Contemporary proximity extension assays (PEAs) are used for qualitative proteinquantifications in serological samples, with possibilities for scaling assays in multiplex. Medical research can however benefit from robust immunoassays functional for assessingprotein levels in other types of biospecimens. Formalin-fixed paraffin embedded (FFPE)tissues have long been used for morphological studies. The proteome encapsulated byextensive cross-linking from formalin fixation has however impeded the development ofproteomic analysis from the vast biorepositories FFPE-tissues constitute. In this study, Ipresent a proof of concept for assessing FFPE-samples in multiplex format through PEA.Furthermore, a homogenization and protein extraction protocol for assessing fresh-frozentissue with PEA is presented, together with a novel sample buffer for which remarkable risesin protein detection can be seen in several protein assays. Together, these findings extend theapplication area of PEA to tissues together with improved quantification characteristics.

RNA molecules isolated from FFPE samples are highly fragmented and modified, and generally deemed unsuitable for downstream gene expression profiling. With the development of molecular biology, there has been growing interest in profiling archival FFPE samples. Successful profiling of transcripts from FFPE samples would greatly expand tissue sources for large scale gene expression studies; also it would pave the way for future applications on the type of tissue readily available in the clinical setting. So far, there is a lack of systemic studies evaluating the quality of RNA isolated from routinely processed FFPE samples, and it has remained difficult to assess how well FFPE-derived RNA mirrors the status of RNA isolated before fixation. In this project, the similarity of miRNA and mRNA profiles between matched frozen and FFPE lymphoid hyperplasia tissues (N=7 for miRNA comparison, N=4 for mRNA comparison) were evaluated. We found consistently good correlation (mean of Pearson coefficient=0.939, mean of Spearman coefficient=0.905, mean of Kendall tau=0.744) between matched frozen and FFPE-derived miRNA profiles, suggesting FFPE samples may retain miRNA expression information quite well. This has major positive implications for research using FFPE samples, as miRNA profiling becomes more prominent in bioprofiling studies. On the contrary, mRNA isolated from FFPE samples showed less correlation (Spearman coefficient less than 0.75) with its frozen counterpart on the Agilent microarray platform. With a post extraction heat treatment aimed at reversing base modifications and cross linking structures, obvious global mRNA quality improvement was observed in cases where samples appeared to be heavily cross linked, but was less effective and even detrimental in cases where cross linking was less prominent. This research suggests that the extent of cross linking may be critical in terms of determining whether a particular FFPE tissue will become a useful source of mRNA for global profiling studies / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2008-09-26 10:49:50.044

Formalin-fixed, paraffin embedded (FFPE) tissue is the most abundant, accessible, and versatile tissue sample type available for genetic research and clinical applications. However, FFPE DNA extraction presents unique challenges and requires lengthy incubation periods, which can be impractical for certain applications. Here, we propose the use of focused ultrasound extraction (FUSE) technology for improved DNA extraction from FFPE tissue. FUSE generates a dense bubble cloud of acoustic cavitation capable of ablating tissue into an acellular lysate. FUSE treatment was applied to de-paraffinized porcine pancreas FFPE scrolls, followed by heated incubation for formaldehyde-induced DNA-protein crosslink reversal. When applied for 30 minutes, FUSE was found to successfully extract DNA from FFPE tissue as defined by increased DNA yield and improved purity ratios compared to conventional methods. DNA extracted via FUSE showed comparable fragmentation to conventional methods, and three out of four samples successfully amplified via PCR, indicating suitability for downstream analysis. These findings suggest that FUSE has the potential to increase the efficiency and effectiveness of DNA extraction from FFPE tissue. Further development and optimization of this protocol could develop a streamlined, easy to use extraction method that would simplify FFPE DNA extraction methods and address the primary time constraints which currently make FFPE DNA extraction time-consuming and impracticable for high-throughput applications. / Master of Science / Formalin-fixed, paraffin embedding (FFPE) has historically been the most popular method of biological tissue preservation, as it allows tissue to remain shelf stable for decades. As such, FFPE tissue is the most abundant, accessible, and versatile tissue sample type available for genetic research applications. Here, we propose the use of focused ultrasound extraction (FUSE) technology for improved DNA extraction from FFPE tissue. FUSE treatment applies rapid, focused ultrasound waves to tissue, resulting in the mechanical breakdown of cells and subsequent release of DNA. FUSE treatment was applied to pig pancreatic FFPE samples. When applied for 30 minutes, FUSE was found to successfully extract DNA from FFPE tissue as defined by increased DNA yield and improved purity compared to conventional methods. Three out of four DNA samples extracted via FUSE were successfully amplified, and DNA fragment lengths were comparable between FUSE and conventional methods, showing that FUSE did not fragment DNA beyond useful fragment lengths. These findings suggest that FUSE has the potential to increase the efficiency and effectiveness of DNA extraction from FFPE tissue. Further development and optimization of this protocol could develop a streamlined, easy to use extraction method that would simplify FFPE DNA extraction methods and address the primary time constraints which currently make FFPE DNA extraction time-consuming and impracticable for high-throughput applications.

Formaldehyde is widely used fixative. Its advantages are low cost, simplicity of use and good fixation traits, which are fast tissue penetration, good preservation of morphological structures and compatibility with downstream histological applications. Formaldehyde disadvantages are negative effects on nucleic acids. Formaldehyde solutions modify primary structure of deoxyribonucleic acid (DNA), fragment DNA and create protein-DNA covalent bonds that hinder DNA isolation procedures. Level of negative effects of formaldehyde is dependent on many factors. Effect of formaldehyde chemical composition (formaldehyde dilution, presence of buffer or formic acid) and effect of fixation length were studied in this work. On DNA extracted from fixed tissues, DNA quantity and level of DNA fragmentation were studied by quantitative polymerase chain reaction, fluorescence assay for DNA quantification and by on- chip electrophoresis on bioanalyzer Agilent 2100. Quality and quantity of acquired DNA were tested by DNA profile determination for identification purposes using STR (short tandem repeats) analysis. Results show that of all tested fixatives, buffered 4% formaldehyde is the most suited solution in regards of sufficient amount of DNA and sufficient DNA quality. Other formaldehyde variants (non-buffered 4%...

O câncer colorretal (CCR) é a segunda de causa morte relacionada ao câncer, com aproximadamente 500.000 mortes por ano em países desenvolvidos. Os pacientes com doença localmente avançada ou metastática possuem um prognóstico ruim e, desta forma, a detecção precoce é necessária para reduzir a alta mortalidade associada à essa doença. Ainda, os métodos de rastreamento utilizados no momento apresentam boa exatidão, porém são de alto custo, consomem muito tempo e são incômodos para os pacientes. A colonoscopia é o método mais comumente utilizado para a detecção de CCR. Entretanto, apresentam duas limitações importantes: i) necessidade da luz intestinal estar limpa, sem resíduos fecais, o que só é conseguido após um preparo rigoroso, com dieta e laxativos, nos dias que antecedem a realização do exame, e, ii) é examinador-dependente, ou seja, dependem da experiência do profissional em localizar as alterações anatômicas, e ainda da habilidade em selecionar o melhor local para a realização da biópsia. Portanto, o desenvolvimento de um método rápido e sensível para o diagnóstico do câncer colorretal é extremamente importante.<br /> A proposta desta dissertação foi desenvolver um método eficaz para o preparo dos tecidos parafinizados e extração de proteínas para experimentos em eletroforese bidimensional (2DE). Tecidos fixados em formaldeído e embebidos em parafina apresentam como vantagens a preservação da qualidade morfológica, abundância e facilidade de armazenamento, o que permite estudos futuros. Entretanto, para análises proteômicas, a recuperação do material biológico é ineficiente. A partir deste estudo, 156 proteínas foram obtidas de tecidos parafinizados, sendo possível identificar por espectrometria de massas, levando a estudos posteriores para identificação de possíveis biomarcadores relacionados ao CCR. / Colorectal cancer (CRC) is the second cause of cancer-related death, with approximately 500,000 deaths per year in developed countries. Patients with locally advanced or metastatic disease have a poor prognosis and, thus, early detection is needed to reduce the high mortality associated with this disease. Yet, the screening methods used at the time have good accuracy, but are expensive, time consuming and are uncomfortable for patients. Colonoscopy is the most commonly used method for the detection of CRC. However, present two major limitations: i) the need to be clean intestinal lumen without fecal waste, which is achieved only after rigorous preparation with laxatives and diet, in the days before the exam, and ii) it is examiner-dependent, i.e., depends on the professional\'s experience in locating anatomical changes, and even the ability to select the best site for biopsy. Therefore, the development of a rapid and sensitive method for the diagnosis of colorectal cancer is extremely important. The purpose of this dissertation was to develop an effective method for the preparation of tissues and protein extraction experiments for two-dimensional electrophoresis (2DE). Tissues fixed in formaldehyde and embedded in paraffin have the advantage of preserving the morphological quality, abundance and storage facility, which allows for further studies. However, for proteomic analysis, the recovery of biological material is inefficient. From this study, 156 proteins were obtained from paraffinized tissues, and can be identified by mass spectrometry, leading to further studies to identify potential biomarkers related to CCR.

O câncer colorretal (CCR) é a segunda de causa morte relacionada ao câncer, com aproximadamente 500.000 mortes por ano em países desenvolvidos. Os pacientes com doença localmente avançada ou metastática possuem um prognóstico ruim e, desta forma, a detecção precoce é necessária para reduzir a alta mortalidade associada à essa doença. Ainda, os métodos de rastreamento utilizados no momento apresentam boa exatidão, porém são de alto custo, consomem muito tempo e são incômodos para os pacientes. A colonoscopia é o método mais comumente utilizado para a detecção de CCR. Entretanto, apresentam duas limitações importantes: i) necessidade da luz intestinal estar limpa, sem resíduos fecais, o que só é conseguido após um preparo rigoroso, com dieta e laxativos, nos dias que antecedem a realização do exame, e, ii) é examinador-dependente, ou seja, dependem da experiência do profissional em localizar as alterações anatômicas, e ainda da habilidade em selecionar o melhor local para a realização da biópsia. Portanto, o desenvolvimento de um método rápido e sensível para o diagnóstico do câncer colorretal é extremamente importante.<br /> A proposta desta dissertação foi desenvolver um método eficaz para o preparo dos tecidos parafinizados e extração de proteínas para experimentos em eletroforese bidimensional (2DE). Tecidos fixados em formaldeído e embebidos em parafina apresentam como vantagens a preservação da qualidade morfológica, abundância e facilidade de armazenamento, o que permite estudos futuros. Entretanto, para análises proteômicas, a recuperação do material biológico é ineficiente. A partir deste estudo, 156 proteínas foram obtidas de tecidos parafinizados, sendo possível identificar por espectrometria de massas, levando a estudos posteriores para identificação de possíveis biomarcadores relacionados ao CCR. / Colorectal cancer (CRC) is the second cause of cancer-related death, with approximately 500,000 deaths per year in developed countries. Patients with locally advanced or metastatic disease have a poor prognosis and, thus, early detection is needed to reduce the high mortality associated with this disease. Yet, the screening methods used at the time have good accuracy, but are expensive, time consuming and are uncomfortable for patients. Colonoscopy is the most commonly used method for the detection of CRC. However, present two major limitations: i) the need to be clean intestinal lumen without fecal waste, which is achieved only after rigorous preparation with laxatives and diet, in the days before the exam, and ii) it is examiner-dependent, i.e., depends on the professional\'s experience in locating anatomical changes, and even the ability to select the best site for biopsy. Therefore, the development of a rapid and sensitive method for the diagnosis of colorectal cancer is extremely important. The purpose of this dissertation was to develop an effective method for the preparation of tissues and protein extraction experiments for two-dimensional electrophoresis (2DE). Tissues fixed in formaldehyde and embedded in paraffin have the advantage of preserving the morphological quality, abundance and storage facility, which allows for further studies. However, for proteomic analysis, the recovery of biological material is inefficient. From this study, 156 proteins were obtained from paraffinized tissues, and can be identified by mass spectrometry, leading to further studies to identify potential biomarkers related to CCR.

Formaldehyde is widely used fixative. Its advantages are low cost, simplicity of use and good fixation traits, which are fast tissue penetration, good preservation of morphological structures and compatibility with downstream histological applications. Formaldehyde disadvantages are negative effects on nucleic acids. Formaldehyde solutions modify primary structure of deoxyribonucleic acid (DNA), fragment DNA and create protein- DNA covalent bonds that hinder DNA isolation procedures. Level of negative effects of formaldehyde is dependent on many factors. Effect of formaldehyde chemical composition (formaldehyde dilution, presence of buffer or formic acid) and effect of fixation length were studied in this work. On DNA extracted from fixed tissues, DNA quantity and level of DNA fragmentation were studied by quantitative polymerase chain reaction, fluorescence assay for DNA quantification and by on-chip electrophoresis on bioanalyzer Agilent 2100. Quality and quantity of acquired DNA were tested by DNA profile determination for identification purposes using STR (short tandem repeats) analysis. Results show that of all tested fixatives, buffered 4% formaldehyde is the most suited solution in regards of sufficient amount of DNA and sufficient DNA quality. Other formaldehyde variants (non-buffered 4%...

Head and neck cancer is the 6th most common malignancy worldwide, with tumours of the tongue being one of the most prevalent sites. Despite advances in surgery and radiotherapy, the five-year survival has not changed during the last decades and remains at approximately 50%. Identification of novel biomarkers for more personalized treatment is important for increasing survival in these patients. One of the most commonly used methods in the search for new biomarkers is microarray analysis. A substantial limitation with this technique is the requirement for fresh frozen samples from which high quality RNA can be extracted. This becomes particularly problematic when attempting to discover differences associated with individual sub-types or rare cancers. Recent developments, including the DASL microarray platform, have provided the possibility of analysing RNA of poorer quality from formalin fixed paraffin embedded (FFPE) samples. FFPE is the standard way of preserving tissue from patients and millions of samples are stored around the world. In this thesis we have evaluated the use of FFPE samples and global gene expression profiling for increasing basic knowledge in a subgroup of oral cancer patients with tumours of the tongue. As confirmation of microarray results using qPCR is of outmost importance for conclusive data evaluation, we first aimed at finding a housekeeping gene stably expressed across malignant and non-malignant FFPE oral tissue. TUBA6, which belongs to the tubulin family was detected as being the most stable out of eight possible genes and was thus used for qPCR normalization throughout the following studies. We have performed three separate microarray experiments. Initially only a focused DASL array covering 502 cancer related genes was available and we used it to analyze a smaller cohort of patients and controls (n=36). A similar cohort (n=29) was also analyzed for expression of 836 micoRNAs. In 2009 a whole genome DASL array was launched, covering over 20,000 genes, and all tongue tumour samples available between 1997 and 2010 (n=87) were analysed using this array. Similar to other research groups we observed very high replicate reproducibility using both DASL arrays. When using the microRNA array and the whole genome DASL array an effect of sample quality on the detected expression level of individual genes was noticed. While the expression of some genes severely decreased with a decrease in sample quality others were not changed. This will impair normalization, leading to a residual non-biological variation within the data. Based on our findings we have presented some recommendations for minimizing the effect of sample quality and maximizing the level of biologically relevant information obtained from these experiments, e.g. ensuring that samples in groups to be compared are of the same quality range. For the microRNA data we also introduced an additional normalization step to the standard normalizations. We could show that lists of differentially expressed genes generated when taking these precautions were enriched for genes involved in cancer related processes and contained for tongue carcinoma previously identified changes. A number of differentially expressed genes, novel for tongue carcinoma, were also confirmed in high quality fresh frozen samples, including BCL2A1 (apoptosis), CXCL10 (immune response), SLC2A6 (energy transport) and miR-424 (angiogenesis). In conclusion microarrays can be used to analyze FFPE samples but should be performed with care. Standard normalization methods will not remove the variation introduced by samples being of different quality, leading to spurious results. Taking a few precautions, however, led to the identification of differentially expressed genes relevant in tumour development and maintenance. The recommendations we make can facilitate design of future studies using FFPE samples. The genes we identified as being differentially expressed in tumour tissue now need to be further evaluated for their potential as biomarkers in tongue carcinoma.

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Philosophiae Doctor - PhD / Tumour-specific protein markers are usually present at elevated concentrations in patient biopsy tissue; therefore tumour tissue is an ideal biological material for studying cancer proteomics and biomarker discovery studies. To understand and elucidate cancer pathogenesis and its mechanisms at the molecular level, the collection and characterisation of a large number of individual patient tissue cohorts are required. Since most pathology institutes routinely preserve biopsy tissues by standardised methods of formalin fixation and paraffin embedment, these archived, FFPE tissues are important collections of pathology material, often accompanied by important metadata, such as patient medical history and treatments. FFPE tissue blocks are conveniently stored under ambient conditions for decades, while retaining cellular morphology due to the modifications induced by formalin. / 2022