conclusion. / <p>-</p> / -