Oncolytic Adenoviruses with Radiation Therapy for Treatment of Prostate Cancer

Prostate cancer is the most common cancer in males. Although many patients with localized disease can be cured with surgery and radiotherapy, advanced disease and especially castration resistant metastatic disease remains incurable, with a median life expectancy of less than 18 months. Oncolytic adenoviruses (Ads) are a new promising treatment against cancer due to their innate capacity to kill cancer cells. Viral replication in tumor cells leads to oncolysis and production of a multiplicity of new virions that are capable of further destroying cancerous tissue. Oncolytic Ads can be modified for tumor targeted infection and replication and be armed with therapeutic transgenes to maximize the oncolytic effect. Worldwide, clinical trials with oncolytic Ads have demonstrated good safety while the antitumor efficacy remains to be improved. Importantly, the best responses have been reported when oncolytic adenoviruses have been combined with standard cancer treatments, such as chemotherapy and radiation. Further, a challenge in many virotherapy approaches has been the monitoring of virus replication in vivo. Reporter genes have been extensively used as transgenes to evaluate the biodistribution of the virus and activity of specific promoters. However, these techniques are often limited to preclinical evaluation and not amenable to human use. The aim of the thesis was to find and develop new oncolytic Ads with maximum efficacy against metastatic, castration resistant prostate cancer and study them in vitro and in vivo combined to different forms of radiation therapy. Using combination therapy, we were aiming for better antitumor efficacy with reduced side effects. Capsid modified Ads for enhanced transduction were studied. Serotype 3 targeted chimera, Ad5/3, was found to have enhanced infectivity for prostate cancer and was used for developing new viruses for the study. Correlation between Ad-encoded marker peptide secretion and simultaneous viral replication was evaluated and the effects of radiotherapy on viral replication were studied in detail. We found that the repair of double strand breaks caused by ionizing radiation was inhibited by adenoviral proteins and led to autophagic cell death. Both subcutaneous models and intrapulmonary tumor models mimicking metastatic, aggressive disease were used in vivo. Virus efficacy was evaluated by intratumoral injections. Also, intravenous administration was evaluated to study the effectiveness in metastatic disease. Oncolytic adenovirus treatment led to significant tumor growth control and increased the survival rate of the mice. These results were further improved when oncolytic Ads were combined with radiation therapy. Oncolytic Ads expressing human sodium/iodide transporter (hNIS) as a transgene were evaluated for their oncolytic potency and for the functionality of hNIS in vitro and in vivo. Monitoring of viral replication was also assessed using different imaging modalities relative to clinical use. SPECT imaging of tumor-bearing mice was evaluated and combined with simultaneous CT-scanning to obtain important anatomical information on biodistribution, also in a three-dimensional form. It was shown that hNIS-expressing adenoviruses could harbour a bi-functional transgene allowing for localization and imaging of viral replication. Targeted radiotherapy was applied by systemic radioiodide administration and resulted in iodide accumulation into Ad-infected tumor. The combination treatment showed significantly enhanced antitumor efficacy in mice bearing prostate cancer tumors. In summary, the results presented above aim to provide new treatment modalities for castration resistant prostate cancer. Molecular insights were provided for better understanding of the benefits of combined radiation therapy and oncolytic adenoviruses, which will hopefully facilitate the translation of the approach into clinical use for humans.

Methicillin-resistant Staphylococcus aureus in Finland: recent changes in the epidemiology, long-term facility aspects, and phenotypic and molecular detection of isolates

Staphylococcus aureus is one of the most important bacteria that cause disease in humans, and methicillin-resistant S. aureus (MRSA) has become the most commonly identified antibiotic-resistant pathogen in many parts of the world. MRSA rates have been stable for many years in the Nordic countries and the Netherlands with a low MRSA prevalence in Europe, but in the recent decades, MRSA rates have increased in those low-prevalence countries as well. MRSA has been established as a major hospital pathogen, but has also been found increasingly in long-term facilities (LTF) and in communities of persons with no connections to the health-care setting. In Finland, the annual number of MRSA isolates reported to the National Infectious Disease Register (NIDR) has constantly increased, especially outside the Helsinki metropolitan area. Molecular typing has revealed numerous outbreak strains of MRSA, some of which have previously been associated with community acquisition. In this work, data on MRSA cases notified to the NIDR and on MRSA strain types identified with pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and staphylococcal cassette chromosome mec (SCCmec) typing at the National Reference Laboratory (NRL) in Finland from 1997 to 2004 were analyzed. An increasing trend in MRSA incidence in Finland from 1997 to 2004 was shown. In addition, non-multi-drug resistant (NMDR) MRSA isolates, especially those resistant only to methicillin/oxacillin, showed an emerging trend. The predominant MRSA strains changed over time and place, but two internationally spread epidemic strains of MRSA, FIN-16 and FIN-21, were related to the increase detected most recently. Those strains were also one cause of the strikingly increasing invasive MRSA findings. The rise of MRSA strains with SCCmec types IV or V, possible community-acquired MRSA was also detected. With questionnaires, the diagnostic methods used for MRSA identification in Finnish microbiology laboratories and the number of MRSA screening specimens studied were reviewed. Surveys, which focused on the MRSA situation in long-term facilities in 2001 and on the background information of MRSA-positive persons in 2001-2003, were also carried out. The rates of MRSA and screening practices varied widely across geographic regions. Part of the NMDR MRSA strains could remain undetected in some laboratories because of insufficient diagnostic techniques used. The increasing proportion of elderly population carrying MRSA suggests that MRSA is an emerging problem in Finnish long-term facilities. Among the patients, 50% of the specimens were taken on a clinical basis, 43% on a screening basis after exposure to MRSA, 3% on a screening basis because of hospital contact abroad, and 4% for other reasons. In response to an outbreak of MRSA possessing a new genotype that occurred in a health care ward and in an associated nursing home of a small municipality in Northern Finland in autumn 2003, a point-prevalence survey was performed six months later. In the same study, the molecular epidemiology of MRSA and methicillin-sensitive S. aureus (MSSA) strains were also assessed, the results to the national strain collection compared, and the difficulties of MRSA screening with low-level oxacillin-resistant isolates encountered. The original MRSA outbreak in LTF, which consisted of isolates possessing a nationally new PFGE profile (FIN-22) and internationally rare MLST type (ST-27), was confined. Another previously unrecognized MRSA strain was found with additional screening, possibly indicating that current routine MRSA screening methods may be insufficiently sensitive for strains possessing low-level oxacillin resistance. Most of the MSSA strains found were genotypically related to the epidemic MRSA strains, but only a few of them had received the SCCmec element, and all those strains possessed the new SCCmec type V. In the second largest nursing home in Finland, the colonization of S. aureus and MRSA, and the role of screening sites along with broth enrichment culture on the sensitivity to detect S. aureus were studied. Combining the use of enrichment broth and perineal swabbing, in addition to nostrils and skin lesions swabbing, may be an alternative for throat swabs in the nursing home setting, especially when residents are uncooperative. Finally, in order to evaluate adequate phenotypic and genotypic methods needed for reliable laboratory diagnostics of MRSA, oxacillin disk diffusion and MIC tests to the cefoxitin disk diffusion method at both +35°C and +30°C, both with or without an addition of sodium chloride (NaCl) to the Müller Hinton test medium, and in-house PCR to two commercial molecular methods (the GenoType® MRSA test and the EVIGENETM MRSA Detection test) with different bacterial species in addition to S. aureus were compared. The cefoxitin disk diffusion method was superior to that of oxacillin disk diffusion and to the MIC tests in predicting mecA-mediated resistance in S. aureus when incubating at +35°C with or without the addition of NaCl to the test medium. Both the Geno Type® MRSA and EVIGENETM MRSA Detection tests are usable, accurate, cost-effective, and sufficiently fast methods for rapid MRSA confirmation from a pure culture.

Characterisation of antibiotic-resistant psychrotrophic bacteria in raw milk

Dynamics of raw milk associated bacteria during cold storage of raw milk and their antibiotic resistance was reviewed, with focus on psychrotrophic bacteria. This study aimed to investigate the significance of cold storage of raw milk on antibiotic-resistant bacterial population and analyse the antibiotic resistance of the Gram-negative antibiotic-resistant psychrotrophic bacteria isolated from the cold-stored raw milk samples. Twenty-four raw milk samples, six at a time, were obtained from lorries that collected milk from Finnish farms and were stored at 4°C/4 d, 6°C/3 d and 6°C/4 d. Antibiotics representing four classes of antibiotics (gentamicin, ceftazidime, levofloxacin and trimethoprim-sulfamethoxazole) were used to determine the antibiotic resistance of mesophilic and psychrotrophic bacteria during the storage period. A representative number of antibiotic-resistant Gram-negative isolates retrieved from the cold-stored raw milk samples were identified by the phenotypic API 20 NE system and a few isolates by the 16S rDNA gene sequencing. Some of the isolates were further evaluated for their antibiotic resistance by the ATB PSE 5 and HiComb system. The initial average mesophilic counts were found below 105 CFU/mL, suggesting that the raw milk samples were of good quality. However, the mesophilic and psychrotrophic population increased when stored at 4°C/4 d, 6°C/3 d and 6°C/4 d. Gentamicin- and levofloxacin-resistant bacteria increased moderately (P < 0.05) while there was a considerable rise (P < 0.05) of ceftazidime- and trimethoprim-sulfamethoxazole-resistant population during the cold storage. Of the 50.9 % (28) of resistant isolates (total 55) identified by API 20 NE, the majority were Sphingomonas paucimobilis (8), Pseudomonas putida (5), Sphingobacterium spiritivorum (3) and Acinetobacter baumanii (2). The analysis by ATB PSE 5 system suggested that 57.1% of the isolates (total 49) were multiresistant. This study showed that the dairy environment harbours multidrug-resistant Gramnegative psychrotrophic bacteria and the cold chain of raw milk storage amplifies the antibioticresistant psychrotrophic bacterial population.

Genomic alterations in Myeloproliferative Neoplasms and Myelodysplastic Syndromes

Myeloproliferative neoplasms (MPN) and myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic disorders whose etiology and molecular pathogenesis are poorly understood. During the past decade, enormous developments in microarray technology and bioinformatics methods have made it possible to mine novel molecular alterations in a large number of malignancies, including MPN and MDS, which has facilitated the detection of new prognostic, predictive and therapeutic biomarkers for disease stratification. By applying novel microarray techniques, we profiled copy number alterations and microRNA (miRNA) expression changes in bone marrow aspirate and blood samples. In addition, we set up and validated an miRNA expression test for bone marrow core biopsies in order to utilize the large archive material available in many laboratories. We also tested JAK2 mutation status and compare it with the in vitro growth pattern of hematologic progenitors cells. In the study focusing on 100 MPN cases, we detected a Janus kinase 2 (JAK2) mutation in 71 cases. We observed spontaneous erythroid colony growth in all mutation-positive cases in addition to nine mutation negative cases. Interestingly, seven JAK2V167F negative ET cases showed spontaneous megakaryocyte colony formation, one case of which also harbored a myeloproliferative leukemia virus oncogene (MPL) mutation. We studied copy number alterations in 35 MPN and 37 MDS cases by using oligonucleotide-based array comparative hybridization (array CGH). Only one essential thrombocythemia (ET) case presented copy number alterations in chromosomes 1q and 13q. In contrast, MDS cases were characterized by numerous novel cryptic chromosomal aberrations with the most common copy number losses at 5q21.3q33.1 and 7q22.1q33, while the most common copy number gain was trisomy 8. As for the study of the bone marrow core biopsy samples, we showed that even though these samples were embedded in paraffin and underwent decalcification, they were reliable sources of miRNA and suitable for array expression analysis. Further, when studying the miRNA expression profiles of the 19 MDS cases, we found that, compared to controls, two miRNAs (one human Epstein-Barr virus (miR-BART13) miRNA and one human (has-miR-671-5p) miRNA) were downregulated, whereas two other miRNAs (hsa-miR-720 and hsa-miR-21) were upregulated. However, we could find no correlation between copy number alterations and microRNA expression when integrating these two data. This thesis brings to light new information about genomic changes implicated in the development of MPN and MDS, and also underlines the power of applying genome-wide array screening techniques in neoplasias. Rapid advances in molecular techniques and the integration of different genomic data will enable the discovery of the biological contexts of many complex disorders, including myeloid neoplasias.

Molecular methods for the epidemiological analysis of methicillin-resistant Staphylococcus aureus (MRSA) and Streptococcus pneumoniae

Methicillin-resistant Staphylococcus aureus (MRSA) and Streptococcus pneumoniae are major health problems worldwide, both found in symptomless carriage but also causing even life-threatening infections. The aim of this thesis was to characterise MRSA and S. pneumoniae in detail by using several molecular typing methods for various epidemiological purposes: clonality analysis, epidemiological surveillance, outbreak investigation, and virulence factor analysis. The characteristics of MRSA isolates from the strain collection of the Finnish National Infectious Disease Register (NIDR) and pneumococcal isolates collected from military recruits and children with acute otitis media (AOM) were analysed using various typing techniques. Antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), spa typing, staphylococcal cassette chromosome mec (SCCmec) typing, and the detection of Panton-Valentine leukocidin (PVL) genes were performed for MRSA isolates. Pneumococcal isolates were analysed using antimicrobial susceptibility testing, serotyping, MLST, and by detecting pilus islet 1 (PI-1) and 2 (PI-2) genes. Several international community- and hospital-associated MRSA clones were recognised in Finland. The genetic diversity among MRSA FIN-4 isolates and among FIN-16 isolates was low. Overall, MRSA blood isolates from 1997 to 2006 were genetically diverse. spa typing was found to be a highly discriminatory, rapid and accurate typing method and it also qualifies as the primary typing method in countries with a long history of PFGE-based MRSA strain nomenclature. However, additional typing by another method, e.g. PFGE, is needed in certain situations to be able to provide adequate discrimination for epidemiological surveillance and outbreak investigation. An outbreak of pneumonia was associated with one pneumococcal strain among military recruits, previously healthy young men living in a crowded setting. The pneumococcal carriage rate after the outbreak was found to be exceptionally high. PI-1 genes were detected at a rather low prevalence among pneumococcal isolates from children with AOM. However, the study demonstrated that PI-1 has existed among pneumococcal isolates prior to pneumococcal conjugate vaccine and the increased antimicrobial resistance era. Moreover, PI-1 was found to associate with the serotype rather than the genotype. This study adds to our understanding of the molecular epidemiology of MRSA strains in Finland and the importance of an appropriate genotyping method to be able to perform high-level laboratory-based surveillance of MRSA. Epidemiological and molecular analyses of S. pneumoniae add to our knowledge of the characteristics of pneumococcal strains in Finland.