Activator Protein-1 and Epidermal Growth Factor Receptor Interplay : In Vivo & In Vitro Studies

Critical cellular decisions such as should the cell proliferate, migrate or differentiate, are regulated by stimulatory signals from the extracellular environment, like growth factors. These signals are transformed to cellular responses through their binding to specific receptors present at the surface of the recipient cell. The epidermal growth factor receptor (EGF-R/ErbB) pathway plays key roles in governing these signals to intracellular events and cell-to-cell communication. The EGF-R forms a signaling network that participates in the specification of cell fate and coordinates cell proliferation. Ligand binding triggers receptor dimerization leading to the recruitment of kinases and adaptor proteins. This step simultaneously initiates multiple signal transduction pathways, which result in activation of transcription factors and other target proteins, leading to cellular alterations. It is known that mutations of EGF-R or in the components of these pathways, such as Ras and Raf, are commonly involved in human cancer. The four best characterized signaling pathways induced by EGF-R are the mitogen-activated protein kinase cascades (MAPKs), the lipid kinase phosphatidylinositol 3 kinase (PI3K), a group of transcription factors called Signal Transducers and Activator of Transcription (STAT), and the phospholipase Cγ; (PLCγ) pathways. The activation of each cascade culminates in kinase translocation to the nucleus to stimulate various transcription factors including activator protein 1 (AP-1). AP-1 family proteins are basic leucine zipper (bZIP) transcription factors that are implicated in the regulation of a variety of cellular processes (proliferation and survival, growth, differentiation, apoptosis, cell migration, transformation). Therefore, the regulation of AP-1 activity is critical for the decision of cell fate and their deregulated expression is widely associated with many types of cancers, such as breast and prostate cancers. The aims of this study were to characterize the roles of EGF-R signaling during normal development and malignant growth in vitro and in vivo using different cell lines and tissue samples. We show here that EGF-R regulates cell proliferation but is also required for regulation of AP-1 target gene expression in fibroblasts in a MAP-kinase mediated manner. Furthermore, EGF-R signaling is essential for enterocyte proliferation and migration during intestinal maturation. EGF-R signaling network, especially PI3-K-Akt pathway mediated AP-1 activity is involved in cellular survival in response to ionizing radiation. Taken together, these results elucidate the connection of EGF-R and AP-1 in various cellular contexts and show their importance in the regulation of cellular behaviour presenting new treatment cues for intestinal perforations and cancer therapy.

Resistance Mechanisms of Non-Hodgkin Lymphomas against Rituximab Treatment

Rituximab, a monoclonal antibody against B-cell specific CD20 antigen, is used for the treatment of non-Hodgkin lymphomas (NHL) and chronic lymphatic leukemia. In combination with chemotherapeutics rituximab has remarkably improved the outcome of NHL patients, but a vast variation in the lengths of remissions remains and the outcome of individual patients is difficult to predict. This thesis has searched for an explanation for this by studying the effector mechanisms of rituximab and by comparing gene expression in lymphoma tissue samples of patients with long- and short-term survival. This work demonstrated that activation of complement (C) system is in vitro more efficient effector mechanism of rituximab than cellular mechanisms or apoptosis. Activation of the C system was also shown in vivo during rituximab treatment. However, intravenously administered rituximab could not enter the cerebrospinal fluid, and neither C activation nor removal of lymphoma cells was observed in central nervous system. In vitro cytotoxicity assays showed that rituximab-induced cell killing could be markedly improved with simultaneous neutralization of the C regulatory proteins CD46 (Membrane cofactor protein), CD55 (Decay-accelerating factor), and CD59 (protectin). In a retrospective study of follicular lymphoma (FL) patients, low lymphoma tissue mRNA expressions of CD59 and CD55 were associated with a good prognosis and in a progressive flow cytometry study high expression of CD20 relative to CD55 was correlated to a longer progression free survival. Gene expression profile analysis revealed that expression of certain often cell cycle, signal transduction or immune response related genes correlate with clinical outcome of FL patients. Emphasizing the role of tumor microenvironment the best differentiating genes Smad1 and EphA1 were demonstrated to be mainly expressed in the non-malignant cells of tumors. In conclusion, this thesis shows that activation of the C system is a clinically important effector mechanism of rituximab and that microenvironment factor in tumors and expression of C regulatory proteins affect markedly the efficacy of immunochemotherapy. This data can be used to identify more accurately the patients for whom immunochemotherapy is given. It may also be beneficial in development of rituximab-containing and other monoclonal antibody therapies against cancer.

F-18 Labelling Synthesis, Radioanalysis and Evaluation of A Dopamine Transporter and A Hypoxia Tracer

Positron emission tomography (PET) is an imaging technique in which radioactive positron-emitting tracers are used to study biochemical and physiological functions in humans and in animal experiments. The use of PET imaging has increased rapidly in recent years, as have special requirements in the fields of neurology and oncology for the development of syntheses for new, more specific and selective radiotracers. Synthesis development and automation are necessary when high amounts of radioactivity are needed for multiple PET studies. In addition, preclinical studies using experimental animal models are necessary for evaluating the suitability of new PET tracers for humans. For purification and analysing the labelled end-product, an effective radioanalytical method combined with an optimal radioactivity detection technique is of great importance. In this study, a fluorine-18 labelling synthesis method for two tracers was developed and optimized, and the usefulness of these tracers for possible prospective human studies was evaluated. N-(3-[18F]fluoropropyl)-2β-carbomethoxy-3β-(4-fluorophenyl)nortropane ([18F]β-CFT-FP) is a candidate PET tracer for the dopamine transporter (DAT), and 1H-1-(3-[18F]fluoro-2-hydroxypropyl)-2-nitroimidazole ([18F]FMISO) is a well-known hypoxia marker for hypoxic but viable cells in tumours. The methodological aim of this thesis was to evaluate the status of thin-layer chromatography (TLC) combined with proper radioactivity detection measurement systems as a radioanalytical method. Three different detection methods of radioactivity were compared: radioactivity scanning, film autoradiography, and digital photostimulated luminescence (PSL) autoradiography. The fluorine-18 labelling synthesis for [18F]β-CFT-FP was developed and carbon-11 labelled [11C]β-CFT-FP was used to study the specificity of β-CFT-FP for the DAT sites in human post-mortem brain slices. These in vitro studies showed that β-CFT-FP binds to the caudate-putamen, an area rich of DAT. The synthesis of fluorine-18 labelled [18F]FMISO was optimized, and the tracer was prepared using an automated system with good and reproducible yields. In preclinical studies, the action of the radiation sensitizer estramustine phosphate on the radiation treatment and uptake of [18F]FMISO was evaluated, with results of great importance for later human studies. The methodological part of this thesis showed that radioTLC is the method of choice when combined with an appropriate radioactivity detection technique. Digital PSL autoradiography proved to be the most appropriate when compared to the radioactivity scanning and film autoradiography methods. The very high sensitivity, good resolution, and wide dynamic range of digital PSL autoradiography are its advantages in detection of β-emitting radiolabelled substances.

Prognostic molecular factors and algorithms in diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) is the most common of the non-Hodgkin lymphomas. As DLBCL is characterized by heterogeneous clinical and biological features, its prognosis varies. To date, the International Prognostic Index has been the strongest predictor of outcome for DLBCL patients. However, no biological characters of the disease are taken into account. Gene expression profiling studies have identified two major cell-of-origin phenotypes in DLBCL with different prognoses, the favourable germinal centre B-cell-like (GCB) and the unfavourable activated B-cell-like (ABC) phenotypes. However, results of the prognostic impact of the immunohistochemically defined GCB and non-GCB distinction are controversial. Furthermore, since the addition of the CD20 antibody rituximab to chemotherapy has been established as the standard treatment of DLBCL, all molecular markers need to be evaluated in the post-rituximab era. In this study, we aimed to evaluate the predictive value of immunohistochemically defined cell-of-origin classification in DLBCL patients. The GCB and non-GCB phenotypes were defined according to the Hans algorithm (CD10, BCL6 and MUM1/IRF4) among 90 immunochemotherapy- and 104 chemotherapy-treated DLBCL patients. In the chemotherapy group, we observed a significant difference in survival between GCB and non-GCB patients, with a good and a poor prognosis, respectively. However, in the rituximab group, no prognostic value of the GCB phenotype was observed. Likewise, among 29 high-risk de novo DLBCL patients receiving high-dose chemotherapy and autologous stem cell transplantation, the survival of non-GCB patients was improved, but no difference in outcome was seen between GCB and non-GCB subgroups. Since the results suggested that the Hans algorithm was not applicable in immunochemotherapy-treated DLBCL patients, we aimed to further focus on algorithms based on ABC markers. We examined the modified activated B-cell-like algorithm based (MUM1/IRF4 and FOXP1), as well as a previously reported Muris algorithm (BCL2, CD10 and MUM1/IRF4) among 88 DLBCL patients uniformly treated with immunochemotherapy. Both algorithms distinguished the unfavourable ABC-like subgroup with a significantly inferior failure-free survival relative to the GCB-like DLBCL patients. Similarly, the results of the individual predictive molecular markers transcription factor FOXP1 and anti-apoptotic protein BCL2 have been inconsistent and should be assessed in immunochemotherapy-treated DLBCL patients. The markers were evaluated in a cohort of 117 patients treated with rituximab and chemotherapy. FOXP1 expression could not distinguish between patients, with favourable and those with poor outcomes. In contrast, BCL2-negative DLBCL patients had significantly superior survival relative to BCL2-positive patients. Our results indicate that the immunohistochemically defined cell-of-origin classification in DLBCL has a prognostic impact in the immunochemotherapy era, when the identifying algorithms are based on ABC-associated markers. We also propose that BCL2 negativity is predictive of a favourable outcome. Further investigational efforts are, however, warranted to identify the molecular features of DLBCL that could enable individualized cancer therapy in routine patient care.

Hematopoietic Stem Cell Development and Transcriptional Regulation

The continuous production of blood cells, a process termed hematopoiesis, is sustained throughout the lifetime of an individual by a relatively small population of cells known as hematopoietic stem cells (HSCs). HSCs are unique cells characterized by their ability to self-renew and give rise to all types of mature blood cells. Given their high proliferative potential, HSCs need to be tightly regulated on the cellular and molecular levels or could otherwise turn malignant. On the other hand, the tight regulatory control of HSC function also translates into difficulties in culturing and expanding HSCs in vitro. In fact, it is currently not possible to maintain or expand HSCs ex vivo without rapid loss of self-renewal. Increased knowledge of the unique features of important HSC niches and of key transcriptional regulatory programs that govern HSC behavior is thus needed. Additional insight in the mechanisms of stem cell formation could enable us to recapitulate the processes of HSC formation and self-renewal/expansion ex vivo with the ultimate goal of creating an unlimited supply of HSCs from e.g. human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPS) to be used in therapy. We thus asked: How are hematopoietic stem cells formed and in what cellular niches does this happen (Papers I, II)? What are the molecular mechanisms that govern hematopoietic stem cell development and differentiation (Papers III, IV)? Importantly, we could show that placenta is a major fetal hematopoietic niche that harbors a large number of HSCs during midgestation (Paper I)(Gekas et al., 2005). In order to address whether the HSCs found in placenta were formed there we utilized the Runx1-LacZ knock-in and Ncx1 knockout mouse models (Paper II). Importantly, we could show that HSCs emerge de novo in the placental vasculature in the absence of circulation (Rhodes et al., 2008). Furthermore, we could identify defined microenvironmental niches within the placenta with distinct roles in hematopoiesis: the large vessels of the chorioallantoic mesenchyme serve as sites of HSC generation whereas the placental labyrinth is a niche supporting HSC expansion (Rhodes et al., 2008). Overall, these studies illustrate the importance of distinct milieus in the emergence and subsequent maturation of HSCs. To ensure proper function of HSCs several regulatory mechanisms are in place. The microenvironment in which HSCs reside provides soluble factors and cell-cell interactions. In the cell-nucleus, these cell-extrinsic cues are interpreted in the context of cell-intrinsic developmental programs which are governed by transcription factors. An essential transcription factor for initiation of hematopoiesis is Scl/Tal1 (stem cell leukemia gene/T-cell acute leukemia gene 1). Loss of Scl results in early embryonic death and total lack of all blood cells, yet deactivation of Scl in the adult does not affect HSC function (Mikkola et al., 2003b. In order to define the temporal window of Scl requirement during fetal hematopoietic development, we deactivated Scl in all hematopoietic lineages shortly after hematopoietic specification in the embryo . Interestingly, maturation, expansion and function of fetal HSCs was unaffected, and, as in the adult, red blood cell and platelet differentiation was impaired (Paper III)(Schlaeger et al., 2005). These findings highlight that, once specified, the hematopoietic fate is stable even in the absence of Scl and is maintained through mechanisms that are distinct from those required for the initial fate choice. As the critical downstream targets of Scl remain unknown, we sought to identify and characterize target genes of Scl (Paper IV). We could identify transcription factor Mef2C (myocyte enhancer factor 2 C) as a novel direct target gene of Scl specifically in the megakaryocyte lineage which largely explains the megakaryocyte defect observed in Scl deficient mice. In addition, we observed an Scl-independent requirement of Mef2C in the B-cell compartment, as loss of Mef2C leads to accelerated B-cell aging (Gekas et al. Submitted). Taken together, these studies identify key extracellular microenvironments and intracellular transcriptional regulators that dictate different stages of HSC development, from emergence to lineage choice to aging.

Auger-emitter radiochemotherapy in squamous cell cancers : in vitro and in vivo experiments with In-111-BLMC

Head and neck squamous cell cancer (HNSCC) is the sixth most common cancer worldwide. Despite advances in combined modality therapy (surgery, radiotherapy, chemotherapy) the 5-year survival rate in stage III and IV disease remains at 40% - 60%. Short-range Auger-electron emitters, such as In-111 and In-114m, tagged with a drug, molecule, peptide, protein or nanoparticles brought in close proximity to nuclear DNA represent a fascinating alternative for treating cancer. In this thesis, we studied the usefulness of Indium-111-bleomycin complex (In-111-BLMC) in the diagnostics and potential therapy of HNSCC using in vitro HNSCC cell lines, in vivo nude mice, and in vivo HNSCC patients. In in vitro experiments with HNSCC cell lines, the sensitivity to external beam radiation, BLM, In-111-BLMC, and In-111-Cl3 was studied using the 96-well plate clonogenic assay. The influence of BLM and In-111-BLMC on the cell cycle was measured with flow cytometry. In in vivo nude mice xenograft studies, the activity ratios of In-111-BLMC were obtained in gamma camera images. The effect of In-111-BLMC in HNSCC xenografts was studied. In in vivo patient studies, we determined the tumor uptake of In-111-BLMC with gamma camera and the radioactivity from tumor samples using In-111-BLMC with specific activity of 75, 175, or 375 MBq/mg BLM. The S values, i.e. absorbed dose in a target organ per cumulated activity in a source organ, were simulated for In-111 and In-114m. In vitro studies showed the variation of sensitivity for external beam radiation, BLM, and In-111-BLMC between HNSCC cell lines. IC50 values for BLM were 1.6-, 1.8-, and 2.1-fold higher than In-111-BLMC (40 MBq/mg BLM) in three HNSCC cell lines. Specific In-111 activity of 40 MBq/mgBLM was more effective in killing cells than specific In-111 activity of 195MBq/mgBLM (p=0.0023). In-111-Cl3 alone had no killing effect. The percentage of cells in the G2/M phase increased after exposure to BLM and especially to In-111-BLMC in the three cell lines studied, indicating a G2/M block. The tumor-seeking behavior was shown in the in vivo imaging study of xenografted mice. BLM and In-111-BLMC were more effective than NaCl in reducing xenografted tumor size in HNSCC. The uptake ratios received from gamma images in the in vivo patient study varied from 1.2 to 2.8 in malignant tumors. However, the uptake of In-111-BLMC was unaffected by increasing the injected activity. A positive correlation existed between In-111-BLMC uptake, Ki-67/MIB activity, and number of mitoses. Regarding the S values, In-114m delivered a 4-fold absorbed radiation dose into the tumor compared with In-111, and thus, In-114m-BLMC might be more effective than In-111-BLMC at the DNA level. Auger-electron emitters, such as In-111 and In-114m, might have potential in the treatment of HNSCC. Further studies are needed to develop a radiopharmaceutical agent with appropriate physical properties of the radionuclide and a suitable carrier to bring it to the targeted tissue.

Cyclins in Breast Cancer

Breast cancer is the most common malignancy in women in Western countries. It is a heterogeneous disease with varying biological characteristics and aggressiveness. Family history is one of the strongest predisposing factors for breast cancer. The known susceptibility genes explain only around 25% of all familial breast cancers. At least part of the unknown familial aggregation may be caused by several low-penetrance variants that occur commonly in the general population. Cyclins are cell cycle-regulating proteins. Cyclin expression oscillates during the cell cycle and is under strict control. In cancer cells, cyclin expression often becomes deregulated, leading to uncontrolled cell division and proliferation, one of the hallmarks of cancer. In this study, we investigated the role of cyclins in breast cancer predisposition, pathogenesis, and tumor behavior. Cyclin A immunohistochemistry was evaluated both on traditional large sections and on tissue microarrays (TMA). The concordance of the results was good, indicating that TMA is a reliable method for studying cyclin expression in breast cancer. The expression of cyclins D1, E, and B1 was studied among 1348 invasive breast cancers on TMA. Familial BRCA1/2-mutation negative tumors had significantly more often low cyclin E and high cyclin D1 expression than BRCA1/2 related or sporadic tumors. Unique cyclin E and D1 expression patterns among familial non-BRCA1/2 breast cancers may reflect different predisposition and pathogenesis in these groups and help to differentiate mutation-positive from mutation-negative familial cancers. High cyclin E expression was associated with an aggressive breast cancer phenotype and was an independent marker of poor metastasis-free survival. High cyclin D1 was associated with high grade and high proliferation among estrogen receptor (ER)-positive but with low grade and low proliferation among ER-negative breast cancers. Among ER-positive cancers not treated with chemotherapy, high cyclin D1 showed a trend towards shorter metastasis-free survival. These results suggest that different mechanisms may drive proliferation in ER-negative and -positive breast cancers and that cyclin D1 has a particularly important role in tumorigenesis of hormone receptor-positive breast cancer. High cyclin B1 expression was associated with aggressive breast cancer features and had an independent impact on survival. The results suggest that cyclin B1 immunohistochemistry is a method that could easily be adapted for routine use and is an independent prognostic factor, adding specificity to prognostic evaluation conducted with traditional markers. A commonly occurring cyclin D1 gene polymorphism A870G was associated with increased breast cancer risk in a large material of Finnish and Canadian breast cancer patients. The interaction of the high-activity alleles of cyclin D1 gene and estrogen metabolism gene COMT conferred an even higher risk. These results show that cyclin D1 and COMT act synergistically to contribute to breast cancer progression and that individual risk for breast cancer can be altered by the combined effect of polymorphisms with low-penetrance alleles. By investigating critical cell cycle regulator protein cyclins, we revealed new aspects of breast cancer predisposition, pathogenesis, and clinical course.

Iodine Brachytherapy for Large Uveal Melanomas

Uveal melanoma is the most common primary intraocular malignancy in adults. Vision in the affected eye is threatened by both the tumor and side-effects from the treatments currently available. Poor prognosis for saving vision increases with tumor size and, consequently, enucleation has been the treatment of choice for large uveal melanomas in most centers. However, increasing evidence suggests that no survival benefit is gained (nor lost) by enucleation as compared to eye-conserving methods. The Helsinki University Eye Hospital has since 1990 offered episcleral iodine-125 plaque brachytherapy (IBT) for all patients unwilling to undergo enucleation for a large uveal melanoma. The primary aim of this study was to assess survival, local tumor recurrence and preservation of the eye and vision after IBT in a population-based series of 97 patients with uveal melanomas classified as large by the Collaborative Ocular Melanoma Study (COMS) criteria. Further aims included reporting the incidence of side-effects and assessing the role of intraocular dose distribution and clinical risk factors in their development. Finally, means to improve the current treatment were investigated by using computer models to compare existing plaques with collimating ones and by comparing the outcome of a subgroup of 54 IBT patients with very thick tumors with 33 patients with similarly-sized tumors managed with transscleral local resection (TSR) in Liverpool, United Kingdom. Kaplan-Meier estimates of all-cause and melanoma-specific survival at 5 years after IBT were 62% and 65%, respectively, and visually comparable with the survival experience of patients reported after enucleation by the COMS. Local recurrence developed in 6% of eyes and 84% of eyes were conserved at 5 years. Visual prognosis was guarded with 11% avoiding loss of 20/70 vision and 26% avoiding loss of 20/400 vision in the tumor eye at 2 years. Large tumor height and short distance from the posterior pole were independently associated with loss of vision. Using cumulative incidence analysis to account for competing risks, such as enucleation and metastatic death, the 5-year incidence of cataract after IBT was 79%, glaucoma 60%, optic neuropathy 46%, maculopathy 52%, persistent or recurring retinal detachment (RD) 25%, and vitreous hemorrhage 36%. In multivariate competing risks regression models, increasing tumor height was associated with cataract, iris neovascularization and RD. Maculopathy and optic neuropathy were associated with distance from the tumor to the respective structure. Median doses to the tumor apex, macula and optic disc were 81 Gy (range, 40-158), 79 Gy (range, 12-632), and 83 Gy (range, 10-377), respectively. Dose to the optic disc was independently associated with optic neuropathy, and both dose to the optic disc and dose to the macula predicted vision loss after IBT. Simulated treatment using collimating plaques resulted in clinically meaningful reduction in both optic disc (median reduction, 30 Gy) and macular (median reduction, 36 Gy) doses as compared to the actual treatment with standard plaques. In the subgroup of patients with uveal melanomas classified as large because of tumor height, cumulative incidence analysis revealed that while long-term preservation of 20/70 vision was rare after both IBT and TSR, preservation of 20/400 vision was better after TSR (32% vs. 5% at 5 years). In multivariate logistic regression models, TSR was independently associated with better preservation of 20/400 vision (OR 0.03 at 2 years, P=0.005) No cases of secondary glaucoma were observed after TSR and optic neuropathy was rare. However, local tumor recurrence was more common after TSR than it was after IBT (Cumulative incidence 41% vs. 7% at 5 years, respectively). In terms of survival, IBT seems to be a safe alternative to enucleation in managing large uveal melanomas. Local tumor control is no worse than with medium-sized tumors and the chances of avoiding secondary enucleation are good. Unfortunately, side-effects from radiotherapy are frequent, especially in thick tumors, and long-term prognosis of saving vision is consequently guarded. Some complications can be limited by using collimating plaques and by managing uveal melanomas that are large because of tumor height with TSR instead of IBT. However, the patient must be willing to accept a substantial risk of local tumor recurrence after TSR and it is best suited for cases in which the preservation of vision in the tumor eye is critical.

Accuracy of absorbed dose in external photon beam radiotherapy : what level is sufficient and how to approach it?

Radiation therapy (RT) plays currently significant role in curative treatments of several cancers. External beam RT is carried out mostly by using megavoltage beams of linear accelerators. Tumor eradication and normal tissue complications correlate to dose absorbed in tissues. Normally this dependence is steep and it is crucial that actual dose within patient accurately correspond to the planned dose. All factors in a RT procedure contain uncertainties requiring strict quality assurance. From hospital physicist´s point of a view, technical quality control (QC), dose calculations and methods for verification of correct treatment location are the most important subjects. Most important factor in technical QC is the verification that radiation production of an accelerator, called output, is within narrow acceptable limits. The output measurements are carried out according to a locally chosen dosimetric QC program defining measurement time interval and action levels. Dose calculation algorithms need to be configured for the accelerators by using measured beam data. The uncertainty of such data sets limits for best achievable calculation accuracy. All these dosimetric measurements require good experience, are workful, take up resources needed for treatments and are prone to several random and systematic sources of errors. Appropriate verification of treatment location is more important in intensity modulated radiation therapy (IMRT) than in conventional RT. This is due to steep dose gradients produced within or close to healthy tissues locating only a few millimetres from the targeted volume. The thesis was concentrated in investigation of the quality of dosimetric measurements, the efficacy of dosimetric QC programs, the verification of measured beam data and the effect of positional errors on the dose received by the major salivary glands in head and neck IMRT. A method was developed for the estimation of the effect of the use of different dosimetric QC programs on the overall uncertainty of dose. Data were provided to facilitate the choice of a sufficient QC program. The method takes into account local output stability and reproducibility of the dosimetric QC measurements. A method based on the model fitting of the results of the QC measurements was proposed for the estimation of both of these factors. The reduction of random measurement errors and optimization of QC procedure were also investigated. A method and suggestions were presented for these purposes. The accuracy of beam data was evaluated in Finnish RT centres. Sufficient accuracy level was estimated for the beam data. A method based on the use of reference beam data was developed for the QC of beam data. Dosimetric and geometric accuracy requirements were evaluated for head and neck IMRT when function of the major salivary glands is intended to be spared. These criteria are based on the dose response obtained for the glands. Random measurement errors could be reduced enabling lowering of action levels and prolongation of measurement time interval from 1 month to even 6 months simultaneously maintaining dose accuracy. The combined effect of the proposed methods, suggestions and criteria was found to facilitate the avoidance of maximal dose errors of up to even about 8 %. In addition, their use may make the strictest recommended overall dose accuracy level of 3 % (1SD) achievable.

Prognostic importance of the tumour microenvironment in follicular lymphoma patients treated with immunochemotherapy

Follicular lymphoma (FL) is the second most common non-Hodgkin lymphoma. It is an indolent and clinically heterogeneous disease, which is generally considered incurable. Currently, immunochemotherapy has significantly improved the outcome of FL patients. This is based on the combination of rituximab, a monoclonal anti-CD20 antibody, with chemotherapy, and is used at present as a standard first-line therapy in FL. Thus far, however, patients have been selected for treatment based on clinical risk factors and indices that were developed before the rituximab era. Therefore, there is a growing need to understand the molecular mechanisms underlying the disease, which would not only provide information to predict survival in the rituximab era, but also enable the design of more targeted therapeutic strategies. In this study, our aim was to identify genes predicting the outcome in FL patients treated with immunochemotherapy. Thus, we performed a cDNA microarray with 24 FL patients. When gene expression differences from diagnostic tumour samples were related to the clinical outcome, we identified novel genes with a prognostic impact on survival. The expression of selected genes was further characterized with quantitative PCR and immunohistochemistry (IHC). Interestingly, the prognostic influence of these genes was often associated with their expression in non-malignant cells instead of tumour cells. Based on the observed gene expression patterns, we analyzed the abundance and prognostic value of non-malignant immune cells in 95-98 FL patients treated with immunochemotherapy. We observed that a high content of tumour-associated macrophages was a marker of a favourable prognosis. In contrast, the accumulation of mast cells correlated with a poor outcome and was further associated with tumour vascularity. Increased microvessel density also correlated with an inferior outcome. In addition, we used the same microarray data with a systems biology approach to identify signalling pathways or groups of genes capable of separating patients with favourable or adverse outcomes. Among the transcripts, there were many genes associated with signal transducers and activators of the transcription (STAT5a) pathway. When IHC was used as validation, STAT5a expression was mostly observed in T-cells and follicular dendritic cells, and expression was found to predict a favourable outcome. In cell cultures, rituximab was observed to induce the expression of STAT5a-associated interleukins in human lymphoma cell lines, which might provide a possible link for the cross-talk between rituximab-induced FL cells and their microenvironment. In conclusion, we have demonstrated that the microenvironment has a prognostic role in FL patients treated with immunochemotherapy. The results also address the importance of re-evaluating the prognostic markers in the rituximab era of lymphoma therapies.

Estramustine and its Derivatives in Potentiating Radiotherapy of Prostate Cancer

The purpose of this study was to deepen our knowledge of the combined use of estramustine and radiotherapy in the treatment of prostate cancer. Prostate cancer is a common disease, with a high variability between subjects in its malignant potential. In many cases, the disease is an incidental finding with little or no clinical significance. In other cases, however, prostate cancer may be an aggressive malignant disease, which, if the initial treatment fails, lacks an effective cure and may lead to severe symptoms, metastasis, and death despite all treatment. In many cases, the methods of treatment available at the moment provide cure or significant regression of symptoms, but often at the cost of considerable side effects. Estramustine, a cytostatic drug used for treating advanced cancer of the prostate, has been shown to inhibit prostate cancer progression and also to increase the sensitivity of cancer cells to radiotherapy. The goals of this study were, first, to find out whether it is possible to use either estramustine or an antibody against estramustine binding protein as carrier molecules for bringing therapeutic radioisotopes into prostate cancer cells, and, secondly, to gain more understanding of the mechanisms behind the known radiosensitising effect of estramustine. Estramustine and estramustine binding protein antibody were labelled with iodine-125 to study the biodistribution of these substances in mice. In the first experiment, both of the substances accumulated in the prostate, but radioiodinated estramustine also showed affinity to the liver and the lungs. Since the radiolabelled antibody was found out to accumulate more selectively to the prostate, we studied its biodistribution in nude mice with DU-145 human prostate cancer implants. In this experiment, the prostate and the tumour accumulated more radioactivity than other organs, but we concluded that the difference in the dose of radiation compared to other organs was not sufficient for the radioiodinated antibody to be advocated as a carrier molecule for treating prostate cancer. Mice with similar DU-145 prostate cancer implants were then treated with estramustine and external beam irradiation, with and without neoadjuvant estramustine treatment. The tumours responded to the treatment as expected, showing the radiation potentiating effect of estramustine. In the third experiment, this effect was found without an increase in the amount of apoptosis in the tumour cells, despite previous suggestions to the contrary. In the fourth experiment, we gave a similar treatment to the mice with DU-145 tumours. A reduction in proliferation was found in the groups treated with radiotherapy, and an increased amount of tumour hypoxia and tumour necrosis in the group treated with both neoadjuvant estramustine and radiation. This finding is contradictory to the suggestion that the radiation sensitising effect of estramustine could be attributed to its angiogenic activity.

Relative Contribution of Cell Proliferation and Microvascularity to Metastasis in Malignant Uveal Melanoma

Uveal melanoma (UM) is the second most common primary intraocular cancer worldwide. It is a relatively rare cancer, but still the second most common type of primary malignant melanoma in humans. UM is a slowly growing tumor, and gives rise to distant metastasis mainly to the liver via the bloodstream. About 40% of patients with UM die of metastatic disease within 10 years of diagnosis, irrespective of the type of treatment. During the last decade, two main lines of research have aimed to achieve enhanced understanding of the metastasis process and accurate prognosis of patients with UM. One emphasizes the characteristics of tumor cells, particularly their nucleoli, and markers of proliferation, and the other the characteristics of tumor blood vessels. Of several morphometric measurements, the mean diameter of the ten largest nucleoli (MLN) has become the most widely applied. A large MLN has consistently been associated with high likelihood of dying from UM. Blood vessels are of paramount importance in metastasis of UM. Different extravascular matrix patterns can be seen in UM, like loops and networks. This presence is associated with death from metastatic melanoma. However, the density of microvessels is also of prognostic importance. This study was undertaken to help understanding some histopathological factors which might contribute to developing metastasis in UM patients. Factors which could be related to tumor progression to metastasis disease, namely nucleolar size, MLN, microvascular density (MVD), cell proliferation, and The Insulin-like Growth Factor 1 Receptor(IGF-1R), were investigated. The primary aim of this thesis was to study the relationship between prognostic factors such as tumor cell nucleolar size, proliferation, extravascular matrix patterns, and dissemination of UM, and to assess to what extent there is a relationship to metastasis. The secondary goal was to develop a multivariate model which includes MLN and cell proliferation in addition to MVD, and which would fit better with population-based, melanoma-related survival data than previous models. I studied 167 patients with UM, who developed metastasis even after a very long time following removal of the eye, metastatic disease was the main cause of death, as documented in the Finnish Cancer Registry and on death certificates. Using an independent population-based data set, it was confirmed that MLN and extravascular matrix loops and networks were unrelated, independent predictors of survival in UM. Also, it has been found that multivariate models including MVD in addition to MLN fitted significantly better with survival data than models which excluded MVD. This supports the idea that both the characteristics of the blood vessels and the cells are important, and the future direction would be to look for the gene expression profile, whether it is associated more with MVD or MLN. The former relates to the host response to the tumor and may not be as tightly associated with the gene expression profile, yet most likely involved in the process of hematogenous metastasis. Because fresh tumor material is needed for reliable genetic analysis, such analysis could not be performed Although noninvasive detection of certain extravascular matrix patterns is now technically possible,in managing patients with UM, this study and tumor genetics suggest that such noninvasive methods will not fully capture the process of clinical metastasis. Progress in resection and biopsy techniques is likely in the near future to result in fresh material for the ophthalmic pathologist to correlate angiographic data, histopathological characteristics such as MLN, and genetic data. This study supported the theory that tumors containing epithelioid cells grow faster and have poorer prognosis when studied by cell proliferation in UM based on Ki-67 immunoreactivity. Cell proliferation index fitted best with the survival data when combined with MVD, MLN, and presence of epithelioid cells. Analogous with the finding that high MVD in primary UM is associated with shorter time to metastasis than low MVD, high MVD in hepatic metastasis tends to be associated with shorter survival after diagnosis of metastasis. Because the liver is the main organ for metastasis from UM, growth factors largely produced in the liver hepatocyte growth factor, epidermal growth factor and insulin-like growth factor-1 (IGF-1) together with their receptors may have a role in the homing and survival of metastatic cells. Therefore the association between immunoreactivity for IGF-1R in primary UM and metastatic death was studied. It was found that immunoreactivity for IGF-IR did not independently predict metastasis from primary UM in my series.

Optimal Use of Treatment Modalities and Treatment Results of Osteosarcoma and Soft Tissue Sarcomas

Soft tissue sarcomas are malignant tumours of mesenchymal origin. Because of infiltrative growth pattern, simple enucleation of the tumour causes a high rate of local recurrence. Instead, these tumours should be resected with a rim of normal tissue around the tumour. Data on the adequate margin width are scarce. At Helsinki University Central Hospital (HUCH) a multidisciplinary treatment group started in 1987. Surgical resection with a wide margin (2.5 cm) is the primary aim. In case of narrower margin radiation therapy is necessary. The role of adjuvant chemotherapy remains unclear. Our aims were to study local control by the surgical margin and to develop a new prognostic tool to aid decision-making on which patients should receive adjuvant chemotherapy. Patients with soft tissue sarcoma of the extremity or the trunk wall referred to HUCH during 1987-2002 form material in Studies I and II. External validation material comes from the Lund university sarcoma registry. The smallest surgical margin of at least 2.5 centimetres yielded local control of 89 per cent at five years. Amputation rate was 9 per cent. The proposed prognostic model with necrosis, vascular invasion, size on a continuous scale, depth, location and grade worked well both in Helsinki material and in the validation material, and it also showed good calibration. Based on the present study, we recommend the smallest surgical margin of 2-3 centimetres in soft tissue sarcoma irrespective of grade. Improvement in local control was present but modest in margins wider than 1 centimetre. In cases where gaining a wider margin would lead to a considerable loss of function, smaller margin is to be considered combined to radiation therapy. Patients treated with inadequate margins should be offered radiation therapy irrespective of tumour grade. Our new prognostic model to estimate 10-year survival probability in patients with soft tissue sarcoma of the extremities or trunk wall showed good dicscrimination and calibration. For time being the prognostic model is available for scientific use and further validations. In the future, the model may aid in clinical decision-making. For operable osteosarcoma, neoadjuvant multidrug chemotherapy followed by delayed surgery and multidrug adjuvant chemotherapy is the treatment of choice. Overall survival rates at five years are approximately 75 per cent in modern trials with classical osteosarcoma. All patients diagnosed and reported to the Finnish Cancer Registry with osteosarcoma in Finland during 1971-2005 form the material in Studies III and IV. Limb-salvage rate increased from 23 per cent to 78 per cent during 1971-2005. The 10-year sarcoma-specific survival for the whole study population improved from 32 per cent to 62 per cent. It was 75 per cent for patients with a local high-grade osteosarcoma of the extremity diagnosed during 1991-2005. This study outlines the improved prognosis of osteosarcoma patients in Finland with modern chemotherapy. The 10-year survival rates are good also in an international scale. Nonetheless, their limb-salvage rate remains inferior to those seen for highly selected patient series. Overall, the centralisation of osteosarcoma treatment would most likely improve both survival and limb-salvage rates even further.