Background: Lung cancer causes more deaths than any other cancer. Smoking causes roughly 90% of lung cancer cases. Concurrent chemoradiation therapy is the standard of care for stage IIIb patients with performance status (PS) 0-1. A less toxic approach is warranted for less fit patients. To optimize care, the understanding of common clinical variables such as haematological responses to inflammation could be much improved. Adherence to guidelines for proper clinical work-up is vital to ensure patients’ optimal care, especially for predictive assays. Screening of high-risk patients is now being implemented internationally. Chronic pulmonary obstructive disease (COPD) patients, a group at high risk to develop lung cancer, could be of interest for screening. Methods: Patient cohorts collected nationally and regionally by manual search in patient records or automated search in electronic patient records and national registries were analysed in relation to overall survival, comorbidities, medication, treatment, smoking status, biomarkers and adherence to guidelines. Standard statistics were applied to adjust for confounding factors. Results: Induction chemotherapy results in longer overall survival than radiotherapy alone (15.6 and 11.6 months respectively). The overall survival for patients with combined anaemia, leucocytosis and thrombocytosis at diagnosis is half of what could be anticipated if blood samples are normal (8.0 and 16.0 months respectively). Fifty percent of patients were overlooked in the routine work-up with EGFR analysis. Less than 40% of the patients received EGFR-tyrosine kinase inhibitors in first-line therapy. The frequency of EGFR mutation was 9.9%. COPD patients with asthma and medicating with inhaled corticosteroids, specific serotonin reuptake inhibitors (SSRI) or beta-blockers have a significantly decreased risk of lung cancer. Conclusions: Patients unfit to receive chemoradiation therapy should be considered for induction chemotherapy sequentially to radiotherapy. A patient that presents with pathological blood samples is likely to have poor prognosis and diagnostic work-up should be thorough to optimize outcome. Inadequate adherence to the national guidelines regarding treatment and EGFR analysis was shown. COPD patients medicating with ICS, beta-blockers or SSRI and with a concurrent asthma diagnosis have a decreased risk of lung cancer.

Introduction HNF-1β is a transcription factor that plays a crucial role in the ontogenesis, regulates expression of multiple genes involved in cell cycle modulation and seems to be involved in cancerogenesis of various tumors. HNF-1β protein is coded by the HNF1B gene. Genetic and epigenetic changes of HNF1B play role in tumorigenesis and these changes can be accompanied by loss of expression or increased protein expression as detected by immunohistochemistry. In gynecopathology, expression of HNF-1β was considered as specific marker of clear cell carcinomas of the ovary and endometrium. However, more recent studies described HNF-1β expression also in tumors of other histogenesis. Aims: Our study focused on the immunohistochemical and molecular analysis of HNF1B in the normal tissue, various types of tumors and non-neoplastic lesions of the female genital tract. The goals of our study were: 1. Analysis of HNF-1β expression in cervical carcinomas. 2 Analysis of HNF-1β expression in endometrial carcinomas and non-neoplastic tissues of the female genital tract. 3. Analysis of epigenetic and genetic changes of HNF1B in endometrioid carcinomas and ovarian clear cell carcinomas. 4. Comprehensive analysis of atypical polypoid adenomyomas. Material and methods: A total of 574 samples including 399...

Introduction HNF-1β is a transcription factor that plays a crucial role in the ontogenesis, regulates expression of multiple genes involved in cell cycle modulation and seems to be involved in cancerogenesis of various tumors. HNF-1β protein is coded by the HNF1B gene. Genetic and epigenetic changes of HNF1B play role in tumorigenesis and these changes can be accompanied by loss of expression or increased protein expression as detected by immunohistochemistry. In gynecopathology, expression of HNF-1β was considered as specific marker of clear cell carcinomas of the ovary and endometrium. However, more recent studies described HNF-1β expression also in tumors of other histogenesis. Aims: Our study focused on the immunohistochemical and molecular analysis of HNF1B in the normal tissue, various types of tumors and non-neoplastic lesions of the female genital tract. The goals of our study were: 1. Analysis of HNF-1β expression in cervical carcinomas. 2 Analysis of HNF-1β expression in endometrial carcinomas and non-neoplastic tissues of the female genital tract. 3. Analysis of epigenetic and genetic changes of HNF1B in endometrioid carcinomas and ovarian clear cell carcinomas. 4. Comprehensive analysis of atypical polypoid adenomyomas. Material and methods: A total of 574 samples including 399...

Malaria remains one of the world's major health problems, especially in developing countries. A better understanding of the pathology and pathophysiology of severe malaria is key to develop new treatments. Different approaches have been used in malaria research including the in vitro co-culture models with endothelial cells and both murine and simian animal models. However these are open to controversy due to disagreement on their representativeness of human disease. Using human post-mortem tissue in malaria research is another important approach but is practically challenging, limiting the availability of post mortem samples from malaria patients. The work in this thesis had two main themes. First I examined the role of the endothelial signalling Angiopoetin-Tie-2 receptor pathway in malaria. Ang-2 has been shown to be a significant biomarker of severe and fatal malaria. I examined the tissue specific expression of proteins from this pathway in post-mortem brain tissues from fatal malaria cases, but found no difference between cerebral malaria and non-cerebral malaria cases. Ang-2 correlated with the severity of malaria in these patients. An attempt to examine the interaction of hypoxia and the Ang-Tie-2 pathway in vitro using a co-culture model of human brain endothelial cells was unsuccessful due to contamination of the cell line. The second part of the thesis aimed to utilise molecular pathology techniques including miRNA and whole-genome microarrays. I have shown for the first time that these can be successfully applied to human post-mortem tissue in malaria. First I used archival tissues to examine the microRNA signature in the kidney of patients with malaria associated renal failure. Second I optimised a protocol to preserve post mortem tissue for molecular pathology, from an autopsy study in Mozambique. Using the subsequent total mRNA transcriptomic data and bioinformatics analysis this work has expanded our knowledge of differential gene expression and the families of genes which are dysregulated in the brain in response to malaria infection.

Introdução: As malformações congênitas (MCs) são a segunda causa de mortes fetais e infantis no Brasil e, em grande parte dos casos, a sua etiologia não é bem definida. Devido às consequências clínicas das MCs, alguns pacientes falecem sem tempo hábil para uma investigação etiológica acurada. Dessa forma, a maioria dos casos permanece sem uma confirmação molecular das suspeitas clínicas, dificultando o aconselhamento genético para as famílias. Objetivos: O presente trabalho utilizou técnicas citogenômicas para caracterizar molecularmente a presença de anormalidades no DNA, desde aneuploidias até a variação do número de cópias gênicas (CNVs) em diferentes tecidos de pacientes falecidos portadores de MC encaminhados ao Serviço de Verificação de Óbitos para avaliação anatomopatológica. Casuística e Métodos: Foram avaliadas amostras de 30 pacientes portadores de MC submetidos à necropsia. O DNA foi extraido de diferentes tecidos (cérebro, coração, fígado, pele e diafragma) previamente conservados em RNA later, formol ou emblocados em parafina. Foram utilizadas as técnicas de Multiplex Ligation-dependent Probe Amplification (MLPA) com os kits P095, P064 e P070 (MRC-Holland®), Marcadores Microssatélites (MMS) com o kit MiniFiler (Life Technologies®), a Fluorescence in Situ Hybridization (FISH), a técnica de array (Infinium® CytoSNP-850K BeadChip - Illumina) e o Sequenciamento Bidirecional por Sanger. A interpretação dos resultados foi realizada utilizando os softwares GeneMarker, Coffalyser, BlueFuse Multi, Sequencher e com os bancos de dados Database of Genomic Variants (DGV - http://projects.tcag.ca/variation/), Database of Chromosomal Imbalance and Phenotype in Humans Using Ensembl Resources (DECIPHER - http://decipher.sanger.ac.uk/), UCSC Genome Bioinformatics (http://genome.ucsc.edu) e Mutation Taster. Resultados: Dos 30 pacientes avaliados, 13 apresentaram alterações patogênicas. Entre eles, oito apresentaram aneuploidias envolvendo os cromossomos 13, 18, 21, X e Y, sendo dois deles com mosaicismo intratecidual. Quatro pacientes apresentaram microdeleções ou microduplicações envolvendo diferentes genes, sendo um paciente com duplicação do gene TYMS em 18p11.32; um com deleção do gene CHL1 em 3p26.3; um com deleção para o gene HIC1 em 17p13.3 e um paciente com deleção do gene TOM1L2 em 17p11.2; um paciente apresentou mutação de base única, patogênica, g.8535C > G (c.746C > G) no éxon 7 do gene FGFR3 compatível com Displasia Tanatofórica tipo I. Por fim, dois pacientes com doenças do desenvolvimento sexual apresentaram resultados dos testes citogenômicos normais. Discussão: Sugere-se que todas as alterações encontradas estão relacionadas ao fenótipo clínico ou participam na via de sinalização de genes correlatos. As técnicas de MLPA e MMS mostraram viabilidade e eficiência para a detecção de alterações genômicas em tecidos de pacientes falecidos, contudo são dependentes da integridade e quantidade do DNA obtido. Conclusão: O estudo citogenômico post-mortem é importante para a elucidação diagnóstica de casos sem etiologia definida, para o aconselhamento genético familiar, para a caracterização de mosaicismo inter e intratecidual e para a compreensão da patogênese das MCs / Introduction: Congenital malformations (CMs) are the second leading cause of fetal and infant deaths in Brazil and in most cases the etiology is not well defined. Also, the patients remain without a conclusive diagnostic making difficult the genetic counseling. Objectives: This study applied cytogenomics techniques in order to characterize the presence of DNA abnormalities, as well as, aneuploidies and genomic copy number variations (CNVs) in different tissues from deceased patients with CM from \"Serviço de Verificação de Óbitos\". Patients and Methods: We evaluated samples from 30 patients undergoing necropsy. The DNA was extracted from different tissues (brain, heart, liver, skin and diaphragm) stored in RNA later, formaldehyde and embedded in paraffin. We performed Multiplex Ligation-dependent Probe Amplification (MLPA) with P095 kits, P064 and P070 (MRC-Holland®), microsatellite markers (MMS) with MiniFiler kit (Life Technologies), Fluorescence In Situ Hybridization (FISH), array technique (Infinium® CytoSNP-850K BeadChip - Illumina) and bidirectional sequencing by Sanger. The results was analyzed using different softwares: GeneMarker, Coffalyser, BlueFuse Multi Sequencher and databases Database of Genomic Variants (DGV - http://projects.tcag.ca/variation/) Database of Chromosomal Imbalance and Phenotype in Humans Using Ensembl Resources (Decipher - http://decipher.sanger.ac.uk/), UCSC Genome Bioinformatics (http://genome.ucsc.edu) and Mutation Taster. Results: The results showed 13 patients with pathogenic CNVs, and among them, eight presented aneuploidies involving chromosomes 13, 18, 21, X and Y. Two of them presented intra-tissue mosaicism. Also four patients showed several different microdeletions or microduplications: duplication of TYMS gene (18p11.32); deletion of CHL1 gene (3p26.3); deletion of HIC1 gene (17p13.3); deletion of TOM1L2 gene (17p11.2 ). One patient showed a pathogenic missense mutation of g.8535C>G (c.746C > G) in exon 7 from FGFR3 gene compatible with Thanatophoric Dysplasia type I. And two patients presented sexual development disorders and normal molecular results. Discussion: We conclude that the genomic abnormalities found in different tissues are pathogenic and associated to clinic manifestations in all patients studied. Besides, the cytogenomic techniques applied were efficient to help in the conclusive diagnostic; however, there are dependent of integrity and quality of DNA. Conclusion: Indeed the post-mortem cytogenomic study is crucial to genetic counseling, to characterize the presence of intra-tissue mosaicism and also to better understand the pathogenesis of congenital malformations

Introdução: As malformações congênitas (MCs) são a segunda causa de mortes fetais e infantis no Brasil e, em grande parte dos casos, a sua etiologia não é bem definida. Devido às consequências clínicas das MCs, alguns pacientes falecem sem tempo hábil para uma investigação etiológica acurada. Dessa forma, a maioria dos casos permanece sem uma confirmação molecular das suspeitas clínicas, dificultando o aconselhamento genético para as famílias. Objetivos: O presente trabalho utilizou técnicas citogenômicas para caracterizar molecularmente a presença de anormalidades no DNA, desde aneuploidias até a variação do número de cópias gênicas (CNVs) em diferentes tecidos de pacientes falecidos portadores de MC encaminhados ao Serviço de Verificação de Óbitos para avaliação anatomopatológica. Casuística e Métodos: Foram avaliadas amostras de 30 pacientes portadores de MC submetidos à necropsia. O DNA foi extraido de diferentes tecidos (cérebro, coração, fígado, pele e diafragma) previamente conservados em RNA later, formol ou emblocados em parafina. Foram utilizadas as técnicas de Multiplex Ligation-dependent Probe Amplification (MLPA) com os kits P095, P064 e P070 (MRC-Holland®), Marcadores Microssatélites (MMS) com o kit MiniFiler (Life Technologies®), a Fluorescence in Situ Hybridization (FISH), a técnica de array (Infinium® CytoSNP-850K BeadChip - Illumina) e o Sequenciamento Bidirecional por Sanger. A interpretação dos resultados foi realizada utilizando os softwares GeneMarker, Coffalyser, BlueFuse Multi, Sequencher e com os bancos de dados Database of Genomic Variants (DGV - http://projects.tcag.ca/variation/), Database of Chromosomal Imbalance and Phenotype in Humans Using Ensembl Resources (DECIPHER - http://decipher.sanger.ac.uk/), UCSC Genome Bioinformatics (http://genome.ucsc.edu) e Mutation Taster. Resultados: Dos 30 pacientes avaliados, 13 apresentaram alterações patogênicas. Entre eles, oito apresentaram aneuploidias envolvendo os cromossomos 13, 18, 21, X e Y, sendo dois deles com mosaicismo intratecidual. Quatro pacientes apresentaram microdeleções ou microduplicações envolvendo diferentes genes, sendo um paciente com duplicação do gene TYMS em 18p11.32; um com deleção do gene CHL1 em 3p26.3; um com deleção para o gene HIC1 em 17p13.3 e um paciente com deleção do gene TOM1L2 em 17p11.2; um paciente apresentou mutação de base única, patogênica, g.8535C > G (c.746C > G) no éxon 7 do gene FGFR3 compatível com Displasia Tanatofórica tipo I. Por fim, dois pacientes com doenças do desenvolvimento sexual apresentaram resultados dos testes citogenômicos normais. Discussão: Sugere-se que todas as alterações encontradas estão relacionadas ao fenótipo clínico ou participam na via de sinalização de genes correlatos. As técnicas de MLPA e MMS mostraram viabilidade e eficiência para a detecção de alterações genômicas em tecidos de pacientes falecidos, contudo são dependentes da integridade e quantidade do DNA obtido. Conclusão: O estudo citogenômico post-mortem é importante para a elucidação diagnóstica de casos sem etiologia definida, para o aconselhamento genético familiar, para a caracterização de mosaicismo inter e intratecidual e para a compreensão da patogênese das MCs / Introduction: Congenital malformations (CMs) are the second leading cause of fetal and infant deaths in Brazil and in most cases the etiology is not well defined. Also, the patients remain without a conclusive diagnostic making difficult the genetic counseling. Objectives: This study applied cytogenomics techniques in order to characterize the presence of DNA abnormalities, as well as, aneuploidies and genomic copy number variations (CNVs) in different tissues from deceased patients with CM from \"Serviço de Verificação de Óbitos\". Patients and Methods: We evaluated samples from 30 patients undergoing necropsy. The DNA was extracted from different tissues (brain, heart, liver, skin and diaphragm) stored in RNA later, formaldehyde and embedded in paraffin. We performed Multiplex Ligation-dependent Probe Amplification (MLPA) with P095 kits, P064 and P070 (MRC-Holland®), microsatellite markers (MMS) with MiniFiler kit (Life Technologies), Fluorescence In Situ Hybridization (FISH), array technique (Infinium® CytoSNP-850K BeadChip - Illumina) and bidirectional sequencing by Sanger. The results was analyzed using different softwares: GeneMarker, Coffalyser, BlueFuse Multi Sequencher and databases Database of Genomic Variants (DGV - http://projects.tcag.ca/variation/) Database of Chromosomal Imbalance and Phenotype in Humans Using Ensembl Resources (Decipher - http://decipher.sanger.ac.uk/), UCSC Genome Bioinformatics (http://genome.ucsc.edu) and Mutation Taster. Results: The results showed 13 patients with pathogenic CNVs, and among them, eight presented aneuploidies involving chromosomes 13, 18, 21, X and Y. Two of them presented intra-tissue mosaicism. Also four patients showed several different microdeletions or microduplications: duplication of TYMS gene (18p11.32); deletion of CHL1 gene (3p26.3); deletion of HIC1 gene (17p13.3); deletion of TOM1L2 gene (17p11.2 ). One patient showed a pathogenic missense mutation of g.8535C>G (c.746C > G) in exon 7 from FGFR3 gene compatible with Thanatophoric Dysplasia type I. And two patients presented sexual development disorders and normal molecular results. Discussion: We conclude that the genomic abnormalities found in different tissues are pathogenic and associated to clinic manifestations in all patients studied. Besides, the cytogenomic techniques applied were efficient to help in the conclusive diagnostic; however, there are dependent of integrity and quality of DNA. Conclusion: Indeed the post-mortem cytogenomic study is crucial to genetic counseling, to characterize the presence of intra-tissue mosaicism and also to better understand the pathogenesis of congenital malformations

<div> Advancements in cancer treatments have increased rapidly in recent years, but cures remain elusive. Surgical tumor resection is a central treatment for many solid malignancies. Residual tumor at surgical margins leads to tumor recurrence. Novel tools for assessing residual tumor at surgical margins could improve surgical outcomes by helping to maximize the extent of resection. Ambient ionization-mass spectrometry (MS) methods generate and analyze ions from minimally prepared samples in near-real-time (e.g. seconds to minutes). These methods leverage the high sensitivity and specificity of mass spectrometry for analyzing gas phase ions and generating those ions quickly and with minimal sample preparation. Recent work has shown that differential profiles of ions, corresponding to phospholipids and small metabolites, are detected from cancerous and their respective normal tissue with ambient ionization-MS methods. When properly implemented, ambient ionization-MS could be used to assess for tumor at surgical margins and provide a molecular diagnosis during surgery. </div><div><br></div><div>The research herein reports efforts in developing rapid intraoperative ambient ionization-MS methods for the molecular assessment of cancerous tissues. Touch spray (TS) ionization and desorption electrospray ionization (DESI) were utilized to analyze kidney cancer and brain cancer.</div><div><br></div><div> As a demonstration of the applicability of TS-MS to provide diagnostic information from fresh surgical tissues, TS-MS was used to rapidly analyze renal cell carcinoma and healthy renal tissue biopsies obtained from human subjects undergoing nephrectomy surgery. Differential phospholipid profiles were identified using principal component analysis (PCA), and the significant ions were characterized using multiple stages of mass spectrometry and high resolution/exact mass MS. The same TS-MS analyzed renal tissues were subsequently analyzed with DESI-MS imaging to corroborate the TS-MS results, and the significant DESI-MS ions were also characterized with MS.</div><div><br></div><div>Significant efforts were made in developing and evaluating a standalone intraoperative DESI-MS system for analyzing brain tissue biopsies during brain tumor surgery. The intraoperative DESI-MS system consists of a linear trap quadrupole mass spectrometer placed on a custom-machined cart that contains all hardware for operating the mass spectrometer. This instrument was operated in the neurosurgical suites at Indiana University School of Medicine to rapidly analyze brain tissue biopsies obtained from glioma resection surgeries. A DESI-MS library of normal brain tissue and glioma was used to statistically classify the brain tissue biopsies collected in the operating room. Multivariate statistical methodologies were employed to predict the disease state and tumor cell percentage of the samples. A DESI-MS assay for detecting 2-hydroxyglutarate (2HG), the oncometabolic product of the isocitrate dehydrogenase (IDH) mutation (a key glioma prognostic marker), was developed and applied to determine the IDH mutation status during the surgical resection. The strengths, weaknesses, and areas of future work in this field are discussed. </div><div><br></div>

<p>My research has focused on the development and translation of ambient ionization mass spectrometry (MS)-based platforms in clinical and surgical settings, specifically in the area of brain cancer diagnostics and surgical decision making. Ambient ionization MS methods, such as those described herein, generate and analyze gas phase ions with high sensitivity and specificity from minimally prepared samples in near-real-time, on the order of seconds to minutes, rendering them well suited to point-of-care applications. We used ambient ionization MS methods, specifically desorption electrospray ionization mass spectrometry (DESI-MS) and extraction nanoelectrospray ionization mass spectrometry (nESI-MS) to molecularly characterize brain cancer biopsies. The characterization was made using diagnostic compounds identified as markers of disease state, tissue composition, tumor type, and genotype in human brain tissue. Methods were developed and validated offline in the laboratory and translated to clinical and surgical settings, thereby generating chemical information on prognostic features intraoperatively and providing valuable information that would be otherwise unavailable. We believe that, with approval, the methodologies described can assist physicians and improve patient outcomes by providing analytical tools and molecular information that can inform surgical decision making and adjuvant treatment strategies, complementing and not interfering with standard of care protocols.</p> <p><br></p> <p>We have successfully demonstrated the use of desorption electrospray ionization mass spectrometry (DESI-MS) for the expedient molecular assessment of human glioma tissue biopsies based on lipid profiles and prognostic metabolites, both at the tumor core and near surgical margins, in two small-scale, clinical studies. Maximal surgical resection of gliomas that avoids non-infiltrated tissue is associated with survival benefit in patients with glioma. The infiltrative nature of gliomas, as well as their morphological and genetic diversity, renders treatment difficult and demands an integrated imaging and diagnostic approach during surgery to guide clinicians in achieving maximal tumor resection. Further, the estimation of tumor cell percentage (TCP), a measure of tumor infiltration at surgical margins, is not routinely assessed intraoperatively. </p> <p>We have previously shown that rapid, offline molecular assessment of tumor infiltration in tissue biopsies is possible and believe that the same assessment performed intraoperatively in biopsied tissue near surgical margins could improve resection and better inform patient management strategies, including postoperative radiotherapy. Using a DESI-MS spectral library of normal brain tissue and glioma biopsies to generate a statistical model to classify brain tissue biopsies intraoperatively, multivariate statistical approaches were used to predict the disease state and tumor cell percentage (TCP) of each biopsy, thereby providing an measure of tumor infiltration at surgical margins via molecular indicators. In addition to assessment of tumor infiltration, we have developed DESI-MS assays for detecting the oncometabolite 2-Hydroxyglutarate (2HG) to detect isocitrate dehydrogenase (IDH) mutations in gliomas intraoperatively. Knowledge of IDH genotypes at the time of surgical resection could improve patient outcomes, as more aggressive tumor resection of IDH-mutated gliomas is associated with increased survival. While assessments of IDH genotype are typically not available until days after surgery, we have demonstrated the ability to provide this information is less than five minutes. An intraoperative DESI-MS system has successfully been used in a proof-of-concept clinical study and intraoperative performance validation of this platform is ongoing. The findings of these two studies as well as strengths, weaknesses, and areas of improvement for upcoming future iterations of the research are discussed.</p> <p><br></p> <p>Point-of-care applications necessitate the adaptation of MS methodologies to smaller devices. Miniature mass spectrometers (Mini MS) boast small footprints, simple operation, and low power consumption, noise levels, and cost, making them attractive candidates for point-of-care use. In a small-scale clinical study, we demonstrated the first application of a Mini MS for determination of IDH mutation status in gliomas intraoperatively. This study paves a path forward for the application of Mini MS in the OR. With its small footprint and low power consumption and noise level, this application of miniature mass spectrometers represents a simple and cost-effective platform for an important intraoperative measurement. </p> <p><br></p> <p>While MS-based methods of tissue analysis can detect molecular features of interest and rapidly produce large quantities of data, their inherent speed is rarely utilized because they are traditionally coupled with time-consuming separation techniques (e.g., chromatography). Ambient ionization MS, specifically DESI-MS, is well suited for high-throughput applications due to its lack of sample preparation and purification techniques. In an attempt to rapidly characterize microarrays of tissue biopsies, we developed a high-throughput DESI-MS (HT-DESI-MS) method for the rapid characterization of disease state, human brain tumor type, glioma classification, and detection of IDH mutations in tissue microarrays (TMA) of banked and fresh human brain tissue biopsies. We anticipate that HT-DESI-MS analysis of TMAs could become a standard tool for the generation of spectral libraries for sample classification, the identification of biomarkers through large-scale studies, the correlation of molecular features with anatomical features when coupled to digital pathology, and the assessment of drug efficacy. </p>