Effect of long-wave UV radiation on mouse melanoma: an in vitro and in vivo study

The skin cancer incidence has increased substantially over the past decades and the role of ultraviolet (UV) radiation in the etiology of skin cancer is well established. Ultraviolet B radiation (280-320 nm) is commonly considered as the more harmful part of the UV-spectrum due to its DNA-damaging potential and well-known carcinogenic effects. Ultraviolet A radiation (320-400 nm) is still regarded as a relatively low health hazard. However, UVA radiation is the predominant component in sunlight, constituting more than 90% of the environmentally relevant solar ultraviolet radiation. In the light of the recent scientific evidence, UVA has been shown to have genotoxic and immunologic effects, and it has been proposed that UVA plays a significant role in the development of skin cancer. Due to the popularity of skin tanning lamps, which emit high intensity UVA radiation and because of the prolonged sun tanning periods with the help of effective UVB blockers, the potential deleterious effects of UVA has emerged as a source of concern for public health. The possibility that UV radiation may affect melanoma metastasis has not been addressed before. UVA radiation can modulate various cellular processes, some of which might affect the metastatic potential of melanoma cells. The aim of the present study was to investigate the possible role of UVA irradiation on the metastatic capacity of mouse melanoma both in vitro and in vivo. The in vitro part of the study dealt with the enhancement of the intercellular interactions occurring either between tumor cells or between tumor cells and endothelial cells after UVA irradiation. The use of the mouse melanoma/endothelium in vitro model showed that a single-dose of UVA to melanoma cells causes an increase in melanoma cell adhesiveness to non-irradiated endothelium after 24-h irradiation. Multiple-dose irradiation of melanoma cells already increased adhesion at a 1-h time-point, which suggests the possible cumulative effect of multiple doses of UVA irradiation. This enhancement of adhesiveness might lead to an increase in binding tumor cells to the endothelial lining of vasculature in various internal organs if occurring also in vivo. A further novel observation is that UVA induced both decline in the expression of E-cadherin adhesion molecule and increase in the expression of the N-cadherin adhesion molecule. In addition, a significant decline in homotypic melanoma-melanoma adhesion (clustering) was observed, which might result in the reduction of E-cadherin expression. The aim of the in vivo animal study was to confirm the physiological significance of previously obtained in vitro results and to determine whether UVA radiation might increase melanoma metastasis in vivo. The use of C57BL/6 mice and syngeneic melanoma cell lines B16-F1 and B16-F10 showed that mice, which were i.v. injected with B16-F1 melanoma cells and thereafter exposed to UVA developed significantly more lung metastases when compared with the non-UVA-exposed group. To study the mechanism behind this phenomenon, the direct effect of UVA-induced lung colonization capacity was examined by the in vitro exposure of B16-F1 cells. Alternatively, the UVA-induced immunosuppression, which might be involved in increased melanoma metastasis, was measured by standard contact hypersensitivity assay (CHS). It appears that the UVA-induced increase of metastasis in vivo might be caused by a combination of UVA-induced systemic immunosuppression, and to the lesser extent, it might be caused by the increased adhesiveness of UVA irradiated melanoma cells. Finally, the UVA effect on gene expression in mouse melanoma was determined by a cDNA array, which revealed UVA-induced changes in the 9 differentially expressed genes that are involved in angiogenesis, cell cycle, stress-response, and cell motility. These results suggest that observed genes might be involved in cellular response to UVA and a physiologically relevant UVA dose have previously unknown cellular implications. The novel results presented in this thesis offer evidence that UVA exposure might increase the metastatic potential of the melanoma cells present in blood circulation. Considering the wellknown UVA-induced deleterious effects on cellular level, this study further supports the notion that UVA radiation might have more potential impact on health than previously suggested. The possibility of the pro-metastatic effects of UVA exposure might not be of very high significance for daily exposures. However, UVA effects might gain physiological significance following extensive sunbathing or solaria tanning periods. Whether similar UVA-induced pro-metastatic effects occur in people sunbathing or using solaria remains to be determined. In the light of the results presented in this thesis, the avoidance of solaria use could be well justified.

Conjunctival Melanoma in Finland 1967-2000: a population-based study

Eighty-five new cases of conjunctival melanoma (CM) were diagnosed in Finland between 1967 and 2000. The annual crude incidence of CM was 0.51 per million inhabitants. The average age-adjusted incidence of 0.54 doubled during the study period, analogous to the increase in the incidence of cutaneous malignant melanoma during this period, suggesting a possible role for ultraviolet radiation in its pathogenesis. Nonlimbal tumors were more likely than limbal ones to recur and they were associated with decreased survival. Increasing tumor thickness and recurrence of the primary tumor were other clinical factors related to death from CM. The histopathologic specimens of 85 patients with CM melanoma were studied for cell type, mitotic count, tumor-infiltrating lymphocytes and macrophages, mean vascular density, extravascular matrix loops and networks, and mean diameter of the ten largest nucleoli (MLN). The absence of epithelioid cells, increasing mitotic count and small MLN were associated with shorter time to recurrence according to the Cox univariate regression. None of the histopathologic variables was associated with mortality from CM. Four (5%) patients had a CM limited to the cornea without evidence of a tumor other than primary acquired melanosis of the conjunctiva. Because there are no melanocytes in the cornea, the origin of these melanomas most likely is the limbal conjunctiva. All four corneally displaced CM were limited to the epithelium, and none of the patients developed metastases. An anatomic sub-classification based on my patients and world literature was developed for corneally displaced CM. In 20 patients the metastatic pattern could be determined. Ten patients had initial systemic metastases detected, nine had initial regional metastases, and in one case the two types were detected simultaneously. The patients most likely to develop either type of initial metastases were those with nonlimbal conjunctival melanoma, those with a primary tumor more than 2 mm thick, and those with a recurrent conjunctival melanoma. Approximately two thirds of the patients had limbal CM, a location associated with good prognosis. One third, however, had a primary CM originating outside the limbus. In these patients the chance of developing local recurrences as well as systemic metastases was significantly higher than in patients with limbal CM. Each recurrence accompanies an increased risk of developing metastases, and recurrences contribute to death along with increasing tumor thickness and nonlimbal tumor location. In my data, an equal number of patients with initial locoregional and systemic metastasis existed. Patients with limbal primary tumors less than 2 mm in thickness rarely experienced metastases, unless the tumor recurred. Consequently, the patients most likely to benefit from sentinel lymph node biopsy are those who have nonlimbal tumors, CM that are over 2 mm thick, or recurrent CM. The histopathology of CM differs from that of uveal melanoma. Microvascular factors did not prove to be of prognostic importance, possibly due to the fact that CM at least as often disseminates first to the regional lymph nodes, unlike uveal melanoma that almost always disseminates hematogenously.

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.

Macrophages in regressed and progressed uveal melanoma

Uveal melanoma (UM) is the most common primary ocular malignancy in adults. In Finland, approximately 50 new cases are diagnosed yearly. Up to 50% of UM metastasize, mostly to the liver, although other organs are also affected. Despite improvements in the management of the primary tumour, the survival rates of patients with metastatic UM are poor. Until the 1970s, UMs were treated by enucleation i.e. removal of the eye. Currently, UM is usually treated by brachytherapy, which is known to influence tumour cells and blood vessels. UMs enucleated both primarily and secondarily after brachytherapy contain tumour-infiltrating macrophages, and a high number of macrophages in primary UM is associated with a shorter survival and a higher microvascular density (MVD) within the tumour tissue. The latter is independently associated with a shorter time to metastatic death. Macrophages have several diverse roles depending on their response to variable signals from the surrounding microenvironment. They function as scavengers, as producers of angiogenic and growth factors as well as proteases, which modulate extracellular matrix. Thus, tumour invasiveness and the risk for metastasis increase with increasing macrophage density. The aim of this study was to evaluate the effects of regression and progression of UM on macrophage numbers and microcirculation factors. Tumour regression is induced by primary brachytherapy, and tumour progression is evidenced by the development of metastases. Understanding the biological behaviour of UMs in the both states may help us in finding new treatment modalities against this disease. To achieve these aims case-control analyses of irradiated UMs and primarily-enucleated eyes (34 matched pairs) were performed. UMs were stained immunohistochemically to detect macrophages, extravascular matrix (EVM) loops and networks, and MVD. Following brachytherapy, a lower MVD was observed. The average number of macrophages remained unchanged. Considering that irradiated melanomas may still contain proliferating tumour cells, a clinically-relevant consequence of my study would be the reassurance that the risk for metastasis is likely to be reduced, given that the low MVD in untreated UMs indicates a favourable prognosis. The effect of progression on macrophages was studied in a paired analysis of primarily-enucleated UM and their corresponding hepatic metastases (48 pairs). A cross-sectional histopathological analysis of these pairs was carried out by staining both specimens in a similar way to the first study. MVD was greater in hepatic metastases than in corresponding primary tumours, and the survival of the patient tended to be shorter if hepatic metastases had a higher MVD. Hepatic metastases had also more dendritic macrophages than the primary UMs. Thus, the progression to metastasis seems to alter the inflammatory status within the tumour. Furthermore, determining MVD of biopsied hepatic metastases may serve as a supplementary tool in estimating the prognosis of patients with metastatic uveal melanoma. After irradiation, the majority of treated eyes have been clinically observed to have pigmented episcleral deposits. A noncomparative clinical case series of 211 irradiated UM eyes were studied by recording the number and location of pigmented episcleral deposits during follow-up visits after brachytherapy. For the first time, the study described pigmented episcleral deposits, which are found in the most UM eyes after brachytherapy, and proved them to consist of macrophages full with engulfed melanin particles. This knowledge may save patients from unnecessary enucleation, because episcleral pigmented deposits might be mistaken for extrascleral tumour growth. The presence of pigmented macrophage-related episcleral deposits was associated with plaque size and isotope rather than with tumour size, suggesting that, in addition to tumour regression, radiation atrophy of retinal pigment epithelium and choroid contributes to the formation of the deposits. In the paired (the same 34 pairs as in the first study) cross-sectional study of irradiated and non-irradiated UMs, clinically-visible episcleral deposits and migrating macrophages in other extratumoral tissues were studied histopathologically. Resident macrophages were present in extratumoral tissues in eyes with both irradiated and non-irradiated UM. Irradiation increased both the number of CD68+ macrophages in the sclera beneath the tumour and the number of clinically-observed episcleral macrophages aggregates. Brachytherapy seemed to alter the route of migration of macrophages: after irradiation, macrophages migrated preferentially through the sclera while in non-irradiated UMs they seemed to migrate more along the choroid. In order to understand the influence of these routes on tumour progression and regression in the future, labelling and tracking of activated macrophages in vivo is required.

Latent TGF-beta Binding Proteins : Adhesive functions and matrix association of LTBP-2 and potential functions of LTBP-1 and LTBP-3 in mesothelioma

Latent transforming growth factor-beta (TGF-beta) binding proteins (LTBPs) -1, -3 and -4 are ECM components whose major function is to augment the secretion and matrix targeting of TGF-beta, a multipotent cytokine. LTBP-2 does not bind small latent TGF-beta but has suggested functions as a structural protein in ECM microfibrils. In the current work we focused on analyzing possible adhesive functions of LTBP-2 as well as on characterizing the kinetics and regulation of LTBP-2 secretion and ECM deposition. We also explored the role of TGF-beta binding LTBPs in endothelial cells activated to mimic angiogenesis as well as in malignant mesothelioma. We found that, unlike most adherent cells, several melanoma cell lines efficiently adhered to purified recombinant LTBP-2. Further characterization revealed that the adhesion was mediated by alpha3beta1 and alpha6beta1 integrins. Heparin also inhibited the melanoma cell adhesion suggesting a role for heparan sulphate proteoglycans. LTBP-2 was also identified as a haptotactic substrate for melanoma cell migration. We used cultured human embryonic lung fibroblasts to analyze the temporal and spatial association of LTBP-2 into ECM. By We found that LTBP-2 was efficiently assembled to the ECM only in confluent cultures following the deposition of fibronectin (FN) and fibrillin-1. In early, subconfluent cultures it remained primarily in soluble form after secretion. LTBP-2 colocalized transiently with FN and fibrillin-1. Silencing of fibrillin-1 expression by lentiviral shRNAs profoundly disrupted the deposition of LTBP-2 indicating that the ECM association of LTBP-2 depends on a pre-formed fibrillin-1 network. Considering the established role of TGF-beta as a regulator of angiogenesis we induced morphological activation of endothelial cells by phorbol 12-myristate 13-acetate (PMA) and followed the fate of LTBP-1 in the endothelial ECM. This resulted in profound proteolytic processing of LTBP-1 and release of latent TGF-beta complexes from the ECM. The processing was coupled with increased activation of MT-MMPs and specific upregulation of MT1-MMP. The major role of MT1-MMP in the proteolysis of LTBP-1 was confirmed by suppressing the expression with lentivirally induced short-hairpin RNAs as well as by various metalloproteinases inhibitors. TGF-beta can promote tumorigenesis of malignant mesothelioma (MM), which is an aggressive tumor of the pleura with poor prognosis. TGF-beta activity was analyzed in a panel of MM tumors by immunohistochemical staining of phosphorylated Smad-2 (P-Smad2). The tumor cells were strongly positive for P-Smad2 whereas LTBP-1 immunoreactivity was abundant in the stroma, and there was a negative correlation between LTBP-1 and P-Smad2 staining. In addition, the high P-Smad2 immunoreactivity correlated with shorter survival of patients. mRNA analysis revealed that TGF-beta1 was the most highly expressed isoform in both normal human pleura and MM tissue. LTBP-1 and LTBP-3 were both abundantly expressed. LTBP-1 was the predominant isoform in established MM cell lines whereas the expression of LTBP-3 was high in control cells. Suppression of LTBP-3 expression by siRNAs resulted in increased TGF-beta activity in MM cell lines accompanied by decreased proliferation. Our results suggest that decreased expression of LTBP-3 in MM could alter the targeting of TGF-beta to the ECM and lead to its increased activation. The current work emphasizes the coordinated process of the assembly and appropriate targeting of LTBPs with distinct adhesive or cytokine harboring properties into the ECM. The hierarchical assembly may have implications in the modulation of signaling events during morphogenesis and tissue remodeling.

Construction of a global map of human gene expression : the process, tools and analysis

This thesis studies human gene expression space using high throughput gene expression data from DNA microarrays. In molecular biology, high throughput techniques allow numerical measurements of expression of tens of thousands of genes simultaneously. In a single study, this data is traditionally obtained from a limited number of sample types with a small number of replicates. For organism-wide analysis, this data has been largely unavailable and the global structure of human transcriptome has remained unknown. This thesis introduces a human transcriptome map of different biological entities and analysis of its general structure. The map is constructed from gene expression data from the two largest public microarray data repositories, GEO and ArrayExpress. The creation of this map contributed to the development of ArrayExpress by identifying and retrofitting the previously unusable and missing data and by improving the access to its data. It also contributed to creation of several new tools for microarray data manipulation and establishment of data exchange between GEO and ArrayExpress. The data integration for the global map required creation of a new large ontology of human cell types, disease states, organism parts and cell lines. The ontology was used in a new text mining and decision tree based method for automatic conversion of human readable free text microarray data annotations into categorised format. The data comparability and minimisation of the systematic measurement errors that are characteristic to each lab- oratory in this large cross-laboratories integrated dataset, was ensured by computation of a range of microarray data quality metrics and exclusion of incomparable data. The structure of a global map of human gene expression was then explored by principal component analysis and hierarchical clustering using heuristics and help from another purpose built sample ontology. A preface and motivation to the construction and analysis of a global map of human gene expression is given by analysis of two microarray datasets of human malignant melanoma. The analysis of these sets incorporate indirect comparison of statistical methods for finding differentially expressed genes and point to the need to study gene expression on a global level.

Long-term clinical outcome after liver transplantation

With transplant rejection rendered a minor concern and survival rates after liver transplantation (LT) steadily improving, long-term complications are attracting more attention. Current immunosuppressive therapies, together with other factors, are accompanied by considerable long-term toxicity, which clinically manifests as renal dysfunction, high risk for cardiovascular disease, and cancer. This thesis investigates the incidence, causes, and risk factors for such renal dysfunction, cardiovascular risk, and cancer after LT. Long-term effects of LT are further addressed by surveying the quality of life and employment status of LT recipients. The consecutive patients included had undergone LT at Helsinki University Hospital from 1982 onwards. Data regarding renal function – creatinine and estimated glomerular filtration rate (GFR) – were recorded before and repeatedly after LT in 396 patients. The presence of hypertension, dyslipidemia, diabetes, impaired fasting glucose, and overweight/obesity before and 5 years after LT was determined among 77 patients transplanted for acute liver failure. The entire cohort of LT patients (540 patients), including both children and adults, was linked with the Finnish Cancer Registry, and numbers of cancers observed were compared to site-specific expected numbers based on national cancer incidence rates stratified by age, gender, and calendar time. Health-related quality of life (HRQoL), measured by the 15D instrument, and employment status were surveyed among all adult patients alive in 2007 (401 patients). The response rate was 89%. Posttransplant cardiovascular risk factor prevalence and HRQoL were compared with that in the age- and gender-matched Finnish general population. The cumulative risk for chronic kidney disease increased from 10% at 5 years to 16% at 10 years following LT. GFR up to 10 years after LT could be predicted by the GFR at 1 year. In patients transplanted for chronic liver disease, a moderate correlation of pretransplant GFR with later GFR was also evident, whereas in acute liver failure patients after LT, even severe pretransplant renal dysfunction often recovered. By 5 years after LT, 71% of acute liver failure patients were receiving antihypertensive medications, 61% were exhibiting dyslipidemia, 10% were diabetic, 32% were overweight, and 13% obese. Compared with the general population, only hypertension displayed a significantly elevated prevalence among patients – 2.7-fold – whereas patients exhibited 30% less dyslipidemia and 71% less impaired fasting glucose. The cumulative incidence of cancer was 5% at 5 years and 13% at 10. Compared with the general population, patients were subject to a 2.6-fold cancer risk, with non-melanoma skin cancer (standardized incidence ratio, SIR, 38.5) and non-Hodgkin lymphoma (SIR 13.9) being the predominant malignancies. Non-Hodgkin lymphoma was associated with male gender, young age, and the immediate posttransplant period, whereas old age and antibody induction therapy raised skin-cancer risk. HRQoL deviated clinically unimportantly from the values in the general population, but significant deficits among patients were evident in some physical domains. HRQoL did not seem to decrease with longer follow-up. Although 87% of patients reported improved working capacity, data on return to working life showed marked age-dependency: Among patients aged less than 40 at LT, 70 to 80% returned to work, among those aged 40 to 50, 55%, and among those above 50, 15% to 28%. The most common cause for unemployment was early retirement before LT. Those patients employed exhibited better HRQoL than those unemployed. In conclusion, although renal impairment, hypertension, and cancer are evidently common after LT and increase with time, patients’ quality of life remains comparable with that of the general population.

Matrix metalloproteinases as biomarkers in premalignant and malignant tumors of the human skin

The incidence of non-melanoma skin cancer is increasing worldwide. Basal cell carcinoma followed by squamous cell carcinoma and malignant melanoma are the most frequent skin tumors. Immunosuppressed patients have an increased risk of neoplasia, of which non-melanoma skin cancer is the most common. Matrix metalloproteinases (MMPs) are proteolytic enzymes that collectively are capable of degrading virtually all components of the extracellular matrix. MMPs can also process substrates distinct from extracellular matrix proteins and influence cell proliferation, differentiation, angiogenesis, and apoptosis. MMP activity is regulated by their natural inhibitors, tissue inhibitors of metallopro-teinases (TIMPs). In this study, the expression patterns of MMPs, TIMPs, and certain cancer-related molecules were investigated in premalignant and malignant lesions of the human skin. As methods were used immunohistochemisty, in situ hybridization, and reverse transcriptase polymerase chain reaction (RT-PCR) from the cell cultures. Our aim was to evaluate the expression pattern of MMPs in extramammary Paget's disease in order to find markers for more advanced tumors, as well as to shed light on the origin of this rare neoplasm. Novel MMPs -21, -26, and -28 were studied in melanoma cell culture, in primary cutaneous melanomas, and their sentinel nodes. The MMP expression profile in keratoacanthomas and well-differentiated squamous cell carcinomas was analyzed to find markers to differentiate benign keratinocyte hyperproliferation from malignantly transformed cells. Squamous cell carcinomas of immunosuppressed organ transplant recipients were compared to squamous cell carcinomas of matched immunocompetent controls to investigate the factors explaining their more aggressive nature. We found that MMP-7 and -19 proteins are abundant in extramammary Paget's disease and that their presence may predict an underlying adenocarcinoma in these patients. In melanomas, MMP-21 was upregulated in early phases of melanoma progression, but disappeared from the more aggressive tumors with lymph node metastases. The presence of MMP-13 in primary melanomas and lymph node metastases may relate to more aggressive disease. In keratoacanthomas, the expression of MMP-7 and -9 is rare and therefore should raise a suspicion of well-differentiated squamous cell carcinomas. Furthermore, MMP-19 and p16 were observed in benign keratinocyte hyperproliferation of keratoacanthomas, whereas they were generally lost from malignant keratinocytes of SCCs. MMP-26 staining was significantly stronger in squamous cell carcinomas and Bowen s disease samples of organ transplant recipients and it may contribute to the more aggressive nature of squamous cell carcinomas in immunosuppressed patients. In addition, the staining for MMP-9 was significantly stronger in macrophages surrounding the tumors of the immunocompetent group and in neutrophils of those patients on cyclosporin medication. In conclusion, based on our studies, MMP-7 and -19 might serve as biomarkers for more aggressive extramammary Paget's disease and MMP-21 for malignant transformation of melanocytes. MMP -7, -9, and -26, however, could play an important role in the pathobiology of keratinocyte derived malignancies.

The Risk of Cancer Associated with Immunosuppressive Therapy for Skin Diseases

The possible carcinogenic risk of immunosuppressive therapies is an important issue in everyday clinical practise. Carcinogenesis is a slow multi step procedure, thus a long latency period is needed before cancer develops. PUVA therapy is used for many skin diseases including psoriasis, early stage cutaneous T cell lymphoma, atopic dermatitis, palmoplantar pustulosis and chronic eczema. There has been concern about the increased melanoma risk associated to PUVA therapy, which has previously been associated with an increased risk on non-melanoma skin cancer, especially squamous cell carcinoma. The increased risk of basal cell carcinoma (BCC) is also documented but it is modest compared to squamous cell carcinoma (SCC). This thesis evaluated melanoma and noncutaneous cancer risk associated to PUVA, and the persistence of nonmelanoma cancer risk after the cessation of PUVA treatment. Also, the influence of photochemotherapy to the development of secondary cancers in cutaneous T cell lymphoma and the role of short term cyclosporine in later cancer development in inflammatory skin diseases were evaluated. The first three studies were performed on psoriasis patients. The risk of melanoma started to increase 15 years after the first treatment with PUVA. The risk was highest among persons who had received over 250 treatments compared to those under 250 treatments. In noncutaneous cancer, the overall risk was not increased (RR=1.08,95% CI=0.93-1.24), but significant increases in risk were found in thyroid cancer, breast cancer and in central nervous system neoplasms. These cancers were not associated to PUVA. The increased risk of SCC was associated to high cumulative UVA exposure in the PUVA regimen. The patients with high risk had no substantial exposure to other carcinogens. In BCC there was a similar but more modest tendency. In the two other studies, the risk of all secondary cancers (SIR) in CTCL patients was 1.4 (95% CI=1.0-1.9). In separate sites, the risk of lung cancer, Hodgkin and non-Hodgkin lymphomas were increased. PUVA seemed not to contribute to any extent to the appearance of these cancers. The carcinogenity of short-term cyclosporine was evaluated in inflammatory skin diseases. No increased risk for any type of cancer including the skin cancers was detected. To conclude, our studies confirm the increased skin cancer risk related to PUVA treatment in psoriasis patients. In clinical practice, this has led to a close and permanent follow-up of patients treated with PUVA. In CTCL patients, PUVA treatment did not contribute to the development of secondary cancers. We could not detect any increase in the risk of cancer in patients treated with short term cyclosporine, unlike in organ transplant patients under such long-term therapy.

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.

Digital Photography in the Diagnosis and Follow-up of Ocular Diseases

Purpose: The aim of the present study was to develop and test new digital imaging equipment and methods for diagnosis and follow-up of ocular diseases. Methods: The whole material comprised 398 subjects (469 examined eyes), including 241 patients with melanocytic choroidal tumours, 56 patients with melanocytic iris tumours, 42 patients with diabetes, a 52-year old patient with chronic phase of VKH disease, a 30-year old patient with an old blunt eye injury, and 57 normal healthy subjects. Digital 50° (Topcon TRC 50 IA) and 45° (Canon CR6-45NM) fundus cameras, a new handheld digital colour videocamera for eye examinations (MediTell), a new subtraction method using the Topcon Image Net Program (Topcon corporation, Tokyo, Japan), a new method for digital IRT imaging of the iris we developed, and Zeiss photoslitlamp with a digital camera body were used for digital imaging. Results: Digital 50° red-free imaging had a sensitivity of 97.7% and two-field 45° and 50° colour imaging a sensitivity of 88.9-94%. The specificity of the digital 45°-50° imaging modalities was 98.9-100% versus the reference standard and ungradeable images that were 1.2-1.6%. By using the handheld digital colour video camera only, the optic disc and central fundus located inside 20° from the fovea could be recorded with a sensitivity of 6.9% for detection of at least mild NPDR when compared with the reference standard. Comparative use of digital colour, red-free, and red light imaging showed 85.7% sensitivity, 99% specificity, and 98.2 % exact agreement versus the reference standard in differentiation of small choroidal melanoma from pseudomelanoma. The new subtraction method showed growth in four of 94 melanocytic tumours (4.3%) during a mean ±SD follow-up of 23 ± 11 months. The new digital IRT imaging of the iris showed the sphincter muscle and radial contraction folds of Schwalbe in the pupillary zone and radial structural folds of Schwalbe and circular contraction furrows in the ciliary zone of the iris. The 52-year-old patient with a chronic phase of VKH disease showed extensive atrophy and occasional pigment clumps in the iris stroma, detachment of the ciliary body with severe ocular hypotony, and shallow retinal detachment of the posterior pole in both eyes. Infrared transillumination imaging and fluorescein angiographic findings of the iris showed that IR translucence (p=0.53), complete masking of fluorescence (p=0.69), presence of disorganized vessels (p=0.32), and fluorescein leakage (p=1.0) at the site of the lesion did not differentiate an iris nevus from a melanoma. Conclusions: Digital 50° red-free and two-field 50° or 45° colour imaging were suitable for DR screening, whereas the handheld digital video camera did not fulfill the needs of DR screening. Comparative use of digital colour, red-free and red light imaging was a suitable method in the differentiation of small choroidal melanoma from different pseudomelanomas. The subtraction method may reveal early growth of the melanocytic choroidal tumours. Digital IRT imaging may be used to study changes of the stroma and posterior surface of the iris in various diseases of the uvea. It contributed to the revealment of iris atrophy and serous detachment of the ciliary body with ocular hypotony together with the shallow retinal detachment of the posterior pole as new findings of the chronic phase of VKH disease. Infrared translucence and angiographic findings are useful in differential diagnosis of melanocytic iris tumours, but they cannot be used to determine if the lesion is benign or malignant.

Identification of molecules relevant for the invasiveness of fibrosarcomas and melanomas

Cancer is becoming the leading cause of deaths in the world. As 90% of all deaths from cancer are caused by metastasis, discovery of the mechanisms behind cancer cell invasion and metastasis is of utmost importance. Only new effective therapies targeting cancer progression can reduce cancer mortality rates. The aim of this study was to identify molecules that are relevant for tumor cell invasion and spreading in fibrosarcomas and melanomas, and to analyze their potential for cancer biomarkers or therapeutic targets. First, the gene expression changes of normal cells and transformed cells showing high invasiveness, S-adenosylmethionine decarboxylase (AdoMetDC)-transfected murine fibroblasts and human melanoma cells, were studied by microarray analyses. The function of the identified candidate molecules were then studied in detail in these cell lines. Finally, the physiological relevance of the identified changes was studied by immunohistochemical analyses of human sarcoma and melanoma specimens or by a mouse xenograft model. In fibrosarcoma cells, the most remarkable change detected was a dramatic up-regulation of the actin-sequestering molecule thymosin beta 4 (TB4), which was shown to be important for the transformed phenotype of the AdoMetDC-transfected cells (Amdc-s and -as). A sponge toxin latrunculin A, inhibiting the binding of TB4 to actin, was found to selectively inhibit the migration and invasion of these cells. Further, Amdc-s-induced mouse tumors and human high-grade sarcomas were found to show intense TB4 immunostaining. In addition to TB4, integrin subunits alfa 6 and beta 7 (ItgA6 and ItgB7) were found to be up-regulated in Amdc-s and -as cells. ItgA6 was shown to dimerize mainly with ItgB1 in Amdc-s. Inhibition of ItgA6 or ItgB1 function with neutralizing antibodies fully blocked the invasiveness of Amdc-s cells, and importantly also human HT-1080 fibrosarcoma cells, in three-dimensional (3D)-Matrigel mimicking tumor extracellular matrix (ECM). By immunohistochemical analyses, strong staining for ITGA6 was detected in human high-grade fibrosarcomas and other sarcomas, especially at the invasion fronts of the tumors. In the studied melanoma cell lines, the expression levels of the adhesion-related ECM proteins tenascin-C (TN-C), fibronectin (FN), and transforming growth factor beta-induced (TGFBI) were found to be highly up-regulated. By immunohistochemistry, intense TN-C and FN staining was detected in invasive and metastatic melanoma tumors, showing co-localization (together with procollagen-I) in tubular meshworks and channels around the invading melanoma cells. In vitro, TN-C and FN were further found to directly stimulate the migration of melanoma cells in 3D-collagen-I matrix. The third candidate protein, TGFBI, was found to be an anti-adhesive molecule for melanoma cells, and knockdown of its expression in metastatic melanoma cells (TGFBI-KD cells) led to dramatically impaired tumor growth in immunocompromized mice. Interestingly, the control tumors showed intense TGFBI immunostaining in the invasion fronts, showing partial co-localization with the fibrillar FN staining, whereas the small TGFBI-KD cell-induced tumors displayed amorphous, non-fibrillar FN staining. These data suggest an important role for TGFBI in FN fibrillogenesis and melanoma progression. In conclusion, we have identified several invasion-related molecules, which show potential for cancer diagnostic or prognostic markers, or therapeutic targets. Based on our previous and present fibrosarcoma studies, we propose the possibility of using ITGA6 antagonists (affecting tumor cell adhesion) in combination with TB4 inhibitors (affecting tumor cell migration) and cathepsin L inhibitors (affecting the degradation of basement membrane and ECM proteins) for the treatment of fibrosarcomas and other tumors overexpressing these molecules. With melanoma cells, in turn, we point to the importance of three secreted ECM proteins, TN-C, FN, and TGFBI, in melanoma progression. Of these, especially the potential of TN-C as a prognostic melanoma biomarker and TGFBI as a promising therapeutic target molecule are clearly worth additional studies.