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- Jonasson, Emma, 1987
(författare)
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Tumor cell heterogeneity profiling using single-cell analysis
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cancer is a diverse disease with large variations between tumor types and patients regarding tumor progression and prognosis. Additionally, most individual tumors are heterogeneous, containing subpopulations of cells with various characteristics. Numerous factors affect the differences observed between tumor cells, such as variations in genetics, epigenetics, cellular states and the microenvironment surrounding the individual cells. One clinically relevant subpopulation, commonly referred to as cancer stem cells, consists of cells with stem cell characteristics. These are present in many tumor types and are known to be important for tumor development and treatment resistance. The tumor microenvironment is a key factor affecting the cellular phenotype, including the cancer stem cell subpopulation. Analysis at cell population level will not capture the true variations between individual cells. Instead, single-cell analysis offers new means to study and understand cellular and molecular differences between tumor subpopulations. The main objective of this thesis was to study tumor cell heterogeneity in myxoid liposarcoma and breast cancer with the help of single-cell gene expression analysis methods. We could generate a flexible workflow to measure gene expression, including the assessment of total mRNA amounts in each cell, using several diverse approaches developed from already existing protocols. Subsequently, we combined a number of functional cell culture methods to enrich for tumor cells with characteristic cellular properties together with single-cell gene expression profiling methods, to match phenotype with the corresponding transcription pattern. Single-cell analysis of myxoid liposarcoma cells, sorted based on the cell-cycle, identified a number of genes previously not reported as cell-cycle regulated and defined two subgroups of cells within the G1 phase. In the same tumor type, we identified a subpopulation of cells with cancer stem cell- and chemotherapy resistance properties associated with an active JAK-STAT signaling pathway. Here, a combination treatment of chemotherapy and JAK-STAT inhibition was in vitro shown to be more effective against tumor cells than chemotherapy alone. In breast cancer cells, we identified a number of potential biomarkers overexpressed in a subpopulation of cells with cancer stem cell characteristics. We also developed a new in vivo-like culture system based on decellularized human tumors to study the effect of the microenvironment on breast cancer cells. We demonstrated that the gene expression profiles of cells cultured in these patient-derived scaffolds closely mimic the profiles of in vivo cells. Furthermore, gene expression patterns changed differently depending on the patient-derived scaffold, which could be linked to patient recurrence. In conclusion, we developed single-cell analysis methods as well as a new in vivo-like model system. Furthermore, we identified genes and pathways connected to different subpopulations of myxoid liposarcoma or breast cancer cells that potentially can be used as biomarkers and future drug targets.
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| 2. |
- Milosevic, Jelena, et al.
(författare)
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PPM1D is a neuroblastoma oncogene and therapeutic target in childhood neural tumors
- 2020
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Majority of cancers harbor alterations of the tumor suppressor TP53. However, childhood cancers, including unfavorable neuroblastoma, often lack TP53 mutations despite frequent loss of p53 function, suggesting alternative p53 inactivating mechanisms.Here we show that p53-regulating PPM1D at chromosome 17q22.3 is linked to aggressive tumors and poor prognosis in neuroblastoma. We identified that WIP1-phosphatase encoded by PPM1D, is activated by frequent segmental 17q-gain further accumulated during clonal evolution, gene-amplifications, gene-fusions or gain-of-function somatic and germline mutations. Pharmacological and genetic manipulation established WIP1 as a druggable target in neuroblastoma. Genome-scale CRISPR-Cas9 screening demonstrated PPM1D genetic dependency in TP53 wild-type neuroblastoma cell lines, and shRNA PPM1D knockdown significantly delayed in vivo tumor formation. Establishing a transgenic mouse model overexpressing PPM1D showed that these mice develop cancers phenotypically and genetically similar to tumors arising in mice with dysfunctional p53 when subjected to low-dose irradiation. Tumors include T-cell lymphomas harboring Notch1-mutations, Pten-deletions and p53-accumulation, adenocarcinomas and PHOX2B-expressing neuroblastomas establishing PPM1D as a bona fide oncogene in wtTP53 cancer and childhood neuroblastoma. Pharmacological inhibition of WIP1 suppressed the growth of neural tumors in nude mice proposing WIP1 as a therapeutic target in neural childhood tumors.
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| 3. |
- Afshari, Maryam K.
(författare)
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Transcriptomic and functional studies of fusion oncogene-driven salivary gland tumors
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Fusion genes are potent oncogenic drivers resulting from exchange of regulatory/coding sequences between two genes. They were originally identified in leukemias but are now recognized as key oncogenic events also in many solid tumors, including salivary gland tumors (SGTs). Adenoid cystic carcinoma (ACC) is a highly malignant SGT with no effective treatment for patients with recurrent and/or metastatic disease. The MYB-NFIB fusion is the main genomic hallmark of ACC and a po- tential therapeutic target. Here, oncogenic signaling pathways as well as the molecular consequences and regulation of MYB-NFIB were assessed in cultured ACC cells and in ACC surgical samples. A combination of molecular and functional assays was used including RNAi, qPCR, western blot, phospho-receptor tyrosine kinase (RTK) arrays, proliferation/apoptosis/sphere assays, and gene expression microarrays. ACC patient- derived xenografts (PDX) were used to study the effects of RTK-inhibition on tumor growth. MYB-NFIB was shown to promote proliferation and spherogenesis of ACC cells. The fusion regulated expression of genes involved in DNA replication/repair, cell cycle, and RNA processing, and induced an MYC-like transcriptional program. MYB-NFIB was shown to be regulated by IGF1R through IGF2-activated AKT-signaling and phar- macological inhibition of IGF1R partially reversed the transcriptional program induced by MYB-NFIB. Moreover, IGF1R, EGFR, and MET were co-activated in ACC cells. Combined inhibition of these receptors in ACC cells and PDX-models induced differentiation and synergistic growth inhibition. The results provide new insights about the function and regulation of MYB-NFIB and are the first to show that a druggable cell surface receptor can regulate a fusion oncogene encoding a transcription factor. Importantly, the results also highlight novel potential treatment strategies for ACC patients. Pleomorphic adenoma (PA) is the most common SGT. Although it is a benign tumor, treatment may be complicated by recurrence and/or malignant transformation. Previous studies of PA have revealed recurrent chromosomal rearrangements that activate the key oncogenes PLAG1 and HMGA2 by gene fusion events. Here, detailed studies of previously uncharacterized subsets of PAs with 8;9- or 9;12-rearrangements revealed breakpoints within or in the proximity of either PLAG1 or HMGA2, and NFIB. Further analyses using RNA- seq, RT-PCR, qPCR, and arrayCGH revealed a novel NFIB-PLAG1 fusion in a PA with an ins(9;8) and HMGA2-NFIB fusions in cases with t(9;12). These findings highlight the role of NFIB as a fusion partner gene in both benign and malignant SGTs and indicate that NFIB can activate both PLAG1 and HMGA2 by gene fusion/enhancer hijacking events in PA. Furthermore, RNA-seq based transcriptomic analysis of PAs revealed a high frequency of PLAG1 and HMGA2 fusions (≈80% of the cases) and multiple novel fusion partner genes. The findings indicate that gene fusions are more common in PA than previously documented. Global gene expression and pathway analyses revealed several activated oncogenic signaling pathways and showed that the expression profile reflects certain morphological features typical of PA. Finally, the results showed that PLAG1 and HMGA2 drive tumorigenesis via shared signaling pathways. The results provide further insights into the pathogenesis of PA and reveal new potential therapeutic targets.
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| 4. |
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| 5. |
- Bergman, Daniel
(författare)
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Canine heterophilic antibodies
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Anamnesis, physical examination and laboratory testing are the pillars of the clinical diagnostic procedure. Alas, laboratory tests are not perfect and analytical errors happen, which can lead to misdiagnosis and detrimental consequences for patient care. Immunoassays are commonly used to measure various hormones and disease markers in patient samples. Despite decades of methodological development and technological advances, immunoassays used for clinical diagnosis are still associated with limitations and even some flaws.This thesis focuses on a long-lived immunoassay flaw that has been poorly researched in veterinary medicine. Humans and animals both carry heterophilic antibodies, also called anti-animal antibodies, in their circulation. These antibodies can interfere with immunoassays and cause erroneous results. The mechanism of action is the same for animals as it is for humans; the heterophilic antibodies bind to animal antibodies employed by the immunoassay, usually leading to a falsely increased measurement. Due to the extensive use of mouse IgG for analyte detection in immunoassays, anti-mouse antibodies are of particular concern.Herein, the prevalence of heterophilic antibodies against mouse IgG in a cohort of dog patients is estimated. It is demonstrated that the antibodies can have tangible consequences for patient care as they can interfere with commercial immunoassays used in veterinary laboratories. Falsely increased anti-Müllerian hormone (AMH) measurements were found, which could lead to needless surgery in dog patients. The molecular characteristics of canine heterophilic antibodies were shown to be heterogeneous. They may react with the Fc region or the Fab region of the murine IgG molecule. There is cross-reactivity with IgG from several species, and heterophilic antibodies in dogs are made up of the IgA, IgG and IgM isotypes. The prevalence of the antibodies varies between dog breeds, and the Bernese mountain dog is tentatively predisposed to heterophilic antibodies. The origin of these antibodies remains mostly unclear, but there is occasional cross-reactivity between antibodies to mouse IgG and canine autoantibodies to IgG. Canine heterophilic antibodies can persist for at least two years in serum and represent a risk factor for repeated analytical errors and misdiagnosis in patients with these antibodies.
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| 6. |
- Lager, Malin, 1975-
(författare)
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Molecular and serological tools for clinical diagnostics of Lyme borreliosis - can the laboratory analysis be improved?
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Lyme borreliosis (LB) is caused by spirochetes within the Borrelia burgdorferi sensu lato complex and is the most common tick-transmitted disease in the northern hemisphere. The transmission of the spirochetes to humans in Europe is done by the Ixodes ricinus ticks, which can also transmit the relapsing fever species Borrelia miyamotoi. LB may cause clinical manifestations in the skin, in the central nervous system, in joints, and in the heart. Diagnosis of LB is mainly based on the patient´s medical history, self-described symptoms, and clinical signs in combination with the detection of Borrelia-specific antibodies (serological methods). In some cases/issues, detection of Borrelia-specific deoxyribonucleic acid (molecular methods) may be used as a complement to serology. All diagnosed LB infections are treated with antibiotics to prevent disease progression, and most patients fully recover without further sequelae. The overall aims of this thesis were to evaluate molecular and serological tools for laboratory diagnosis of LB, with a special focus on Lyme neuroborreliosis (LNB), and to identify potential improvements.The results presented in this thesis showed that the immunoglobulin (Ig) G assays, currently in use in northern Europe for detection of antibodies in serum, had high diagnostic sensitivity (88 %) together with comparable results both between and within assays. For the IgM assays, the diagnostic sensitivity was lower (59 %) with more heterogeneous results. Small variations in diagnostic performance for IgM and IgG were mainly presented for samples within the borderline zone. These results support the theory that separate testing of IgM antibodies in serum has low diagnostic value. However, simultaneous detection in serum and cerebrospinal fluid (CSF) for both IgM and IgG antibodies was essential for the diagnosis of LNB, at least for certain assays.So far (to our knowledge), no systematic evaluation and optimisation of the pre-analytical handling of CSF samples before molecular testing has been performed. By use of the precipitate concentrated by moderate centrifugation, extraction of total nucleic acid followed by reversetranscription to complementary deoxyribonucleic acid, in combination with the absence of polymerase chain reaction (PCR) inhibitors, detection of Borrelia garinii, Borrelia afzelii, Borrelia burgdorferi sensu stricto, and B. miyamotoi was possible. These four species are all known to be pathogenic to humans. The results revealed a high analytical sensitivity and specificity for the optimised pre-analytical conditions. The thesis also presents results showing that the real-time PCR protocols currently used in Scandinavia have high analytical sensitivity, specificity, and concordance. This indicates that the low diagnostic sensitivity for detection of Borrelia in CSF was not a result of poorly designed and evaluated PCR protocols, but was possibly due to the low number of spirochetes in the samples. However, to further evaluate the diagnostic performance for detection of Borrelia in CSF by PCR, clinical samples need to be evaluated based on our new recommendations for the pre-analytical handling of CSF samples.In conclusion, this thesis presents results revealing that both molecular and serological tools for detection of Borrelia have, in general high sensitivity and specificity with results comparable between different protocols and different laboratories. It also presents recommendations for pre-analytical handling of CSF samples before PCR-analysis, and shows the benefits in diagnostic performance by simultaneous detection of IgM and IgG antibodies in serum and CSF for accurate diagnosis of LNB. Even though the techniques mentioned above have high analytical performance, the ability to discriminate an active infection from a previous one is limited and further studies need to be carried out. These studies need to focus on finding diagnostic tools that can help physicians to determine ongoing infection to ensure adequate treatment. It is also desirable to improve the standardisation of the diagnostic tools and to find methods that can discriminate between different Borrelia species.
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| 7. |
- Rostami, Elham, 1979-, et al.
(författare)
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Molecular Targets in Craniopharyngioma
- 2020
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Ingår i: Adult Craniopharyngiomas. - Cham : Springer. - 9783030411756 - 9783030411787 - 9783030411763 ; , s. 209-221
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- Craniopharyngiomas have been histologically categorized into adamantinomatous (ACP) and papillary (PCP) subtype of craniopharyngioma. However, recent developments in molecular and genetic analysis have identified specific mutations in each, β-catenin in ACP and BRAF mutation in PCP. Furthermore, these developments have provided a deeper insight into the origin and pathology of this tumour and opened a new field of treatment opportunities. Recent findings indicate connection between stem cells and ACP and suggest a paracrine model in which pituitary stem cells drive neoplastic proliferation of nearby epithelial cells through growth factor signalling. Investigation of molecular and genetic alterations in CPs has identified several biomarkers that have paved the way for new possibility to predict the biological behaviour of this tumour as well as early diagnosis of recurrence and new treatment options. Currently, the most promising adjuvant treatment is offered by dual therapy with BRAF and MEK inhibitors in PCPs expressing BRAFV600E mutation. So far this has been reported as case studies, hence, ongoing and upcoming larger clinical trials are highly anticipated to provide more information on this treatment option and its long-term efficacy.It might be possible that in the future, emerging treatments may be applied to reduce the tumour size and facilitate total surgical removal of the tumours potentially improving the outcome. Or even more exciting, simple analysis of tumour markers in serum and/or cerebrospinal fluid in combination with MR imaging would provide sufficient information on diagnosis and available targeted therapy could be applied precluding any surgical intervention.
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| 8. |
- Starnberg, Karin
(författare)
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Release and Clearance Mechanisms of Cardiac Troponin
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Myocardial infarction (MI) is often suspected when a patient presents with chest pain. MI is defined as cardiac necrosis due to ischemia, most often mediated through impaired coronary perfusion. Cardiac necrosis results in the release of myoglobin, creatine kinase and cardiac troponin (cTn) to the circulation. According to current guidelines, the MI diagnosis is, to a large extent, based on the patient’s levels of cTn. This thesis examines the mechanisms of cTn release and subsequent clearance from the circulation. The trimeric cardiac troponins, troponin T (cTnT), troponin I (cTnI) troponin C (cTnC), bind to each other and via cTnT to insoluble filaments in the cardio¬myocyte. Contrary to the prevailing opinion we found that a large fraction of cTnT could be released in 37°C plasma from necrotic human cardiac tissue without degradation of insoluble filaments. In contrast to myoglobin, which lacks affinity for cardiac tissue, the release of cTnT was highly plasma volume-dependent, which could explain the delayed clearance of cTnT observed in patients with MI. We then examined the clearance of cTnT from the circulation by injecting cardiac extracts containing both myoglobin and cTnT in rats. We also examined the renal extraction of circulating cTnT by comparing the cTnT concentration in blood samples from the renal vein and an artery in heart failure patients. We found high renal extraction of cTnT and that correction for renal clearance makes the cTnT analysis slightly better at finding patients with an MI in the emergency ward. We next examined the difference in release and clearance of cTnT and cTnI using the currently most frequently used clinical assays. We found that most cTnT and cTnI released from human cardiac tissue were degradation products produced by tissue-resident proteases. We also found that cTnI was degraded and released much faster than cTnT, whereas their subsequent clearance, once they reached the circulation, did not differ between cTnT and cTnI in either rats or humans. Our data potentially explain why cTnI reaches higher levels and disappears faster than cTnT in patients with MI.
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