<p>The aim of this thesis was to gain further insight into the biology of normal human skin and basal cell carcinoma (BCC). Morphology in combination with microdissection was used as primary tool for sampling.</p><p>Using the X-chromosome inactivation assay, we found normal human skin to consist of a mosaic of cells, with either the maternal or the paternal X-chromosome inactivated. We believe that each tile is made up of several epidermal proliferative units with identical X-chromosome inactivation patterns. Using the same method, we found BCC to be a monoclonal neoplasm imbedded in polyclonal stroma. However, one tumor displayed clear evidence of being composed of two intermingled monoclonal tumors.</p><p>To better enable molecular analysis of defined cells from tissue sections, we investigated a zinc-based fixative as alternative to neutral-buffered formalin. Zinc-based fixative preserves good quality of genomic DNA, with only slight impairment of morphology. In addition, it partly abrogates the need for antigen retrieval.</p><p>The patched gene is involved in BCC development. We analyzed the distribution of a coding polymorphism (Pro/Leu) at codon 1315 in populations with different skin types. We found a reduced Pro/Pro genotype frequency in populations with lighter pigmentation. This in combination with genotype analyses of patients with multiple BCCs, showed that failure to lose the Pro allele during a shift towards lighter pigmented skin may be associated with an increased risk of developing BCC.</p><p>We compared the expression profile of BCC cells with putative progenitor cells in the basal layer of epidermis. In addition to discovering several unknown genes, we found the Wnt signaling pathway to upregulated. Furthermore, differentiation markers were downregulated together with proteins important for scavenging of oxygen radicals.</p><p>In conclusion, the combination of morphology, microdissection and subsequent molecular applications provided valid information deepening our understanding of normal skin and BCC.</p>

<p>Radioimmunotherapy (RIT) has been shown to be a practicable way to treat head and neck squamous cell carcinoma. A specific antibody recognizes the charasteristic structure of tumour cells when loaded with cytotoxic agents (toxins, drugs, radionuclides, etc). But RIT kills not only tumour cells with attached radionuclides but also adjacent tumour cells due to the “cross fire effect”. To be efficacious, RIT depends closely on suitable monoclonal antibody, on the properties of the chosen radionuclides, and on a suitable labelling method for attaching radionuclide to antibody. </p><p>In this study we initially used radionuclide-labelled cMAB U36, via linker DABI in order to improve the retention of radio-conjugates in the tumour cells. Improved retention is important because the longer the radionuclide remains in tumour cells, the more effective will the tumour cells be eradicated. In the investigation, both normal mice and HNSCC-bearing nude mice were used to compare our form of treatment against other radio-iodination methods. In the biodistribution study, normal mice showed that radioactive uptake in organs diminished with time, irrespectively of whether the conjugate was directly or indirectly labelled. But in thyroid, there was a tenfold greater accumulation of direct-labelled than of indirectly labelled conjugate.</p><p>In tumour-bearing nude mice, by contrast, the results showed promising uptake of radioactivity, but little uptake in direct-labelled conjugate in thyroid. Significant differences were observed on comparing tumour: organ ratios between 131I-cMAb U36 vs. 125I-DABI-cMAb U36.</p><p>In the present study, cMAb U36 labelled with 211Astatine was initially used to treat HNSCC in nude mice. The biodistribution of 211At-cMAb U36 did not reveal any significant difference between an antibody-blocked group and a non-blocked group. But it did highlight the characteristics of a successful targeting conjugate in HNSCC-bearing nude mice.</p><p>In the subcutaneous therapy experiment, most of the treated tumours (n=18) had disappeared by the 26th day, in both U36-blocked and non-blocked groups. Treatment in the intravenous therapy experiment had also proved effective. In the antibody non-blocked group, the smallest tumour volume was 25 mm3 (average 111 mm3) vis-á-vis 65 mm3 (average 145 mm3) in the blocked group. None of tumours grew again following treatment.</p>

<p>Antibody-based targeting techniques play an increasingly important role in cancer research. By targeting a structure that is abundant in tumour cells, but rare in healthy tissues, an antibody can mediate the delivery of radioactivity specifically to tumour cells in the body. This idea is particularly appealing for head and neck squamous cell carcinoma (HNSCC), as the advanced stages have a large fraction of spread disease that is difficult to treat with procedures available today. </p><p>In this thesis, we have investigated possible radioimmunotargeting structures for HNSCC, and found that CD44v6 is a suitable target for antibody-based radiotherapy and diagnostics in this patient group. We have identified radiohalogens as attractive nuclides for such use, and have investigated the possibility of radiohalogenating the anti CD44v6 chimeric monoclonal antibody (cMAb) U36. Several feasible labelling methods were identified, using both direct and indirect labelling. The cMAb U36 was then successfully labelled with ²¹¹At and ¹³¹I, and preclinically evaluated for therapeutic use. Results proved the astatinated conjugate to be most efficient in this context, demonstrating a specific and dose-dependent cytotoxicity. The cMAb U36 was then evaluated for diagnostic use in thyroid anaplastic carcinoma, using ¹²⁴I as the diagnostic nuclide. Results in tumour-bearing mice were promising, with all of the tumours identified in micro-PET studies.</p><p>These results demonstrate how antibody-based radionuclide targeting can provide more sensitive and specific methods for identifying and treating head and neck cancer, and hopefully help improve long-term survival rates for this patient group in the future.</p>

<p>The diagnosis and treatment of patients with advanced tumors in the head and neck is an interesting challenge where there is a need for new approaches in diagnostics and adjuvant treatment. Differences in antigen expression between tumors and normal tissues provide a means for application of antibody-based targeting techniques. By targeting a structure that is abundant on tumor cells and limited on normal cells, radioactivity can be delivered.</p><p>The use of positron emission tomography (PET) in patients with head and neck tumors is evaluated in this thesis. PET using the tracer fluorodeoxyglucose (FDG) is found to play an important diagnostic role and often has a direct clinical impact on planned surgery or other treatment. Possible targeting structures are also investigated in this thesis, and it is concluded that the EGFR and CD44v6 stand out as possible antigens for targeting approaches of squamous cell carcinomas in the head and neck (HNSCC). A radioimmunoassay for quantification of EGFR and CD44v6 is validated and concluded to be a valuable complement to immunohistochemistry for the analysis of tumors and for the planning of radioimmunotherapy. Finally, promising results of radioimmunotherapy in tumor bearing mice with the monoclonal antibody U36 labeled with the alpha emitter astatine-211 are presented.</p><p>These results demonstrate how differences between tumors and normal tissues can be used to improve diagnostic outcomes and indicate that radioimmunotherapy can be a future adjuvant therapy or treatment of residual disease in HNSCC.</p>

Telomeres are protective structures at the end of our chromosomes, composed of multiple repeats of the DNA sequence TTAGGG. They are essential for maintaining chromosomal stability by preventing damage and degradation of the chromosome ends. Telomeres are normally shortened with each cell division until a critical length is reached, at which stage cell cycle arrest is induced. Telomere shortening can be prevented in the presence of the telomere-­‐elongating enzyme telomerase. Telomerase is expressed during embryogenesis and in certain normal cell types, but most somatic cells exhibit undetectable levels of telomerase activity. In contrast, most cancer cells express telomerase enabling them to proliferate indefinitely. There is a search for reliable molecular markers that can be used to help predict cancer risk and outcome. The interest of investigating telomere length as a potential biomarker in malignancy has grown rapidly, and both tumors and normal tissues have been in focus for telomere length measurements. In this thesis, telomere length was investigated in breast cancer patients and in patients with renal cell carcinoma (RCC). The breast cancer patients were found to have significantly longer mean telomere length in peripheral blood cells (i.e. immune cells) compared to a tumor-­‐free control group. Moreover, patients with the longest blood telomere length had a significantly worse outcome compared to patients with shorter blood telomeres. In a patient group with clear cell RCC, telomere length was investigated in peripheral blood cells, in tumors and in corresponding kidney cortex. Again, patients with the longest blood telomere length had a significantly worse prognosis compared to those with shorter blood telomeres. In contrast, telomere length in tumor and kidney cortex tissues did not predict outcome per se. Immunological components may play a role in telomere length dynamics as well as in cancer development. We aimed to investigate a possible association between telomere length and certain immunological parameters, including various cytokines and peripheral levels of a blood cell type with suppressor function [regulatory T cells (Tregs)]. In our patients with clear cell RCC, three cytokines correlated significantly with tumor telomere length, but not with telomere length in peripheral blood cells. In a separate patient group with various RCC tumors, blood telomere length correlated positively with the amount of Tregs. It might be speculated that a subset of patients with long blood telomeres has a less efficient immune response due to high Treg levels, contributing to a worse prognosis. Another aim of this thesis was to explore telomere length changes over time. Evaluation of blood samples collected at a 6-­‐month interval from 50 individuals, showed that half of the participants experienced a decline in mean telomere length during the time period. This group had longer telomere length at baseline compared to those who demonstrated increased/stable telomere length. In a separate group of five blood donors, a remarkable drop in telomere length was detected in one donor over a 6-­‐month period, whereas the other donors exhibited only small fluctuations in telomere length. In conclusion, the results of this thesis indicate that blood telomere length has potential to act as an independent prognostic marker in malignancy. Adding to the complexity is the fact that changes in blood telomere length might occur within relatively short time spans, indicating that telomere length is a dynamic character.

Ultraviolet radiation (UVR) is a major risk factor for development of skin cancer. UVR-induced DNA damage and a dysfunctional p53 protein are important steps in the development of squamous cell carcinoman in human skin (SCC). The aim of the present investigation was to analyze incidence trends of SCC in Sweden, quantify the risk of second primary cancer after SCC and further analyze the effects of UVR and p53 protein in human skin in vivo and in vitro. The effect of photoprotection by sunscreens was also evaluated. We found that the age-standardized incidence rate of SCC in Sweden increased substantially in both men and women during the period 1961-1995, especially in men and at chronically sun-exposed skin sites. Patients with SCC are also at increased risk of developing new primary cancers, especially in the skin, squamous cell epithelium, hematopoietic tissues and respiratory organs. In experimental studies in vivo and in vitro in human skin we observed that repair of UV-induced DNA damage appears to be more efficient in chronically sun-exposed skin despite a less uniform p53 response. Non-sun- exposed skin is more homogeneous with respect to the epidermal p53 response. Keratinocytes in skin exposed frequently to the sun may be prone to react more easily to cytotoxic stress. Two different modalities of photoprotection significantly reduced the amount of DNA damage and the number of p53-positive cells. In addition, we demonstrated that a well-defined system for in vitro culture of explanted skin provides an excellent alternative to in vivo experiments. In conclusion, this study has increased our knowledge of SCC epidemiology in Sweden and of the effects of artificial and solar UVR and sunscreens on chronically sun-exposed and non-sun-exposed sites, respectively, of human skin.

The aim of this thesis was to gain further insight into the biology of normal human skin and basal cell carcinoma (BCC). Morphology in combination with microdissection was used as primary tool for sampling. Using the X-chromosome inactivation assay, we found normal human skin to consist of a mosaic of cells, with either the maternal or the paternal X-chromosome inactivated. We believe that each tile is made up of several epidermal proliferative units with identical X-chromosome inactivation patterns. Using the same method, we found BCC to be a monoclonal neoplasm imbedded in polyclonal stroma. However, one tumor displayed clear evidence of being composed of two intermingled monoclonal tumors. To better enable molecular analysis of defined cells from tissue sections, we investigated a zinc-based fixative as alternative to neutral-buffered formalin. Zinc-based fixative preserves good quality of genomic DNA, with only slight impairment of morphology. In addition, it partly abrogates the need for antigen retrieval. The patched gene is involved in BCC development. We analyzed the distribution of a coding polymorphism (Pro/Leu) at codon 1315 in populations with different skin types. We found a reduced Pro/Pro genotype frequency in populations with lighter pigmentation. This in combination with genotype analyses of patients with multiple BCCs, showed that failure to lose the Pro allele during a shift towards lighter pigmented skin may be associated with an increased risk of developing BCC. We compared the expression profile of BCC cells with putative progenitor cells in the basal layer of epidermis. In addition to discovering several unknown genes, we found the Wnt signaling pathway to upregulated. Furthermore, differentiation markers were downregulated together with proteins important for scavenging of oxygen radicals. In conclusion, the combination of morphology, microdissection and subsequent molecular applications provided valid information deepening our understanding of normal skin and BCC.

Radioimmunotherapy (RIT) has been shown to be a practicable way to treat head and neck squamous cell carcinoma. A specific antibody recognizes the charasteristic structure of tumour cells when loaded with cytotoxic agents (toxins, drugs, radionuclides, etc). But RIT kills not only tumour cells with attached radionuclides but also adjacent tumour cells due to the “cross fire effect”. To be efficacious, RIT depends closely on suitable monoclonal antibody, on the properties of the chosen radionuclides, and on a suitable labelling method for attaching radionuclide to antibody. In this study we initially used radionuclide-labelled cMAB U36, via linker DABI in order to improve the retention of radio-conjugates in the tumour cells. Improved retention is important because the longer the radionuclide remains in tumour cells, the more effective will the tumour cells be eradicated. In the investigation, both normal mice and HNSCC-bearing nude mice were used to compare our form of treatment against other radio-iodination methods. In the biodistribution study, normal mice showed that radioactive uptake in organs diminished with time, irrespectively of whether the conjugate was directly or indirectly labelled. But in thyroid, there was a tenfold greater accumulation of direct-labelled than of indirectly labelled conjugate. In tumour-bearing nude mice, by contrast, the results showed promising uptake of radioactivity, but little uptake in direct-labelled conjugate in thyroid. Significant differences were observed on comparing tumour: organ ratios between 131I-cMAb U36 vs. 125I-DABI-cMAb U36. In the present study, cMAb U36 labelled with 211Astatine was initially used to treat HNSCC in nude mice. The biodistribution of 211At-cMAb U36 did not reveal any significant difference between an antibody-blocked group and a non-blocked group. But it did highlight the characteristics of a successful targeting conjugate in HNSCC-bearing nude mice. In the subcutaneous therapy experiment, most of the treated tumours (n=18) had disappeared by the 26th day, in both U36-blocked and non-blocked groups. Treatment in the intravenous therapy experiment had also proved effective. In the antibody non-blocked group, the smallest tumour volume was 25 mm3 (average 111 mm3) vis-á-vis 65 mm3 (average 145 mm3) in the blocked group. None of tumours grew again following treatment.

The diagnosis and treatment of patients with advanced tumors in the head and neck is an interesting challenge where there is a need for new approaches in diagnostics and adjuvant treatment. Differences in antigen expression between tumors and normal tissues provide a means for application of antibody-based targeting techniques. By targeting a structure that is abundant on tumor cells and limited on normal cells, radioactivity can be delivered. The use of positron emission tomography (PET) in patients with head and neck tumors is evaluated in this thesis. PET using the tracer fluorodeoxyglucose (FDG) is found to play an important diagnostic role and often has a direct clinical impact on planned surgery or other treatment. Possible targeting structures are also investigated in this thesis, and it is concluded that the EGFR and CD44v6 stand out as possible antigens for targeting approaches of squamous cell carcinomas in the head and neck (HNSCC). A radioimmunoassay for quantification of EGFR and CD44v6 is validated and concluded to be a valuable complement to immunohistochemistry for the analysis of tumors and for the planning of radioimmunotherapy. Finally, promising results of radioimmunotherapy in tumor bearing mice with the monoclonal antibody U36 labeled with the alpha emitter astatine-211 are presented. These results demonstrate how differences between tumors and normal tissues can be used to improve diagnostic outcomes and indicate that radioimmunotherapy can be a future adjuvant therapy or treatment of residual disease in HNSCC.

Cutaneous reparative processes, including wound healing, are highly developed procedures in which a chain of actions occurs to reconstitute the function of the wounded tissue. To prevent a delayed or excessive reparative process it is important to understand how this procedure develops and is maintained. One of the major extracellular matrix components of the skin is the glycosaminoglycan hyaluronan (HA). HA contributes to an extracellular environment, which is permissive for cell motility and proliferation, features that may account for HA’s unique properties observed in scarless foetal wound healing. The molecule is found at high concentration whenever proliferation, regeneration and repair of tissue occur. The aims of the present studies were to analyse the distribution of HA and to investigate its possible role in various cutaneous conditions associated with an impaired reparative process like in scar tissue formation in healing wounds, changed skin characteristics in diabetes mellitus and proliferating activity in basal cell carcinomas. Tissue biopsies were obtained from healthy human skin, type-I diabetic skin and various scar tissues. The samples were analysed in the light microscope with a hyaluronan-binding-probe, antibodies for collagen I, III, PCNA and Ki-67. Ultrastructural analyses were performed on the same tissue samples. In normal skin HA was present mainly in the papillary dermis. In epidermis HA was located in between the keratinocytes in the spinous layer. In the different scar tissues the localization of HA varied, with an HA distribution in mature scar type resembling that in normal skin. In keloids the papillary dermis lacked HA, but the thickened epidermis contained more HA than the other scar types. Ultrastructural studies of keloids revealed an altered collagen structure in the dermal layers, with an abundance of thin collagen fibers in the reticular dermis and thicker collagen fibers in the papillary dermis. Furthermore, the keloids displayed epidermal changes, which involved the basement membrane (BM), exhibiting fewer hemidesmosomes, and an altered shape of desmosomes in the entire enlarged spinous layer. These alterations in epidermis are suggested to influence the hydrodynamic and cell regulatory properties of the wounded skin. In diabetic patients, a reduced HA staining in the basement membrane zone was seen. The staining intensity of HA correlated to the physical properties of the skin reflected by their grades of limited joint mobility (LJM). Furthermore, the HA staining correlated with serum concentration of the HbA1c. In basal cell carcinomas (BCC), HA occurred predominantly in the tumour stroma. The distribution was most intense in the highly developed superficial BCC type, and resembled that of the papillary dermis of normal skin. In contrast, in the infiltrative BCC type, the tumour stroma stained weakly in the infiltrative part of the tumour. Moreover, the surrounding dermal layer was deranged and devoid of HA. The findings suggest that the tumour stroma in superficial BCC causes a slow, well-regulated cell growth in which the tumour cells do not substantially disturb the normal skin function. In the infiltrative BCC type, the tumour cells cause a disintegration of the tumour stroma as well as the normal surrounding dermis, which permits further spreading of the tumour. In fact, the behaviour of the infiltrative BCC tumour, growing beyond its boundaries, resembles that of the keloid. The mapping of the distribution of HA could be a useful tool for prognostic information, for evaluating the degree of progress and for deciding the choice of treatment in various diseases of the skin. In skin malignancies such as BCC it can be used to determine the radicality at the surgical excision of the tumour. Keywords: Hyaluronan, scar tissue, diabetes mellitus, basal cell carcinoma, skin, wound healing