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Aspects of Gene Expression Profiling in Disease and Health
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology. (Fredrik Pontén)
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Description
Abstract [en]

The aim of this thesis is to in various ways explore protein expression in human normal tissue and in cancer and to apply that knowledge in biomarker discovery.

In Paper I the prognostic significance of RNA-binding motif protein 3 (RBM3) is explored in malignant melanoma. To further evaluate the prognostic significance of RBM3 expression was assessed in 226 incident cases of malignant melanoma from the prospective populationbased cohort study Malmö Diet and Cancer Study using tissue microarray technique (TMA). RBM3 was shown to be down regulated in metastatic melanoma and high nuclear expression in the primary tumor was an independent marker of prolonged over all survival. As a tool to facilitate clinical biomarker studies the Human Protein Atlas has created a tissue dictionary as an introduction to human histology and histopathology. In Paper II this work is introduced.

A cancer diagnosis can be a complex process with difficulties of establishing tumor type in localized disease or organ of origin in generalized disease. Immunohistochemically assisted diagnosis of cancer is common practice among pathologists where its application combined with known protein expression profiles of different cancer types, can strengthen or help dismiss a suspected diagnosis. In Paper III the diagnostic performance of 27 commonly used antibodies are tested in a predominantly metastatic, multicancer cohort using TMA technique. Overall these 27 diagnostic markers showed a low sensitivity and specificity for its intended use, highlighting the need for novel, more specific markers.

Breast, ovarian, endometrial and ovarian cancers affect predominantly women. Differential diagnostics between these cancer types can be challenging. In Paper IV an algorithm, based on six different IHC markers, to differentiate between these cancer types is presented. A new diagnostic marker for breast cancer, namely ZAG is also introduced.

In Paper V the transcriptomic landscape of the adrenal gland is explored by combining a transcriptomic approach with a immunohistochemistry based proteomic approach. In the adrenal gland we were able to detect 253 genes with an elevated pattern of expression in the adrenal gland, as compared to 31 other normal human tissue types analyzed. This combination of a transcriptomic and immunohistochemical approach provides a foundation for a deeper understanding of the adrenal glands function and physiology.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2017. , p. 43
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1294
Keywords [en]
Cancer, biomarkers, differential diagnostics, immunohistochemistry, transcriptomics, protein profiling, adrenal gland.
National Category
Basic Medicine
Research subject
Pathology
Identifiers
URN: urn:nbn:se:uu:diva-312939ISBN: 978-91-554-9802-3 (print)OAI: oai:DiVA.org:uu-312939DiVA, id: diva2:1065385
Public defence
2017-03-10, Fåhraeussalen, Rudbecklaboratoriet, Dag Hammarskjölds v 20, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2017-02-17 Created: 2017-01-16 Last updated: 2018-01-13
List of papers
1. Low RBM3 protein expression correlates with tumour progression and poor prognosis in malignant melanoma: An analysis of 215 cases from the Malmo Diet and Cancer Study
Open this publication in new window or tab >>Low RBM3 protein expression correlates with tumour progression and poor prognosis in malignant melanoma: An analysis of 215 cases from the Malmo Diet and Cancer Study
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2011 (English)In: Journal of Translational Medicine, ISSN 1479-5876, E-ISSN 1479-5876, Vol. 9, p. 114-Article in journal (Refereed) Published
Abstract [en]

Background: We have previously reported that expression of the RNA-and DNA-binding protein RBM3 is associated with a good prognosis in breast cancer and ovarian cancer. In this study, the prognostic value of immunohistochemical RBM3 expression was assessed in incident cases of malignant melanoma from a prospective population-based cohort study. Methods: Until Dec 31(st) 2008, 264 incident cases of primary invasive melanoma had been registered in the Malmo Diet and Cancer Study. Histopathological and clinical information was obtained for available cases and tissue microarrays (TMAs) constructed from 226 (85.6%) suitable paraffin-embedded tumours and 31 metastases. RBM3 expression was analysed by immunohistochemistry on the TMAs and a subset of full-face sections. Chi-square and Mann-Whitney U tests were used for comparison of RBM3 expression and relevant clinicopathological characteristics. Kaplan Meier analysis and Cox proportional hazards modelling were used to assess the relationship between RBM3 and recurrence free survival (RFS) and overall survival (OS). Results: RBM3 could be assessed in 215/226 (95.1%) of primary tumours and all metastases. Longitudinal analysis revealed that 16/31 (51.6%) of metastases lacked RBM3 expression, in contrast to the primary tumours in which RBM3 was absent in 3/215 (1.4%) cases and strongly expressed in 120/215 (55.8%) cases. Strong nuclear RBM3 expression in the primary tumour was significantly associated with favourable clinicopathological parameters; i. e. non-ulcerated tumours, lower depth of invasion, lower Clark level, less advanced clinical stage, low mitotic activity and non-nodular histological type, and a prolonged RFS (RR = 0.50; 95% CI = 0.27-0.91) and OS (RR = 0.36, 95% CI = 0.20-0.64). Multivariate analysis demonstrated that the beneficial prognostic value of RBM3 remained significant for OS (RR = 0.33; 95% CI = 0.18-0.61). Conclusions: In line with previous in vitro data, we here show that RBM3 is down-regulated in metastatic melanoma and high nuclear RBM3 expression in the primary tumour is an independent marker of a prolonged OS. The potential utility of RBM3 in treatment stratification of patients with melanoma should be pursued in future studies.

Identifiers
urn:nbn:se:uu:diva-158331 (URN)10.1186/1479-5876-9-114 (DOI)000293917900001 ()
Available from: 2011-09-06 Created: 2011-09-06 Last updated: 2017-12-08Bibliographically approved
2. A tool to facilitate clinical biomarker studies - a tissue dictionary based on the Human Protein Atlas
Open this publication in new window or tab >>A tool to facilitate clinical biomarker studies - a tissue dictionary based on the Human Protein Atlas
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2012 (English)In: BMC Medicine, ISSN 1741-7015, E-ISSN 1741-7015, Vol. 10, p. 103-Article in journal (Refereed) Published
Abstract [en]

The complexity of tissue and the alterations that distinguish normal from cancer remain a challenge for translating results from tumor biological studies into clinical medicine. This has generated an unmet need to exploit the findings from studies based on cell lines and model organisms to develop, validate and clinically apply novel diagnostic, prognostic and treatment predictive markers. As one step to meet this challenge, the Human Protein Atlas project has been set up to produce antibodies towards human protein targets corresponding to all human protein coding genes and to map protein expression in normal human tissues, cancer and cells. Here, we present a dictionary based on microscopy images created as an amendment to the Human Protein Atlas. The aim of the dictionary is to facilitate the interpretation and use of the image-based data available in the Human Protein Atlas, but also to serve as a tool for training and understanding tissue histology, pathology and cell biology. The dictionary contains three main parts, normal tissues, cancer tissues and cells, and is based on high-resolution images at different magnifications of full tissue sections stained with H & E. The cell atlas is centered on immunofluorescence and confocal microscopy images, using different color channels to highlight the organelle structure of a cell. Here, we explain how this dictionary can be used as a tool to aid clinicians and scientists in understanding the use of tissue histology and cancer pathology in diagnostics and biomarker studies.

Keywords
Antibody-based proteomics, cancer biomarkers, tissue and cell dictionary, immunohistochemistry, protein expression, histology, pathology
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-192083 (URN)10.1186/1741-7015-10-103 (DOI)000312389800001 ()
Available from: 2013-01-16 Created: 2013-01-16 Last updated: 2017-12-06Bibliographically approved
3. A systematic analysis of commonly used antibodies in cancer diagnostics
Open this publication in new window or tab >>A systematic analysis of commonly used antibodies in cancer diagnostics
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2014 (English)In: Histopathology, ISSN 0309-0167, E-ISSN 1365-2559, Vol. 64, no 2, p. 293-305Article in journal (Refereed) Published
Abstract [en]

AimsImmunohistochemistry plays a pivotal role in cancer differential diagnostics. To identify the primary tumour from a metastasis specimen remains a significant challenge, despite the availability of an increasing number of antibodies. The aim of the present study was to provide evidence-based data on the diagnostic power of antibodies used frequently for clinical differential diagnostics. Methods and resultsA tissue microarray cohort comprising 940 tumour samples, of which 502 were metastatic lesions, representing tumours from 18 different organs and four non-localized cancer types, was analysed using immunohistochemistry with 27 well-established antibodies used in clinical differential diagnostics. Few antibodies, e.g. prostate-specific antigen and thyroglobulin, showed a cancer type-related sensitivity and specificity of more than 95%. A majority of the antibodies showed a low degree of sensitivity and specificity for defined cancer types. Combinations of antibodies provided limited added value for differential diagnostics of cancer types. ConclusionsThe results from analysing 27 diagnostic antibodies on consecutive sections of 940 defined tumours provide a unique repository of data that can empower a more optimal use of clinical immunohistochemistry. Our results highlight the benefit of immunohistochemistry and the unmet need for novel markers to improve differential diagnostics of cancer.

Keywords
biological tumour markers, differential diagnosis, immunohistochemistry, surgical pathology, tissue microarray analysis
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-216054 (URN)10.1111/his.12255 (DOI)000328347800012 ()
Note

De två (2) första författarna delar förstaförfattarskapet.

Available from: 2014-01-20 Created: 2014-01-17 Last updated: 2018-02-01Bibliographically approved
4. A six marker panel for differential diagnostics of female cancers
Open this publication in new window or tab >>A six marker panel for differential diagnostics of female cancers
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Aim: To present a new immunohistochemistry-based panel for clinical differential diagnostics of breast, ovarian, endometrial and cervical cancer.

 

Background: Diagnostics of metastatic ER-positive tumors can present a clinical challenge. Breast and gynecological cancers are to a varying degree ER+, and can have similar growth patterns. The close proximity of the ovaries, endometrium and cervix also renders difficulties to distinguish between primary gynecological cancer in advanced stages.

 

Material and Methods: As a discovery set for the selection of antibodies, tissue microarray (TMA) blocks containing 60 breast, 60 ovarian, 60 endometrial and 60 cervical tumor samples of predominantly metastatic sources were sectioned and immunohistochemically stained using 43 different primary antibodies, including both well accepted diagnostic markers and novel candidate markers. The results were analyzed for best possible differential diagnostic power to discriminate between these forms of female cancer.

 

Results: By the implementation of a decision tree we were able to define a six-marker panel including antibodies detecting the WT1, ZAG, VIM, CK5, GATA3 and PAX8 proteins. This antibody panel enabled differentiation between breast, ovarian, endometrial and cervical cancer with an accuracy of 80%. The selected markers were then examined in a second cohort of 452 cancer samples comprising 60 breast, 48 ovary, 233 endometrium and 111 cervix patients. A decision tree classifier was used to evaluate the performance of various combinations of these markers in differential diagnosis of the female cancers.  The results suggest that this panel could be used in a clinical setting to achieve a more accurate diagnosis and thus provide a basis for further prospective clinical studies.

Keywords
Differential diagnostics, immunohistochemistry
National Category
Clinical Medicine
Research subject
Pathology
Identifiers
urn:nbn:se:uu:diva-312936 (URN)
Projects
Human Protein Atlas
Funder
Knut and Alice Wallenberg Foundation
Available from: 2017-01-16 Created: 2017-01-16 Last updated: 2017-01-16
5. The human adrenal gland proteome defined by transcriptomics and antibody-based profiling
Open this publication in new window or tab >>The human adrenal gland proteome defined by transcriptomics and antibody-based profiling
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2017 (English)In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 158, no 2, p. 239-251Article in journal (Refereed) Published
Abstract [en]

The adrenal gland is a composite endocrine organ with vital functions that include the synthesis and release of glucocorticoids and catecholamines. To define the molecular landscape that underlies the specific functions of the adrenal gland, we combined a genome-wide transcriptomics approach based on mRNA sequencing of human tissues with immunohistochemistry-based protein profiling on tissue microarrays. Approximately two-thirds of all putative protein coding genes were expressed in the adrenal gland and the analysis identified 253 genes with an elevated pattern of expression in the adrenal gland, with only 37 genes showing a markedly higher expression level (>5-fold) in the adrenal gland compared to 31 other normal human tissue types analyzed. The analyses allowed for an assessment of the relative expression levels for well-known proteins involved in adrenal gland function, but also identified previously poorly characterized proteins in the adrenal cortex, such as FERM domain containing 5 (FRMD5) and protein NOV homolog (NOV). In summary, we provide a global analysis of the adrenal gland transcriptome and proteome, with a comprehensive list of genes with elevated expression in the adrenal gland and spatial information with examples of protein expression patterns for corresponding proteins. These genes and proteins constitute important starting points for an improved understanding of the normal function and pathophysiology of the adrenal glands.

National Category
Endocrinology and Diabetes
Identifiers
urn:nbn:se:uu:diva-312934 (URN)10.1210/en.2016-1758 (DOI)000397101700008 ()27901589 (PubMedID)
Funder
Knut and Alice Wallenberg Foundation
Available from: 2017-01-16 Created: 2017-01-16 Last updated: 2018-09-20Bibliographically approved

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