Lääketieteellinen tiedekunta

 

Recent Submissions

  • Järvinen, Riikka (Unigrafia, 2016)
    Non-muscle-invasive bladder carcinoma (NMIBC) should be treated with a transurethral resection of the bladder tumour (TURBT). The need for an adjuvant intravesical treatment is evaluated after TURBT and is based on various tumour characteristics. The aim of the adjuvant intravesical treatment is to reduce the number of recurrences and the risk of progression. This thesis consists of four individual FinnBladder (FB) studies that compared different intravesical regimens of chemo- and immunotherapy among patients with intermediate- or high-risk NMIBC. The present thesis analyses the long-term results of those studies. Patients in these studies belonged to the intermediate- or to the high-risk group of NMIBC. Patients in study I were treated with mitomycin C (MMC) or bacillus Calmette-Guérin (BCG) instillations for up to one year, whereas the patients in study II had one perioperative plus four weekly MMC instillations followed by MMC or alternating MMC and BCG instillations. The patients in study III had one perioperative MMC plus four weekly MMC instillations followed by either BCG or alternating BCG and interferon-α2b (IFN) instillations. The patients of study IV had one perioperative epirubicin (EPI) followed by BCG or combination of EPI and IFN. The follow-up times of the patients that were still alive were between 8.0 (study IV) and 19.4 years (study II). The median follow-up times of all patients were between 7.2 (study II) and 8.6 years (study III). In study I, we found that BCG significantly reduced the time to recurrence but had no effect on progression, disease specific survival or overall survival compared to MMC. In study II, we found no difference between the study groups (one perioperative MMC and four weekly MMC followed by BCG or alternating MMC and BCG). However, the risk of dying of BC appeared to be markedly lower than observed in the series of untreated patients. In study III, we found that one perioperative and four weekly MMC instillations followed by a monthly maintenance with BCG significantly reduced the probability of recurrence compared to maintenance with alternating BCG and IFN. In study IV, we found that one perioperative EPI administration followed by five weekly BCG and a 1- to 2-yr BCG maintenance treatment was significantly better in reducing recurrence than was a combination of EPI and IFN using the same schedule. Two subgroup analyses of our studies revealed a lower probability of recurrence in patients that had been treated with a single perioperative chemotherapy instillation compared to those without the single instillation. The risk of progression was very low in the best treatment groups of our studies after 10 years, which might question the benefit of a recommended life-long follow-up for all patients. Three out of four patients who progressed died of BC despite the relatively meticulous follow-up. Better methods are still needed to recognize the progression in NMIBC early enough to be able to lower the mortality of this disease.
  • Taipale, Kristian (2016)
    In the history of oncology scientists have tried to treat cancer in numerous different ways. Traditionally surgery, chemotherapy and radiotherapy have been regarded as the three main treatment modalities. However, harnessing the power of the human immune system by cancer immunotherapy has recently attracted considerable interest, even though studies on immunotherapy started already more than hundred years ago. The crucial feature of immunotherapy is that it reshapes the immunosuppressive environment created by the tumor. Immunosuppression blocks the activity of body s own immune system, which would normally destroy the transformed cells, and it has been identified as one of the hallmarks of cancer. Oncolytic viruses are a promising form of immunotherapy, and the first viruses have already been approved for clinical use. Oncolytic virotherapy is based on the concept of selective viral replication inside tumor cells. Replication causes tumor cell lysis, release of danger signals and immune cell activation. Viruses can also be modified to stimulate the immune system further, for example by inserting immunostimulatory cytokines into their genomes. One of the most popular viruses used as an oncolytic platform is adenovirus, which is a non-enveloped double-stranded DNA virus. Treatments with oncolytic adenoviruses have been previously shown to be safe and able to activate the anti-tumor immunity in pre-clinical and clinical settings. This thesis investigates the influence of adenoviral immunotherapy on the human immune system. Additionally, predictive and prognostic potential of several immune-related biomarkers and clinical variables is evaluated. The patients included in these studies were treated as a part of the Advanced Therapy Access Program (ATAP), which was a clinical access program in contrast to a clinical trial. The thesis includes analyses from all of the 290 patients treated in ATAP. The first part of the thesis includes findings from the T cell subsets in the peripheral blood and tumors of 50 patients treated with an oncolytic adenovirus. When comparing pre-and post-treatment samples, a significant shift in the ratio of CD4- and CD8-positive T cells in peripheral blood was identified. The proportion of CD4-positive T helper cells decreased in the majority patients, while the proportion CD8-positive cytotoxic T cells increased. Interestingly, the changes in peripheral blood T cell levels were correlated with T cell levels in the tumor biopsies. Moreover, this correlation was found to be inverse for CD4-positive T cells and positive for CD8-positive T cells. The second study of the thesis concerned the pre-treatment tumor biopsies of 27 patients treated in the ATAP program. Several pathways related to immune responses, such as B cell receptor signaling, GM-CSF signaling and leukocyte extravasation signaling, were activated in patients surviving a shorter time than their matched controls. In the level of individual genes, several macrophage markers, complement components and complement receptors were also upregulated in this same patient population. To verify findings on a protein level, immunohistochemical stainings were performed for 19 biopsies. In these analyses, helper T cell marker CD4 and macrophage marker CD163 were significantly higher in patients with poor prognosis. Relating to infiltration of T helper cells, many T cell exhaustion markers, such as TIM-3, PD-L1, PD-L2, CTLA-4, were increased in patients with worse than expected survival, possibly suggesting a presence of exhausted T cell population. Third part focused on identifying predictive and prognostic factors for adenoviral immunotherapy. In this study, absence of pre-treatment neutralizing antibodies and low baseline neutrophil-to-lymphocyte ratio were found to correlate with a significantly longer overall survival, whereas absence of liver metastases was associated with an improved disease control rate. In multivariate Cox regression tumor type, gender and WHO performance status were linked to a lower hazard ratio for tumor related mortality. Interestingly, treatment with viruses that were armed with immunostimulatory cytokines was also associated with a lower hazard ratio. In predictive regression analysis intraperitoneal administration route of the viral treatment was correlated with a significantly higher odds ratio for disease control. In addition to improving the understanding on immunologic phenomena related to adenoviral immunotherapy, these results offer interesting directions for the development of biomarkers and clinical criteria for selecting the right patients for the treatments. Moreover, the findings provide a rationale for combination studies with already approved and emerging immune checkpoint inhibitors. In the future this knowledge could possibly be utilized to improve also the design of the viruses by targeting them more specifically to the immunosuppressive pathways that are activated in cancer.
  • Aakula, Anna (Helsingin yliopisto, 2016)
    MicroRNAs (miRNAs/miRs) impact on cellular signaling pathways and biological processes in physiological and pathological states. The expression of miRNAs is often altered in cancer, but the functions and their targets are less well understood. In this thesis project, the miRNA regulation of genes relevant for PCa proliferation was comprehensively mapped through phenotypic screening and tumor expression data. Given the role of androgen receptor (AR) as a key oncogenic driver in PCa and estrogen receptor α (ERα) playing a similar role in BCa, the regulation of these receptors was investigated here. Systematic gain-of-function screens were conducted with 1129 miRNA molecules and changes in AR, proliferation marker Ki67, and the apoptotic marker cPARP were quantified using reverse-phase protein arrays. The results identify miR-19a, -32, -124a, -130b, -148a, and -583 as regulators of proliferation and, as potential regulators of FLNC, MSRB3, PARVA, PCDH7, PRNP, RAB34 and SORBS1 genes. These aberrantly expressed genes were significantly associated with biochemical recurrence-free survival of 140 PCa patients. Altogether 13 miRNAs that regulate the AR 3'-untranslated region (3'UTR) (miR-135b, -185, -297, -299-3p, -34a, -34c, -371-3p, -421, -449a, -449b, -634, -654-5p and -9) were identified and validated in PCa. Interestingly, the RNA-sequencing data revealed a longer 3 UTR of AR, and most of the AR targeting miRNAs were found to act via this extended AR mRNA. MiR-135b and miR-34c showed an inverse association with AR protein levels in 47 clinical PCa samples. Similarly, miR-135b targeted the ERα 3'UTR in BCa and showed significant inverse correlation with the steady-state ERα protein levels in a cohort of 101 breast tumors. MiR-135b also inhibited the growth of AR-positive PCa and ERα-positive BCa cells, whereas it failed to affect the AR- and ERα-negative cells. Prolonged miR-135b expression in 3D and xenograft experiments, however, seem to confer a growth advantage and putatively induce an epithelial-to-mesenchymal switch in LNCaP cells. Taken together, this study identifies specific miRNAs as important regulators of AR, ERα, and HIF1AN protein levels, as well as novel regulatory molecules, which impact on PCa and BCa proliferation and are aberrantly expressed in tumors. This reveals novel regulatory nodes with potential as diagnostic biomarkers and that could be considered as targets for therapy or as therapy if delivery of miRNA as therapy becomes feasible.
  • Leppälahti, Suvi (Helsingin yliopisto, 2016)
    Teenage pregnancy is recognized by the World Health Organization (WHO) as one of the main public health concerns worldwide. Teenage childbirth and motherhood are associated with substantial socioeconomic and health inequalities. Research concerning those who undergo abortion at a young age remains limited, partly due to lack of reliable data. The present register-based study was designed to assess teenage pregnancy in Finland from 1987 to 2012. The aim was to investigate the trends, determinants and health and social consequences of teenage abortion and childbirth. The trends in teenage abortion between 1987 and 2009 (n=52 968) were examined. The proportion of abortions among all teenage pregnancies remained steady throughout the study period, being 82% among 13- to 15-year-olds and 41% among 18- to 19-year-olds in 2007–2009. Great fluctuation in the incidence of teenage abortion was seen (8.0/1000 in 1993, 11.5/1000 in 2003 and 9.7/1000 in 2009 among all teenagers). Repeat abortions almost doubled among the 16- to 19-year-olds. Obstetric outcomes were compared between all nulliparous teenagers (13–19 years [n=7305], further analyzed in groups by specific age) and control women (25–29 years, n=51 142) with singleton deliveries in 2006–2011. Teenagers faced increased risks of maternal complications during pregnancy, including a risk of urinary tract infection (UTI) (adjusted OR 2.9 [1.8-4.8]), pyelonephritis (6.3 [3.8-10.4]) and eclampsia (3.2 [1.4-7.3]). However, they were more likely to have uncomplicated vaginal deliveries (1.9 [1.7-2.0)]. The 1987 Finnish Birth Cohort was used to analyze the determinants and consequences of teenage pregnancy up to 25 years of age: girls who had experienced underage (<18 years) abortion (n=1041, 3.6%) or childbirth (n=395, 1.4%) were compared with each other and with girls who had no pregnancies. Five percent of all the girls in the cohort experienced underage pregnancy. Early-onset behavioral and emotional disorders and poor socioeconomic background were associated with higher risks of childbirth, and to a lesser degree, abortion. Specific risk factors of underage induced abortion were psychoactive substance use disorders (2.2 [1.3-3.5]), and having a mother who smoked during pregnancy (1.5 [1.3-1.8]) or had undergone induced abortion (1.8 [1.5-2.2.]) When the early adulthood outcomes of those who had experienced underage abortion were compared with those who gave birth, no significant differences in the risks of psychiatric disorders were found. Those who underwent abortion were more likely to achieve higher educational levels (2.4 [1.2-5.0]). When compared with those with no pregnancies, both pregnancy groups had elevated risks of most adverse conditions, but the risks were similar before and after conception. In conclusion, the low incidence of teenage pregnancy in Finland suggests that reproductive health services function comparatively well. To add, abortion or childbirth per se was not associated with mental ill-health. However, a substantial proportion of girls who experience a teenage pregnancy have a disadvantaged background and mostly for that reason, they seem to be a risk group for adverse outcomes in adulthood. These girls should be offered extra attention by social and healthcare professionals to prevent unplanned teenage pregnancies and further marginalization.
  • Martelius, Laura (Helsingin yliopisto, 2016)
    The occurrence and significance of vertical lung ultrasound artifacts known as B-lines were investigated in children. B-lines are a non-specific sign of lung pathology. They can be used to estimate lung liquid and to diagnose lung disease. Healthy term infants, children with congenital heart disease undergoing cardiac surgery, and children undergoing computed tomography of the chest were included in the study. We examined whether the mode of delivery influences B-lines in healthy term infants and compared lung ultrasound with static lung compliance, computed tomography, and chest radiographs. B-lines were more numerous in infants born by elective cesarean section than in those born vaginally. Lung edema scores from lung ultrasound and chest radiographs correlated significantly, but B-lines and static lung compliance did not. A consistent association was present between the number of B-lines in ultrasound and the extent of parenchymal changes in computed tomography. B-lines appear suitable for documenting resolution of lung liquid during postnatal adaptation. They may indicate lung edema after cardiac surgery and detect lung disease in children.
  • Hyvönen, Maija (2016)
    Gliomas, malignant brain tumors, are among the most aggressive cancers in adults. Due to their invasive growth, resistance mechanisms and tendency to relapse, the prognosis of high-grade glioma patients is very poor. Therefore, to improve therapeutic strategies it is essential to study molecular mechanisms underlying the progression of gliomas and identify novel, glioma-specific proteins that could be therapeutically targeted. In this study we first mapped vascular markers of malignant gliomas to find potential candidates for tumor targeting. By using in vivo phage display method we identified a peptide, CooP, which, after systemic delivery, specifically homed to brain tumor satellites and their vasculature in mice. Coop was successfully used in tumor imaging and targeted drug delivery. We also identified mammary-derived growth inhibitor (MDGI) as an interacting partner for CooP in the brain tumor tissue and tumor-associated vasculature. Homing peptide-conjugated nanocarriers have shown great potential in targeting various tumors, including gliomas. Therefore, we estimated the potential of CooP peptide in the surface functionalization and targeted delivery of porous silica nanoparticles in vitro and in vivo. CooP-functionalized particles were shown to be stable, non-toxic and suitable for targeting MDGI expressing subcutanous xenografts. In the last part of this study we characterized the expression and function of MDGI in gliomas. Our immunohistochemical analyses revealed abundant MDGI expression in clinical brain tumor specimens and patient-derived gliospheres. In lower-grade glioma patients MDGI expression correlated with poorer overall survival. Importantly, also endothelium-associated expression of MDGI in the clinical samples was observed. Functional in vitro and ex vivo assays demonstrated that MDGI overexpression enhanced the aggressive growth of glioma cells, whereas even more striking changes occurred when MDGI was genetically silenced. MDGI silencing compromised the growth of human gliospheres, altered the expression of stress-related proteins, disrupted mitochondrial function and eventually caused apoptotic cell death. In addition, mass spectrometric analyses revealed significant changes in the intracellular metabolites after MDGI silencing. Together our results show that the glioma-specific CooP peptide can be utilized both in glioma imaging and targeted drug delivery as well as in biofunctionalization and targeting of nanoparticles in vivo. In addition, MDGI, the interacting partner of CooP, is abundantly expressed in malignant gliomas and essential for glioma cell survival. Since MDGI is reachable via intravenously injected agents, it could be a potential candidate for the targeted treatment of gliomas.
  • Holopainen, Tanja (Helsingin yliopisto, 2016)
    Angiopoietins (Angs) and vascular endothelial growth factors (VEGFs) regulate angiogenesis, the formation of new blood vessels, and lymphangiogenesis, the formation of new lymphatic vessels. Angiogenesis is important in cancer, because for continuous growth a primary tumor needs a supply of oxygen and nutrients delivered via blood vessels. In physiological conditions, the lymphatic vasculature serves to collect interstitial fluid as well as to absorb lipid particles. In the context of cancer, lymphatic vessels serve as a route for the metastatic dissemination of tumor cells. This dissertation aimed to explore the role of vascular endothelial growth factor receptor 3 (VEGFR3) and angiopoietin-2 (Ang2) in cancer progression. In particular, the roles of these proteins in the context of tumor angiogenesis and lymphangiogenesis as well as metastasis were investigated in two separate studies. In addition, the effects of the photodynamic ablation of intralymphatic cancer cells and lymphatic vessels on the development of metastases were explored. Furthermore, the effect of the endothelial bone marrow tyrosine kinase in chromosome X (Bmx) on tumor angiogenesis was investigated. We found that the inhibition of VEGFR3 reduces tumor blood vasculature and primary tumor growth. In the second study we found that in-transit tumor cells can be targeted with verteporfin-induced photodynamic therapy. The photodynamic ablation of lymphatic vessels was improved by combining this strategy with anti-lymphangiogenic therapies via the adenovirally mediated soluble expression of the VEGF-C/D trap. Bmx, a member of the Tec non-receptor tyrosine kinase family, was previously shown to promote tumor cell survival, but relatively little is known about the effect of this kinase on tumor biology. Here, we studied the effects of Bmx tumor angiogenesis by using Bmx gene-deleted mice. Some reduction of primary tumor growth in Bmx-/- mice was detected in several isogenic and oncogenic tumor models, along with some attenuation of tumor angiogenesis. Conversely, overexpression of Bmx resulted in increased tumor progression and angiogenesis. Ang2 is known to be a context-dependent agonist of the tyrosine kinase of the Tie2 receptor. Here, we analyzed the effect of Ang2 on metastatic dissemination into the lungs, finding that the overexpression of Ang2 enhanced lung metastasis. In contrast, Ang2 inhibition decreased the occurrence of lung metastases. In the ultrastructural analysis of the metastatic lungs using transmission electron microscopy, anti-Ang2 treatment attenuated tumor-associated changes in metastasis-associated lung capillaries. This dissertation demonstrates that the blockade of VEGFR3 inhibits tumor angiogenesis, and that the inhibition of Ang2 inhibits lymphatic and lung metastasis and improves endothelial integrity. Furthermore, we demonstrated the ability of photodynamic therapy to eradicate lymphatic vessels and intralymphatic cancer cells. These data provide a rationale for developing new cancer therapies targeting the lymphatics in order to reduce metastasis and tumor progression. Taken together, these results provide new insights into endothelial tyrosine kinase-mediated angiogenesis, tumor lymphangiogenesis, and vascular-based therapeutic strategies in cancer.
  • Lahtela, Jenni (Helsingin Yliopisto, 2016)
    Lung cancer is the leading cause of cancer related deaths worldwide. It is characterised with a high level of intra- and intertumour heterogeneity. Large lung cancer sequencing efforts have identified clear histopathology-specific genetic alteration patterns, which in the cases of lung adenocarcinomas are applied in clinics to direct treatment. Furthermore, lung cancer immunotherapy approaches have recently shown promising results in clinical trials. However, a deeper understanding of the functional importance of novel lung cancer genes as well as the lung cancer-related niche and cell type specific propensities leading to molecular and microenvironmental tumour heterogeneity is needed to better translate the growing amount of information to patient stratified treatments. The first part of this thesis work concentrated on the functional in vitro and in vivo investigation of putative tumour suppressive characteristics of the EPH receptor A3 (EPHA3), a gene commonly mutated in human lung cancers. Our in vitro findings supported the tumour suppressive characteristics of EPHA3 and indicated that EPHA3-mediated tumour suppression was specifically dependent on its kinase activity. However, our in vivo investigation demonstrated that loss of EphA3 does not co-operate with two known genetic alterations of human lung cancer in murine lung tumourigenesis nor it effects lung morphogenesis. Hence, we conclude that our study demonstrates how functional validation of putative cancer genes can be challenged by biological complexity, which may result in acquired compensation or different functional roles in human and mice. The results from the second part of this thesis work showed that cells in the airways of mouse lungs had a higher propensity to develop faster growing and progressing lung tumours than the cells in the distal alveolar space when exposed to known lung cancer genetic alterations, namely expression of oncogenic Kras and loss of Lkb1 (KL). The lung tumours originated from the airways were predominantly classified as adenosquamous carcinomas (ASCs). ASCs showed elevated levels of genes associated with immunosuppression and a notable immune cell infiltration with an increase in the amount of possible myeloid-derived suppressor cells (MDSCs). The KL ASC model may thus represent a relevant preclinical model for the study of anti-MDSC immune therapy as a treatment for ASCs, which in humans represent a rare but aggressive type of lung cancer. Thus, the findings in this thesis work highlight the importance of the functional niche in the progression of lung cancer and, therefore, possibly affecting a response to treatment. Niche-specific investigation of lung cancer genetic alterations thus leads to a more accurate stratification of the preclinical in vivo models, simultaneously revealing relevant molecular mechanisms underlying lung cancer heterogeneity.
  • Saukkonen, Suvi (2015)
    Disruptive behavior is one of the most common psychiatric problems in children and adolescents. Psychopathic-like features designate a group of children and adolescents manifesting a severe, aggressive, and persistent pattern of disruptive behavior. These features have many similarities with adult psychopathy. More research is needed to gain an understanding of the different developmental trajectories for psychopathic-like behavior style in children and adolescents. The current study aimed to investigate cognitive, psychosocial, and personality-related characteristics associated with disruptive behavior in children and adolescents. In addition, psychometric properties of one of the psychopathy measures, Antisocial Process Screening Device Self-Report (APSD-SR), were evaluated. In the first study, the working memory (WM) function of school-aged children with Oppositional Defiant Disorder (ODD)/Conduct Disorder (CD) was compared with age- and gender-matched normative controls. The comorbid diagnosis of Attention-Deficit/Hyperactivity Disorder (ADHD) was controlled for in the patient group. WM function was examined using n-back tasks. The main finding of this study was that patients performed worse on WM tasks than controls, even after controlling for the diagnosis of ADHD. The results suggest that children with disruptive behavior disorders (i.e. ODD and CD) have executive function (EF) deficits, like poor WM, that are not accounted for the comorbid diagnosis of ADHD. The second study, using the data from the Finnish Self-Report Delinquency Study 2012 (FSRD-12), assessed the factor structure and internal consistencies of the APSD-SR in a community sample of 15- to 16-year-old youth (n = 4,855, 51% girls). A three-factor structure of APSD-SR was found to fit the data best, with three meaningful subscales representing narcissism, impulsivity, and callous-unemotional features. The internal consistencies for these subscales were adequate. The results suggest that the self-report version of APSD is a promising screening tool for measuring psychopathic-like features in community youth. The third study used the same FSRD-12 data and examined different types of weapon carrying among youth, and psychosocial and personality-related factors associated with carrying a weapon, especially focusing on the relationship between psychopathic-like features and weapon carrying. The results showed that adolescents carrying weapons had a large cluster of problems in their lives, which were differentiated by the type of weapon carried. In addition, psychopathic-like features were associated with an increased likelihood of carrying any type of weapon (knife, gun, other weapon such as knuckleduster, chain, or pepperspray), but the association was strongest with carrying a gun. This association was significant even after controlling for a variety of psychosocial factors. The findings suggest that psychopathic-like features are strongly related to a higher risk of weapon carrying. Furthermore, the findings underscore the need for a comprehensive evaluation of an adolescent s psychosocial situation, when weapon carrying is identified. Finally, the fourth study investigated the relationship between victimization experiences and psychopathic-like features in youth by using the same FSRD-12 data. The results revealed that victimization was related to psychopathic-like features in youth, even more strongly in girls than in boys. Interestingly, the association between victimization and psychopathic-like features was stronger for recent experiences of victimization than past experiences. The results highlight the need for evaluating victimization experiences when psychopathic-like features are present in youth. Furthermore, some of the juveniles may show psychopathic-like behavior style as a means to cope with trauma.
  • Holma, Tanja (Tanja Holma, 2015)
    Outbreaks of bacteria causing hospital and community acquired infections have dramatically increased in number over the recent years. For example, travelers become colonized by resistant bacteria and may transmit the strains to other people and to medical care settings when they return home. The challenge is new and problematic for laboratories performing bacterial diagnostics. Fast and highly specific tools are needed to identify and characterize resistant bacterial strains in order to prevent outbreaks in local hospitals. This thesis studies the fast identification of virulence and resistance genes of the most important hospital acquired (HA) bacteria. In addition, the applicability of rapid outbreak analysis for HA-bacteria using molecular methods outside of the national reference laboratory was studied. The aim of the study was to establish a sensitive, reliable multiplex-PCR method suitable for daily use in a microbiological diagnostic laboratory and to speed up the reporting of bacteria causing outbreaks. Another aim was to study the usefulness and functionality of a commercial repetitive PCR (DiversiLab) and compare it with reference molecular typing methods. The thesis also gives an overall view of the occurrence of HA-bacteria witnessed in the district of Helsinki and Uusimaa over the recent years. The thesis consists of six studies on four different bacterial species/types causing outbreaks: MRSA (methicillin-resistant Staphylococcus aureus), ESBL (Extended Spectrum Beta-Lactamase)-producing Enterobacteriaceae, Acinetobacter baumannii and Clostridium difficile. Consecutive and retrospective bacterial isolates of the each bacteria were collected and examined. The methods used were conventional multiplex PCR and real-time multiplex PCR. The typing methods were repetitive PCR (DiversiLab), PFGE (pulsed field gel electrophoresis), PCR ribotyping, spa typing, and sequencing-based methods. During this thesis three usable in-house multiplex PCRs were established for the detection of MRSA, carbapenemase genes, and virulence genes in C. difficile. The repetitive PCR method, DiversiLab, was found to be a fast and proprietary typing method, very beneficial in the first-line identification of outbreaks. In addition, the Diversilab system may be used in the comparison of resistant bacterial isolates. The method produced better results with Gram-negatives (ESBL-producing Enterobacteriaceae and A. baumannii). However, the reference methods still serve their purpose in global isolate comparison. In the future, whole genome sequencing will, however, most likely replace contemporary typing methods.
  • Maliniemi, Pilvi (Pilvi Maliniemi, 2015)
    This thesis concentrates on exploring novel agents in the pathogenesis of cutaneous T cell lymphomas (CTCL) and reveals potential new biomarkers and therapeutic targets to improve diagnostics. CTCLs are also known as a heterogeneous group of non-Hodgkin lymphomas. The clinical behavior of these lymphomas varies from benign forms of lymphoproliferative diseases such as lymphomatoid papulosis (LyP) to non-progressive mycosis fungoides (MF) and further to rapidly-progressing leukaemic Sézary syndrome (SS). The incidence of the disease is growing, particularly in Western countries. The mechanisms underlying the disease are still largely unknown and thus far no curative therapy exists. Regarding pathogenesis, it has been suggested that persistent antigen stimulation leads to continuous stimulation of T cells and chronic inflammation, eventually causing the development of a malignant T cell clone. Another hypothesis suggests that a specific viral agent could serve as a triggering factor. The first part of this thesis explores the activity of human endogenous retroviruses (HERVs) in CTCLs. HERVs are retroviral sequences comprising about 8% of the human genome and are derived from ancient retrovirus infections of germ line cells. HERVs have their own promoter areas and genes, some of which encode functional proteins. HERVs may also affect the function of neighbouring genes and influence chromosomal instability (e.g. due to random positioning in the genome). However, HERVs are usually epigenetically silenced, but are reactivated in situtations such as cancer. Healthy tissues also show HERV activity. Moreover, tissue-specificity is typical feature of HERVs. We show that a skin-specific HERV pattern is actively transcribed in MF. We also found that the HERV-W-derived viral protein Syncytin-1 is expressed in morphologically malignant lymphocytes. The second part of this thesis is a unique molecular study on a rare subtype of CTCL. Known as subcutaneous panniculitis-like T cell lymphoma (SPTL), this subtype primarily affects young individuals. The diagnosis of SPTL is challenging and SPTL is usually confused with other panniculitis-related inflammatory diseases. The most important finding of the gene expression profiling was the high expression of indoleamine-2,3-dioxygenase (IDO-1), which catabolises the essential amino acid tryptophan (Trp) into kynurenine (Kyn) metabolites. The enzymatic activity of IDO-1 results in the depletion of Trp in the local microenvironment. Because T cells are particularly sensitive to low Trp levels, the process ultimately leads to immunosuppression and subsequent inhibition of T cell responses. Our results suggest that autoimmune inflammation is underlying the development the disease with the support of an immunosuppressive microenvironment. The last part of this thesis is focused on further studying IDO-1 expression in other CTCL subtypes and lymphoproliferative diseases and also how the tumor microenvironment could be involved in malignant transformation. In this study, we showed that distinct subpopulations of CTCL can be recognized based on IDO1-expressing cells. Especially interesting was the finding that IDO-1 is more extensively expressed in LyP rather than in MF, bearing in mind that LyP is a benign disease. Analyzing the Kyn/Trp ratio from patient sera also showed significant differences between MF and LyP. The Kyn/Trp ratio may therefore serve as a potential prognostic biomarker for evaluating disease activity. Taken together, this study revealed completely new information on gene and protein expression in CTCL. The results will likely be most useful in improving future diagnostics as well as patient care. The results may also be further applied in finding new therapeutic targets and developing novel therapies.
  • Lotsari-Salomaa, Johanna (2015)
    Lynch syndrome (LS) is the most common hereditary colon cancer syndrome with germline mutated DNA mismatch repair (MMR) genes predisposing to early onset colon tumors and various extracolonic tumors. Microsatellite instability (MSI) is a hallmark of the Lynch syndrome, but 15% of sporadic tumors show instability of repeated sequences, too. Endometrial carcinoma is the most common LS related extra-colonial tumor. Whether or not breast carcinoma belongs to the Lynch syndrome tumor spectrum has been under debate because of controversial study results. Epigenetic mechanisms regulate gene expression without altering the DNA sequence. DNA methylation is a well-established epigenetic modification. Colon, endometrial and breast carcinomas and endometrial hyperplasia from 256 Finnish Lynch syndrome families were examined for methylation changes as well as several types of genetic alterations. In addition, Finnish sporadic breast tumors with similar hormone receptor statuses to LS breast carcinomas were analyzed. Sporadic colon tumors with microsatellite unstable (MSI) and microsatellite stable (MSS) subgroups from Finnish and Australian populations were also investigated. The first aim was to investigate whether or not breast tumors belong to the Lynch syndrome tumor spectrum by studying the MMR status and the methylation profiles of several tumor suppressor genes. The second aim was to identify epigenetically regulated miRNA genes relevant for colon and endometrial tumorigenesis. The third aim was to examine tumorigenic mechanisms in the minor subset of colon carcinomas with inactive Wnt signaling. The candidate genes identified in the second and third studies were examined for promoter methylation in patient specimens. Breast carcinomas from LS mutation carriers showed significantly higher frequencies of MSI(35%) and loss of MMR protein expression (65%) compared to breast tumors from proven noncarriers (0%) and sporadic cases (0%). This suggested that breast carcinoma is a likely LS spectrum tumor. MMR deficient breast tumors were diagnosed at age 53. RASSF1, APC, CDH13, and GSTP1 genes were often methylated in LS breast tumors, the same as in tumors from several other organs from LS mutation carriers. The CDKN2B gene showed breast cancer specific methylation. A breast tumor may be the first sign of LS, and therefore female MMR mutation carriers should participate in mammographic surveillance. Novel associations between promoter methylation of tumor-suppressive miRNAs or protein coding tumor suppressor genes and clinical subtypes were observed. miR-132 was frequently hypermethylated in sporadic Finnish and Australian MSI tumors compared to paired normal mucosa. miR-132 hypermethylation correlated with traditional (so called CpG island methylator phenotype; CIMP) markers and were associated with the female gender, later onset, and tumors located in the proximal colon. miR-129-2 hypermethylation was seen in advanced endometrial hyperplasia and may be an early marker for endometrial tumorigenesis. The CMTM3, DGKI, and OPCML genes showed frequent hypermethylation in LS and sporadic colon tumors. The EPCAM and KLK10 genes had LS specific methylation. Colon tumors with inactive Wnt signaling were found to constitute a group of tumors with varying features of chromosomal instability and epigenetic dysregulation.
  • Vlachopoulou, Efthymia (2015)
    The Human Leukocyte Antigen (HLA) region is located on chromosome 6 (6p21.3) and its main function is to regulate the immune system. This region has been associated with several autoimmune and other inflammatory conditions. However, many aspects of these associations remain unknown due to high polymorphism, high linkage disequilibrium (LD) and high gene density of this region that increase the complexity and create computational challenges. In this Thesis, the aim is to investigate the HLA haplotype variation in the Finnish population, to evaluate the HLA imputation using two of the existed softwares and to analyze the HLA association with Acute Coronary Syndrome (ACS). The main question in this study is whether the HLA region varies in the Finnish population compared to other populations and how this variation can influence the results of further analyses. The HLA haplotypes showed great variation in the Finnish population compared to other European populations due to different linkage disequilibrium (LD) structures and this highlights the diversity of the HLA haplotypes among different populations. The most common European haplotype HLA-A*01~HLA-B*08~HLA-DRB1*03 shows a lower frequency in Finland, whereas the enrichment of the haplotype HLA-A*03~HLA-B*35~HLA-DRB1*01 was observed (I). Furthermore, the poor imputation results of the HLA-DRB1 gene enhance the power of the previously mentioned arguments (from I) due to the inaccurate imputation from other European populations. The study emphasizes the need for population-specific reference materials for a successful imputation (II). The diversity of HLA alleles, the variability in the frequencies of Single Nucleotide Polymorphisms (SNP) among populations, the difference of the rare HLA alleles appearing in various populations and the extremely high amount of combinations of HLA alleles (due to high polymorphism) contribute to the low success rates of imputation. The association (OR = 4.43, 95% CI = 3.57-5.50) of a novel BTNL2;HLA-DRA;HLA-DRB1*01-haplotype with the risk of Acute Coronary Syndrome (ACS) was identified (III). The analysis showed the implication of the HLA-DRB1*01 allele and the BTNL2 gene to the inflammation process. These two genes affect the antigen presentation and its immune response. Overall, the associated haplotype appeared approximately in 15% of ACS patients and in 5% of healthy individuals. Furthermore, the survival analysis shows that the HLA-DRB1*01 allele does not affect the survival time after an ACS event. Our results provide more insight into the detailed patterns of the haplotypes in the Finnish population, into the success of the imputation approaches and into the HLA association with ACS. Genetic diversity is depicted on these haplotypes which might lead to different immune responses. These findings help future development of appropriate statistical tools for the HLA region.
  • Rautiola, Juhana (2015)
    Renal cell carcinoma (RCC) is the seventh most common cancer in men and the ninth most common in women and it comprises 2-3% of all malignant tumors in adults. In its localized form, the five-year survival is almost 60%. However, in metastatic renal cell carcinoma (mRCC), the five-year survival has historically remained under 10%. Renal cell carcinoma is highly tolerant to traditional chemotherapy and and recent treatment has instead relied on inhibiting the vascular endothelial growth factor (VEGF) and mammalian target of rapamycin (mTOR) signaling pathways. Since 2005, seven new agents have been approved for the treatment of metastatic RCC. Despite the increased knowledge of RCC, the molecular mechanisms behind acquired and intrinsic resistance to treatment remain largely unknown. Our results demonstrate that hypertension is a strong predictor of good prognosis and response to sunitinib treatment. In addition to hypertension we identified neutropenia and thrombocytopenia as predictive markers of good prognosis. A novel, triple biomarker profile consisting of these adverse events strongly predicts improved survival and outcome in mRCC patients. Results from our investigation of sequential treatment suggest that a newer VEGF receptor inhibitor, pazopanib, has clinically meaningful activity in a patient population where mRCC has progressed through sunitinib treatment and in patients who do not tolerate sunitinib. Encouraged by the fuller understanding of tumor biology, we then investigated the role of an endothelial-cell derived growth factor, angiopoietin-2 (Ang2), on patients outcome. High expression of Ang2 was demonstrated to correlate with a high vessel density and to predict a better response to sunitinib-based first-line therapy.
  • Koskela, Elina (2015)
    Intracranial aneurysms (IAs) affect 2-3% of the population. In Finland, their rupture rate has been exceptionally high. This study aims to evaluate the prevalence and risk factors affecting neuro-ophthalmic findings in patients undergoing treatment for IAs. We also investigate the utility of conventional head computed tomography (CT) in the diagnosis of Terson´s syndrome (TS, vitreous hemorrhage in association with aneurysmal subarachnoid hemorrhage (aSAH)). Patients treated endovascularly or microsurgically for their IAs at the Department of Neurosurgery, Helsinki University Central Hospital, in 2011 prospectively underwent a neuro-ophthalmic examination preoperatively and at 3 days, 14 days, 2 to 4 months, and 6 months postoperatively. Findings suggestive of TS, as independently reviewed by two radiologists, on conventional CT head scans were compared with dilated fundoscopic findings. Participants comprised 121 patients with a ruptured aneurysm and 142 patients with an unruptured intracranial aneurysm (UIA). TS was present on fundoscopy in 13 patients (11%); the overall observed agreement between the two radiologists was 96% (116/121), with a substantial κ of 0.69 (95% CI 0.56-0.82). On average, CT demonstrated sensitivity of 42% and specificity of 97%. Factors independently predicting TS were female gender (OR 5.34, 95% CI 1.05-27.17) and World Federation of Neurosurgical Societies (WFNS) grade (OR 15.05 for grades IV-V vs. I-III, 95% CI 3.07-73.89). For patients with aSAH, the frequencies of a third, fourth, or sixth nerve palsy were 11 (9%; preoperatively), 16 (13%; immediately postoperatively), and 3 (3%; at the last follow-up), compared with corresponding frequencies of 6 (4%), 15 (11%), and 7 (5%) for patients with UIAs. Significant risk factors for postoperative eye movement disorders (EMDs) among patients with UIAs were aneurysm location in the posterior circulation (OR 142.02, 95% CI 20.13-1002.22) and aneurysm size (OR 1.28, for each 1-mm increase in diameter, 95% CI 1.12-1.47). After a follow-up time of 6 months, patients with aSAH presenting with visual field defects (VFDs) for aneurysm- or operation-related reasons numbered 20 (19%); homonymous VFDs were the most prevalent finding, and in logistic regression analysis, they were significantly associated with the Hunt and Hess (H and H) grade (OR 4.45 for grades IV-V vs. I-III, 95% CI 1.21-16.39) and presence of intracranial hemorrhage (OR 8.93, 95% CI 2.14-37.28). By contrast, seven patients (5%) treated for a UIA had VFDs. Poor-grade aSAH (H and H or WFNS grade IV-V) appears to be a strong predictive factor for TS and VFDs. With a high specificity value, conventional head CT scan might prove a useful tool in the diagnosis of TS. Although a common finding in the acute postoperative stage of ruptured and unruptured IAs, EMDs show marked improvement during follow-up.