<p>The most important prognostic factor in breast cancer is the axillary lymph node status. The sentinel node biopsy (SNB) is reported to stage the axilla with an accuracy > 95 % in early breast cancer. Tumour-related perturbation of T-cell function has been observed in patients with malignancies, including breast cancer. The down-regulation of the important T-cell activation molecules CD3-ζ and CD28 may cause T-cell dysfunction, anergy, tolerance and deletion.</p><p>The expression of CD3-ζ and CD28 was evaluated in 25 sentinel node biopsies. The most pronounced down-regulation was seen in the paracortical area, where the best agreement between both parameters was observed. CD28 expression was significantly more suppressed in CD4+ than in CD8+ T-cells.</p><p>From the Swedish sentinel node database, 109 patients with breast cancer > 3 cm planned for both SNB and a subsequent axillary dissection were identified. The false negative rate (FNR) was 12.5%. Thirteen cases of tumour multifocality were detected on postoperative pathology. The FNR in this subgroup was higher (30.8%) than in patients with unifocal disease (7.8%; P = 0.012).</p><p>From the Swedish SNB multicentre cohort trial, 2246 sentinel node-negative patients who had not undergone further axillary surgery were selected for analysis. After a median follow-up time of 37 months (range 0-75), 13 isolated axillary recurrences (13/2246; 0.6%) were found. In another 14 cases, local or distant failure preceded or coincided with axillary relapse (27/2246; 1.2%). </p><p>In conclusion, the immunological analysis of the sentinel node might provide valuable prognostic information and aid selection of patients for immunotherapy. SNB is encouraged in breast cancer larger than 3 cm, if no multifocal growth pattern is present. The axillary recurrence rate after a negative SNB in Sweden is in accordance with international figures. However, a longer follow-up is mandatory before the true failure rate of the SNB can be determined.</p>

Background: Very recently a gene marker panel that allows the mutational analysis of APC, CTNNB1, B-RAF and K-RAS was conceived. The aim of the present study was to use the 4-gene marker panel covering the Wnt and Ras-Raf-MEK-MAPK signalling pathways to determine the percentage of sporadic colorectal carcinomas (CRC) carrying at least one of the four above-mentioned genes in a mutated form alone and/or in combination with microsatellite instability (MSI) and to compare the sensitivity of the gene marker panel used in this study with that of gene marker panels previously reported in the scientific literature. Methods: CTNNB1 and B-RAF were screened by PCR-single-strand conformation polymorphism analysis and K-RAS gene mutations by restriction fragment length polymorphism analysis. For the mutational analysis of the APC gene mutation cluster region (codons 1243–1567) direct DNA sequencing was performed. The U.S. National Cancer Institute microsatellite panel (BAT25, BAT26, D2S123, D5S346 and D17S250) was used for MSI analysis. Results: It could be shown that about 80% of early stage CRC (UICC stages I and II) and over 90% of CRC in the UICC stage IV carried at least one mutated gene and/or showed MSI. No significant increase in the gene mutation frequencies could be determined when comparing tumours in the UICC stage I with those in UICC stage IV. Conclusions: When compared with previously published gene marker panels the 4-gene marker panel used in the present study shows an excellent performance, allowing to detect genetic alterations in 80–90% of human sporadic CRC samples analyzed.

The electrochemical treatment (EChT) of tumours entails thattumour tissue is treated with a continuous direct currentthrough two or more electrodes placed in or near the tumour.Promising results have been reported from clinical trials inChina, where more than ten thousand patients have been treatedwith EChT during the past ten years. Before clinical trials canbe conducted outside of China, the underlying destructionmechanism behind EChT must be clarified and a reliabledose-planning strategy has to be developed. One approach inachieving this is through mathematical modelling. Mathematical models, describing the physicochemical reactionand transport processes of species dissolved in tissuesurrounding platinum anodes and cathodes, during EChT, aredeveloped and visualised in this thesis. The consideredelectrochemical reactions are oxygen and chlorine evolution, atthe anode, and hydrogen evolution at the cathode. Concentrationprofiles of substances dissolved in tissue, and the potentialprofile within the tissue itself, are simulated as functions oftime. In addition to the modelling work, the thesis includes anexperimental EChT study on healthy mammary tissue in rats. Theresults from the experimental study enable an investigation ofthe validity of the mathematical models, as well as of theirapplicability for dose planning. The studies presented in this thesis have given a strongindication of the destruction mechanism involved in EChT. It isshown by the modelling work, in combination with theexperiments, that the most probable cause of tissue destructionis acidification at the anode and alkalisation at the cathode.The pH profiles obtained from the theoretical models have showngood correlation with the experimentally measured destructionzones, assuming that a pH above and below certain values causetissue destruction. This implies that the models presented inthis thesis could be of use in predicting the tumourdestruction produced through EChT, and thereby provide a basisfor a systematic dose planning of clinical treatments.Moreover, the models can serve as valuable tools in optimisingthe operating conditions of EChT. Modelling work of theanode processes has explained the roleof chlorine in the underlying destruction mechanism behindEChT. It is found that the reactions of chlorine with tissueplay important roles as generators of hydrogen ions. Thecontribution of these reactions to the acidification of tissue,surrounding the anode, is strongly dependent on the appliedcurrent density and increases with decreasing currentdensity. <b>Keywords:</b>cancer, direct current, dose planning,electrochemical treatment (EChT), electrotherapy, mathematicalmodelling, tumour.

Tumour targeted radiotherapy is an appealing approach for treatment of disseminated tumour cells. A targeting agent that specifically binds to a structure on tumour cells is then used to transport therapeutically relevant radionuclides. The epidermal growth factor receptor, EGFR, is overexpressed on tumour cells in several malignancies, e.g. highly malignant gliomas. In this thesis, three types of radiolabelled EGF-conjugates, aimed for targeting to EGFR-expressing tumour cells, were developed and studied: EGF-dextran labelled with 125I, EGF labelled with 211At, and two EGF-chelates, DTPA-EGF and Bz-DTPA-EGF, labelled with the radioactive metals 111In and 177Lu. The targeting properties of radioiodinated EGF-dextran were first studied in cultured glioma cells. Radioiodine coupled to the dextran part of EGF-dextran was retained in cells appreciably longer than radioiodine coupled to EGF. This can give about 100 times increased radiation dose to tumour cells. Targeting with 211At-EGF was investigated in combination with the tyrosine kinase inhibitor gefitinib (Iressa™, ZD1839). The uptake of 211At-EGF in EGFR-expressing tumour cells increased with increasing gefitinib concentrations. This was the case for both gefitinib-resistant and gefitinib-sensitive cell lines. The effect of the combined treatment on cell survival, however, differed between the cell lines in an unexpected way. In gefitinib resistant cells, combined treatment decreased cell survival approximately 3.5 times relative to 211At-EGF treatment alone. In gefitinib sensitive cells, however, combined treatment increased the cell survival (i.e. a protective effect). The EGF-chelates studied ([111In]DTPA-EGF, [111In]Bz-DTPA-EGF and [177Lu]Bz-DTPA-EGF) all bound specifically with high affinity (Kd≈2 nM) to EGFR on cultured glioma cells. They were internalised after binding, and the cellular retention of radionuclides was high (60% remained after 45 h). A biodistribution study in mice showed that liver and kidneys accumulated a majority of the radioactivity. The EGF-chelates bound EGFR specifically also in vivo. A tumour-to-blood ratio of 25 was achieved in a preliminary study.

One of the main problems in curing cancer resides in the different microenvironment existing in tumours compared to the normal tissues. The mechanisms of failure are different for radiotherapy and chemotherapy, but they all relate to the poor blood supply known to exist in tumours. It is therefore very important to know the tumour microenvironmental conditions in order to devise techniques that will overcome the problems and will therefore improve the result of the treatment. The aims of the thesis were the modelling of tumour oxygenation and the simulation of polarographic oxygen measurements in order to assess and possibly to improve the accuracy of the electrode in measuring tumour oxygenation. It also aimed to evaluate the implications of tumour microenvironment for the radiotherapy outcome. The project used theoretical modelling as the main tool. The processes of oxygen diffusion and consumption were described mathematically for different conditions, the result being very accurate distributions of oxygen in tissues. A first simple model of tissue oxygenation was based on the oxygen diffusion around a single blood vessel. A more complex model built from the basic physical processes and measurable parameters allowed the simulation of realistical tissues with heterogeneous vasculature. This model also allowed the modelling of the two types of hypoxia known to appear in tumours and their influence on the tumour microenvironment. The computer simulation of tissues was also used for assessing the accuracy of the polarographic technique for measuring tumour oxygenation. The results of this study have shown that it is possible to model theoretically the tissue oxygenation starting from the basic physical processes. The particular application of our theoretical simulation to the polarographic oxygen electrode has shown that this experimental method does not give the oxygen values in individual cells. Because the electrode measures the average oxygenation in a relatively large tissue volume, the resulting oxygen distributions are different from the real ones and the extreme high and low values are not detected. It has further been found that the polarographic electrode cannot make distinction between various types of hypoxia existing in tumours, the geometrical distribution of the hypoxic cells influencing mostly the accuracy of the measurement. It was also shown that because of the averaging implied by the measurement process, electrode results should not be used directly to predict the response to radiation. Thus, the differences between the predictions in clinical tumour control obtained from the real or the measured oxygenations are of the order of tens of percents in absolute value. A method to improve the accuracy of the electrode, i.e. to improve the correlation between the results of the measurements and the actual tissue oxygenation, was proposed. In conclusion, theoretical modelling has been shown to be a very powerful tool for predicting the outcome of radiotherapy and it has the advantage of describing the tumour oxygenation in the least invasive manner. Furthermore it allows the investigation of the invasiveness and the accuracy of various experimental methods.

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 most important prognostic factor in breast cancer is the axillary lymph node status. The sentinel node biopsy (SNB) is reported to stage the axilla with an accuracy &gt; 95 % in early breast cancer. Tumour-related perturbation of T-cell function has been observed in patients with malignancies, including breast cancer. The down-regulation of the important T-cell activation molecules CD3-ζ and CD28 may cause T-cell dysfunction, anergy, tolerance and deletion. The expression of CD3-ζ and CD28 was evaluated in 25 sentinel node biopsies. The most pronounced down-regulation was seen in the paracortical area, where the best agreement between both parameters was observed. CD28 expression was significantly more suppressed in CD4+ than in CD8+ T-cells. From the Swedish sentinel node database, 109 patients with breast cancer &gt; 3 cm planned for both SNB and a subsequent axillary dissection were identified. The false negative rate (FNR) was 12.5%. Thirteen cases of tumour multifocality were detected on postoperative pathology. The FNR in this subgroup was higher (30.8%) than in patients with unifocal disease (7.8%; P = 0.012). From the Swedish SNB multicentre cohort trial, 2246 sentinel node-negative patients who had not undergone further axillary surgery were selected for analysis. After a median follow-up time of 37 months (range 0-75), 13 isolated axillary recurrences (13/2246; 0.6%) were found. In another 14 cases, local or distant failure preceded or coincided with axillary relapse (27/2246; 1.2%). In conclusion, the immunological analysis of the sentinel node might provide valuable prognostic information and aid selection of patients for immunotherapy. SNB is encouraged in breast cancer larger than 3 cm, if no multifocal growth pattern is present. The axillary recurrence rate after a negative SNB in Sweden is in accordance with international figures. However, a longer follow-up is mandatory before the true failure rate of the SNB can be determined.

Squamous cell carcinoma of the head and neck (SCCHN), the 6th most common cancer worldwide, has a low 5-year survival. Disease as well as treatment often causes patients severe functional and aesthetic problems. In order to improve treatment and diagnosis at earlier stages of tumour development it is important to learn more about the molecular mechanisms behind the disease. p63, an important regulator of epithelial formation, has been suggested to play a role in the development of SCCHN. Six different isoforms of p63 have been found and shown to have various functions. The aim of the studies in this thesis was to learn more about the role of p63 and proteins connected to p63 in SCCHN. Expression of p63, Cox-2, EGFR, beta-catenin, PP2A and p53 isoforms was mapped in tumours and normal tumour adjacent tissue from patients with SCCHN using western blot or RT-PCR. Results showed no significant difference between tumours and normal tumour adjacent tissue concerning expression of EGFR and beta-catenin. Cox-2 and PP2A showed significantly higher expression in tumours while p63 was more expressed in normal tumour adjacent tissue. However, expression of all these proteins in normal tumour adjacent tissue differed from tissue from disease-free non-smoking individuals. Smoking in itself did not affect expression of these proteins. The p53 isoforms p53, p53beta, p53gamma, ∆133p53, ∆133p53beta and ∆133p53gamma were expressed at RNA level in samples both from tumours and normal tumour adjacent tissue, though most of them at fairly low levels. The functional properties of the different p63 isoforms have not been fully mapped. By establishing stable cell lines over-expressing the different p63 isoforms we investigated their specific effect on tumour cells from SCCHN. Only the ∆Np63 isoforms could be stably over-expressed, whereas no clones over-expressing TAp63 could be established. Using microarray technique, cell lines stably expressing the ∆Np63 isoforms were studied and CD44, Keratins 4, 6, 14, 19 and Cox-2 were found to be regulated by p63. In conclusion, the present project adds new data to the field of p63 and SCCHN. For example, we have shown that clinically normal tumour adjacent tissue is altered compared to normal oral mucosa in non tumour patients, and that smoking does not change expression of p63, Cox-2, EGFR, beta-catenin or PP2A in oral mucosa. Novel p53 isoforms are expressed in SCCHN, and even though levels are very low they should not be overlooked. Furthermore, CD44, keratins 4, 6, 14, 19 and Cox-2 were identified as p63 targets in SCCHN.

Fibrosis is a sequel of chronic inflammation and is defined as an excessive deposition of collagen that ultimately leads to organ dysfunction. To date there are no effective treatments for fibrosis. The main cell type involved in collagen deposition and organization is the myofibroblast. In the first study we examined how myofibroblasts differentiate in human fibrotic conditions and in experimental animal models. Human tissues were stained with antibodies that recognize integrin receptors and in addition we also stained for α-SMA, a myofibroblast marker. We found a co-localization between these two markers in stromal cells and hypothesized that integrin α1 is important for the acquisition of the myofibroblast phenotype. To tests this hypothesis we used knockout animals for this integrin subunit. These animals showed a reduction of α-SMA positive fibroblasts, indicating that the α1 integrin subunit is required for proper myofibroblast differentiation. In the second study we used a neuroblastoma tumor model to study tumour growth when a drug targeting the synthesis of cellular NAD was administered. In treated animals an expansion of the nonvascular stroma was observed compared to controls. Normalization of the vasculature was observed in treated tumors together with a decrease in hypoxia. Moreover, this was followed by a decrease in stromal PDGF-B and VEGF expression, suggesting a deactivation of the stroma. In the third study the effects of over-expression of the two pro-fibrotic growth factors TGF-β and PDGF-B in skin was evaluated. We observed that both growth factors induced fibrosis. Over time, a decrease in blood vessel density was observed in both treatment groups. Both factors also stimulated an expansion of the connective tissue cell population originating from the microvascular pericyte, but the phenotype of these cells differed in the different treatments with regards to expression of markers. Furthermore, in tissue over-expressing PDGF-B but not TGF-β, the fibrotic process was partially reversible.

Background and aims: Laparoscopic technique was quickly adopted in general surgery because of less pain, quicker recovery and shorter hospital stay. In the 1990´s several reports on port site metastases restrained the enthusiasm to use laparoscopic surgery in malignant diseases. The numerous reports on port site metastases initiated a debate whether laparoscopic surgery would increase the risk of tumour spread and growth. Personal experience of two patients who devloped port site metastases from an incidental gall bladder cancer (GBC) after laparoscopic cholecystectomy (LC), encouraged us to study the incidence of wound metastases from GBC after laparoscopic and open cholecystectomy (OC). Experimentally we examined whether pneumoperitoneum would increase the risk of tumour development. Several studies had demonstrated that minimally invasive procedures exert a less negative influence on the immune system and may have beneficial effects for cancer patients. We wanted to compare the long term survival after OC and LC and if the occurence of port site metastases had any impact on survival. Material and methods: A questionnaire was sent out to all major hospitals in Sweden requesting information obout the number of port site metastases encountered 1991-94. Data on all pateints with verfied GBC were obtained from the Swedish Oncological Centres. Data on all patients with GBC registered with surgical codes for cholecystectomy were collected from the National Board of Health and Welfare (EpC). The patient files were scrutinized and long term survival data was achieved (EpC). In the first experiment on Wistar Fu rats, adenocarcinoma cells were injected intraperitoneally in animals insufflated with air, CO2 and not insufflated controls. In the following studies, rats were similarly insufflated with air,CO2 and compared to not insufflated controls. Laser Doppler blood flow in the abdominal wall was concomitantly measured. To study the effect of reduced blood flow, one rectus muscle was clamped and the other not and laser Doppler Blood flow was measured in both rectus muscles. Adenocarcinoma cells were injected into the rectus muscles in all animals at the induction of pneumoperitoneum/clamping. Results: 14 of 55 patients developed wound metastases from GBC after LC and 12 of 187 after OC. Gallbladder perforation was overrepresented in patients with wound metastases. Improved survival was noted after LC in patients with T3 tumours. Experimentally, air and CO2 equally increased intraperitoneal tumour development, Insufflation with air,CO2 and clamping decreased blood flow in the abdominal wall and increased tumour growth at the same site. Conclusion: Despite a high rate of wound metastases, LC does not seem to worsen the prognosis of GBC and may even have a positive effect on survival. Perforation of the malignant gallbladder seems to be associated with an increased risk of metastatic formation. In the experimental setting, pneumoperitoneum seems to increase tumour development. Other features of laparoscopic surgery such as decreased blood flow in the abdominal wall may contribute to increased risk of tumour progress.