<p>In Sweden approximately 2800 new lung cancer patients are diagnosed every year. Radiotherapy is used with curative intention in certain groups of patients. The aim of this thesis is to study the basis of differences in radioresistance and the possibility to predict response to radiotherapy.</p><p>In the first study we investigated, using the comet assay, four lung cancer cell lines with different sensitivity towards radiation. A clear dose-response relationship for radiation-induced DNA single strand and double strand breaks were found. All cell lines showed a remarkably efficient repair of both the DNA single strand and double strand breaks one hour after irradiation. However, further studies in one radioresistant and one radiosensitive cell line demonstrated that repair during the first 15 min had the best accordance with radiosensitivity measured as surviving fraction.</p><p>In the second and third study, sequencing studies of the p53 gene were performed on cell lines as well as on tumour material. Cell lines that were expressing a mutation in exon 7 were associated with increased radiosensitivity compared with tumor cell lines with mutations in other exons. In the clinical study, 10 patients were found to be mutated in the p53 gene whereas the other 10 patients were not. No correlation to clinical parameters could be drawn.</p><p>In the fourth study, serum from 67 patients with a confirmed diagnosis of non-small cell lung cancer was investigated for the presence of p53 antibodies. P53 antibodies in sera, taken prior to radiation treatment, were associated with increased survival.</p><p>The summary of this thesis indicates that the p53 gene has an impact on the effect of radiotherapy in lung cancer. The presence of p53 antibodies might be of clinical interest for predicting survival after radiotherapy. Further studies on the importance of the p53 gene on early repair are of interest. </p>

<p>Lymphomas are a heterogeneous group of neoplasias originating from B- or T-lymphocytes. In this thesis, we determined the genetic and immunophenotypic characterization of DLBCL and their prognostic impact. Moreover, genomic alterations associated with the transformation to DLBCL from Hodgkin lymphoma (HL) and follicular lymphoma (FL) were elucidated. </p><p>In order to outline the impact of cytogenetic as well as immunophenotypic prognostic markers in DLBCL, we firstly studied a series of 54 DLBCL tumors using comparative genomic hybridization (CGH) and we identified several frequently occurring chromosomal imbalances. Loss of 22q was more often found in the diagnostic tumors with a more advanced clinical stage, while gain of 18q21 was more commonly identified in relapses. Secondly, we correlated the expression patterns of CD10, bcl-6, IRF-4 and bcl-2 with clinical parameters in a series of 173 de novo DLBCL patients. Patients with a germinal center (GC) phenotype displayed a better survival than the non-GC group. Expression of bcl-6 and CD10 was correlated with a better survival while bcl-2 expression was associated with a poor prognosis.</p><p>In approaching the HL transformation, two novel B-cell lines (U-2932 and U-2940), derived from patients with DLBCL following HL, were characterized. Interestingly, a translocation with materials from 2q and 7q as well as loss of material on 6q was found in both cell lines. For FL transformation, we assessed chromosomal alterations in a panel of 28 DLBCL patients with a previous history of FL. The DLBCL tumors displayed more chromosomal imbalances compared to FL tumors. Loss of 6q16-21 and gain of 7pter-q22 were more commonly found in the DLBCL counterparts, suggesting the chromosomal location of putative genes that may be involved in the transformation process.</p>

<p>In contrast-enhanced magnetic resonance angiography (CE-MRA),vascular signal is produced by the acquisition of a T1-weighted magnetic resonance imaging scan while the presence of a contrast agent induces a low T1 in blood. In this thesis,CE-MRA of the aortoiliac arteries was evaluated.Different contrast agents and techniques for synchronisation of the scan with the contrast bolus passage were assessed.</p><p>In 30 patients with clinically suspected iliac artery stenoses,contrast-enhanced magnetic resonance angiography was compared with duplex ultrasound scanning and digital subtraction x-ray angiography (DSA),with intraarterial pressure measurements as reference method. No statistically significant differences in sensitivity or specificity were observed between the techniques regarding the detection of hemodynamically significant iliac stenoses. The use of multiplanar reformats and source images in the MRA examinations was of value for the differentiation between high-grade stenoses and occlusions. With DSA as reference method, MRA had significantly higher sensitivity and specificity than duplex for the detection of ≥50% stenoses.</p><p>In fourteen patients examined with iliac artery MRA, differences in contrast arrival time of up to 7 s was observed between the aorta and the common femoral artery.A dual-station timing technique adjusting for this difference was found feasible. Compared with a fluoroscopically triggered technique (n=13),which is used in clinical rotine, the dual-station technique was more reliable for the visualisation of distal vessels.</p><p>In a clinical phase II study comparing different doses of t he contrast agent gadobenate dimeglumine for the enhancement of iliac artery MRA, a significant improvement in subjective diagnostic quality compared with time-of-flight MRA was found at all doses from 0.025 mmol/kg.An increasing trend with dose was observed up to a dose level of 0.05-0.1 mmol/kg.</p><p>In a phase I clinical study on the intravascular, iron oxide contrast agent NC100150 Injection, a positive dose response was observed for abdominal vascular enhancement, with the highest contrast-to-noise ratio observed at 4.0 mg Fe/kg bw at 1.5 T and at 2.5-4 mg Fe/kg bw at 0.5 T.At 1.5 T higher R2*values were calculated for the aorta than for the IVC.</p>

In contrast-enhanced magnetic resonance angiography (CE-MRA),vascular signal is produced by the acquisition of a T1-weighted magnetic resonance imaging scan while the presence of a contrast agent induces a low T1 in blood. In this thesis,CE-MRA of the aortoiliac arteries was evaluated.Different contrast agents and techniques for synchronisation of the scan with the contrast bolus passage were assessed. In 30 patients with clinically suspected iliac artery stenoses,contrast-enhanced magnetic resonance angiography was compared with duplex ultrasound scanning and digital subtraction x-ray angiography (DSA),with intraarterial pressure measurements as reference method. No statistically significant differences in sensitivity or specificity were observed between the techniques regarding the detection of hemodynamically significant iliac stenoses. The use of multiplanar reformats and source images in the MRA examinations was of value for the differentiation between high-grade stenoses and occlusions. With DSA as reference method, MRA had significantly higher sensitivity and specificity than duplex for the detection of ≥50% stenoses. In fourteen patients examined with iliac artery MRA, differences in contrast arrival time of up to 7 s was observed between the aorta and the common femoral artery.A dual-station timing technique adjusting for this difference was found feasible. Compared with a fluoroscopically triggered technique (n=13),which is used in clinical rotine, the dual-station technique was more reliable for the visualisation of distal vessels. In a clinical phase II study comparing different doses of t he contrast agent gadobenate dimeglumine for the enhancement of iliac artery MRA, a significant improvement in subjective diagnostic quality compared with time-of-flight MRA was found at all doses from 0.025 mmol/kg.An increasing trend with dose was observed up to a dose level of 0.05-0.1 mmol/kg. In a phase I clinical study on the intravascular, iron oxide contrast agent NC100150 Injection, a positive dose response was observed for abdominal vascular enhancement, with the highest contrast-to-noise ratio observed at 4.0 mg Fe/kg bw at 1.5 T and at 2.5-4 mg Fe/kg bw at 0.5 T.At 1.5 T higher R2*values were calculated for the aorta than for the IVC.

Breast cancer is the most common malignancy among females in Sweden. Axillary lymph-node dissection is a standard procedure in the management of breast cancer, aiming at obtaining prognostic information for adjuvant therapy decisions. Axillary dissection entails considerable morbidity. The aims of this study were to establish more selective surgical approaches and to investigate angiogenesis, a potential predictor for lymph-node metastases and prognosis. Clinical nodal status, tumour size and S-phase were associated with nodal metastases in cohort of 1145 women. The proportion of nodal metastases was 13% in the subgroup with the lowest risk. In a study from two registries, 675 and 1035 breast cancers ≤10 mm diagnosed by screening mammography had nodal metastases in 6,5% and 7%, respectively. Clinically detected cancers had a risk of 16% and 14%, respectively. In a study on 415 women, a 5-node biopsy of the axilla had a sensitivity of 97,3% and a false negative rate of 2,7% in comparison with axillary dissection. Six sections from 21 breast cancers were analysed for microvessel density (MVD). The inter-section variation contributed more to the total variance than inter-tumour variation, 45,0% and 37,3%, respectively. In a cohort of 315 women, breast cancers with high MVD more frequently had p53 mutations (27,1%) compared with cases with low MVD (18,4%). This difference was not statistically significant (p=0,075). p53 mutations were associated with a worse outcome, whereas MVD was not. In conclusion, women with screening detected ≤10 mm breast cancers have a low risk of lymph node metastases and some may not need axillary dissection in the future. The 5-node biopsy could be an alternative to axillary dissection. MVD is associated with methodological weaknesses and routine use is not recommended.

Knowledge of the relationship between in vivo contrast agent concentration and magnetic resonance (MR) signal response is an important requirement in contrast enhanced MR imaging in general and in MR based perfusion imaging in particular. This relationship is a complex function of the properties of the contrast agent as well as the structure of the target tissue. The aim of the present work was to quantify the effects of the iron oxide nanoparticle based intravascular contrast agent, NC100150 Injection, on proton relaxation rates in different tissue systems in vivo in a pig model and ex vivo in phantoms containing whole blood. Methods that enabled accurate relaxation rate measurements in these organs were developed, and validated. From these measurements, trans-compartmental water exchange rates and blood volume could be estimated and the MR signal response could be predicted as a function of contrast agent concentration under relevant imaging conditions. Using a 1.5 Tesla clinical MR system, the longitudinal (R1=1/T1) proton relaxation rates in blood, renal cortex, paraspinal muscle and myocardium were measured in vivo as a function of plasma concentration (Cp) of NC100150 Injection. The transverse (R2* = 1/T2*) relaxation rates were measured in vivo in blood, renal cortex and muscle as a function of Cp and ex vivo in blood as a function of Cp and blood oxygenation tension. The proton nuclear MR (NMR) linewidth and lineshape were analysed as a function of Cp and blood oxygen tension ex vivo at 7.05 T. In muscle and renal cortex, there was a linear correlation between R2* and Cp whereas R2* increased as a quadratic function of Cp in blood. The NMR linewidth increased linearly with Cp in fully oxygenated blood whereas in deoxygenated blood the linewidth initially decreased with increasing Cp, reaching a minimum and then increasing again with further increase in Cp. R1 increased linearly with Cp in blood and from the slope of R1 vs. Cp the T1-relaxivity (r1) of NC100150 Injection in blood at 1.5 T was estimated to be (mean ± SD) 13.9 ± 0.9 s-1mM-1. In tissue, the maximum increase in R1 was limited by the rate of water exchange between the intravascular and interstitial tissue compartments. Using a two-compartment exchange-limited relaxation model, the permeability surface area (PS) product was estimated to be 61.9 ± 2.9 mL/min/g in renal cortex and 10.1 ± 1.5 mL/min/g in muscle and the total myocardial water exchange rate, kt, was 13.5 ± 6.4 s-1. The estimated blood volumes obtained from the same model were 19.1 ± 1.4 mL/100 g, 2.4 ± 1.4 mL/100 g and 11.2 ± 2.1 mL/100 g, respectively in renal cortex, muscle and myocardium. Current T2* based first-pass MR perfusion methods assume a linear correlation between R2* and Cp both in blood and tissue and our results therefore suggest that quantitative perfusion values can not easily be obtained with existing tracer kinetic models. The correlation between MR signal response and Cp is further complicated in the kidney by a significant first-pass increase in R1 which may lead to an underestimation of Cp. In T1-based perfusion methods, low concentrations of NC100150 Injection must be used in order to maintain a linear dose-response relationship between R1 and Cp. The effect of blood oxygenation on the NMR linewidth in the presence of NC100150 Injection enabled accurate estimation of magnetic susceptibility of deoxyhemoglobin and the effect can potentially be used to determine blood oxygenation status. In conclusion, NC100150 Injection is well suited as a T2* perfusion agent due to the large magnetisation and intravascular biodistribution of this agent. T1-based perfusion imaging with this agent is limited by water exchange effects and large T2* effects at higher contrast agent concentrations. Quantitative perfusion assessment is unlikely to be feasible with any of these approaches due to the non-linear dose response.

In Sweden approximately 2800 new lung cancer patients are diagnosed every year. Radiotherapy is used with curative intention in certain groups of patients. The aim of this thesis is to study the basis of differences in radioresistance and the possibility to predict response to radiotherapy. In the first study we investigated, using the comet assay, four lung cancer cell lines with different sensitivity towards radiation. A clear dose-response relationship for radiation-induced DNA single strand and double strand breaks were found. All cell lines showed a remarkably efficient repair of both the DNA single strand and double strand breaks one hour after irradiation. However, further studies in one radioresistant and one radiosensitive cell line demonstrated that repair during the first 15 min had the best accordance with radiosensitivity measured as surviving fraction. In the second and third study, sequencing studies of the p53 gene were performed on cell lines as well as on tumour material. Cell lines that were expressing a mutation in exon 7 were associated with increased radiosensitivity compared with tumor cell lines with mutations in other exons. In the clinical study, 10 patients were found to be mutated in the p53 gene whereas the other 10 patients were not. No correlation to clinical parameters could be drawn. In the fourth study, serum from 67 patients with a confirmed diagnosis of non-small cell lung cancer was investigated for the presence of p53 antibodies. P53 antibodies in sera, taken prior to radiation treatment, were associated with increased survival. The summary of this thesis indicates that the p53 gene has an impact on the effect of radiotherapy in lung cancer. The presence of p53 antibodies might be of clinical interest for predicting survival after radiotherapy. Further studies on the importance of the p53 gene on early repair are of interest.

Lymphomas are a heterogeneous group of neoplasias originating from B- or T-lymphocytes. In this thesis, we determined the genetic and immunophenotypic characterization of DLBCL and their prognostic impact. Moreover, genomic alterations associated with the transformation to DLBCL from Hodgkin lymphoma (HL) and follicular lymphoma (FL) were elucidated. In order to outline the impact of cytogenetic as well as immunophenotypic prognostic markers in DLBCL, we firstly studied a series of 54 DLBCL tumors using comparative genomic hybridization (CGH) and we identified several frequently occurring chromosomal imbalances. Loss of 22q was more often found in the diagnostic tumors with a more advanced clinical stage, while gain of 18q21 was more commonly identified in relapses. Secondly, we correlated the expression patterns of CD10, bcl-6, IRF-4 and bcl-2 with clinical parameters in a series of 173 de novo DLBCL patients. Patients with a germinal center (GC) phenotype displayed a better survival than the non-GC group. Expression of bcl-6 and CD10 was correlated with a better survival while bcl-2 expression was associated with a poor prognosis. In approaching the HL transformation, two novel B-cell lines (U-2932 and U-2940), derived from patients with DLBCL following HL, were characterized. Interestingly, a translocation with materials from 2q and 7q as well as loss of material on 6q was found in both cell lines. For FL transformation, we assessed chromosomal alterations in a panel of 28 DLBCL patients with a previous history of FL. The DLBCL tumors displayed more chromosomal imbalances compared to FL tumors. Loss of 6q16-21 and gain of 7pter-q22 were more commonly found in the DLBCL counterparts, suggesting the chromosomal location of putative genes that may be involved in the transformation process.

xHypoxia and repopulation of tumour clonogens are two important determinants of treatment outcome in radiotherapy. In general clinical evidence indicates that loco-regional control may be reduced with long overall treatment times and for tumours with low pre-treatment levels of oxygen. Experimental studies with normobaric carbogen and oxygen showed a two-fold enhancement of the efficacy of radiation in a mouse tumour model when combining oxygen with treatment acceleration. It was then demonstrated that substituting carbogen for oxygen and adding high-dose nicotinamide (NAM) further increased the effect. These findings became the basis for a multi-factorial approach designed to overcome the radioprotective effect of tumour repopulation and that of perfusion–limited and diffusion–limited hypoxia. The strategy, named ARCON, combines Accelerated Radiotherapy with CarbOgen and Nicotinamide. Experimental evaluation of ARCON The therapeutic potential of carbogen combined with NAM (CON) focusing on treatment schedules that use clinically relevant radiation and drug dose levels was evaluated in tumour and normal tissue animal models. Some of the conditions under which ARCON gives the largest degree of tumour radiosensitization and therapeutic benefit were identified. Specifically, NAM-dose level, pharmacokinetics and scheduling, and the effect of NAM on repair processes in vivo were also investigated. The results showed that in conventional and accelerated radiotherapy, carbogen and CON are effective and relatively non-toxic tumour sensitizers. They also demonstrated that tumour sensitization with CON was independent of time of NAM administration but that it was drug dose dependent. Some degree of normal tissue sensitization was observed but even relative to mouse skin a significant therapeutic gain was achieved. The mechanism of action for NAM sensitization originally proposed was that of repair inhibition. In the in vivo mouse models tested, namely skin and kidney, NAM did not alter the rate nor the magnitude of repair of radiation induced damage. Clinical evaluation of ARCON In the early 90s, various centres, particularly in the UK, Sweden, Holland and Switzerland, undertook clinical trials of ARCON. The protocols were designed based on detailed considerations of the rodent and human radiation and pharmacokinetic studies. This document also discusses the findings of a phase II non-randomized trial in advanced bladder cancer of accelerated radiotherapy combined with carbogen alone and ARCON. The aim of the study was to establish the feasibility of administering carbogen and NAM to patients and to determine the extent of early and late normal tissue damage. Historical comparisons suggested no overt increase in normal tissue radiosensitivity and the data indicate that ARCON could achieve a therapeutic gain in advanced bladder cancer.

<p>Colorectal cancer is one of the most common malignant diseases in Sweden – more than 5000 new cases are diagnosed each year. The overall five-year survival is about 60% and in cases of recurrence the prognosis is poor.</p><p>In a phase III study in advanced colorectal cancer the response rate was doubled when 5-FU was given as a bolus injection versus as a short infusion. The toxicity was similar and time to progression was longer in the injection group. However, overall survival was not significantly different. Dose-effect relationships of 5-FU were studied in another phase III study recruiting 312 patients. A decrease from 500 mg/m² to 400 mg/m² worsened the treatment results. A low incidence of severe toxicity was seen in both groups. An increase to 600 mg/m² worsened the toxicity without any improvement of the results.</p><p>A cytotoxic drug sensitivity test in different tumour types, mainly gastrointestinal cancer, poorly predicted treatment outcome in a phase II study.</p><p>The conventional Nordic Flv regimen was split in a phase I/II trial. An escalation of dose was possible and the response rate was 20%.</p><p>Thymidylate synthase (TS) and the gene expression of p53 were investigated by immunohistochemical technique in the primary tumours of 132 patients. None of the markers predicted the later palliative chemotherapy result. However, TS significantly predicted time to recurrence.</p><p>Serum markers were analysed before and during FLv treatment to early predict outcomes among 87 patients. TPS is promising, both as a predictive marker before start of treatment and after a short period of treatment. In the same setting, CEA had lower predictive value. S-VEGF and S-bFGF did not yield any prognostic information of later outcome. In all studies B-haemoglobin values, performance status and subjective response were strong markers, both for prediction of objective response and for survival.</p>