Rectal cancer is one of the three most common malignancies in Sweden with an annual incidence of about 2000 cases. Current treatment consists of surgical resection of the rectum including the loco-regional lymph nodes in the mesorectum. In advanced cases, neoadjuvant chemo-radiotherapy (CRT) prior to the operative treatment reduces local recurrences and enables surgery. The neoadjuvant treatment can also eradicate the tumour completely, i.e. complete response. This research project was designed to investigate the effects of preoperative radiotherapy/ CRT and analyze methods to predict response to CRT. Study I investigated the expression of the FXYD-3 protein with immunohistochemistry in rectal cancer, with or without preoperative radiotherapy. The results from the total cohort showed that, strong FXYD-3 expression was correlated to infiltrative tumour growth (p = 0.02). In the radiotherapy group, strong FXYD-3 expression was related to an unfavourable prognosis (p = 0.02). Tumours with strong FXYD-3 expression had less tumour necrosis (p = 0.02) after radiotherapy. FXYD-3 expression in the primary tumour was increased compared to normal mucosa (p=0.008). We concluded that FXYD-3 expression was a prognostic factor in patients receiving preoperative radiotherapy for rectal cancer. Study II investigated FXYD-3 expression in tumours that developed local recurrences following surgery and compared this with expression in tumours that did not develop local recurrences. There was no difference in the expression of FXYD-3 between the group that developed local recurrences and the group that did not develop local recurrences. There was no difference in survival between those with strong or weak FXYD-3 expression. We concluded that this study could not confirm the findings from study 1 i.e. that FXYD-3 expression has prognostic significance in rectal cancer. Study III was a register-based study on the incidence and effects of complete response to neoadjuvant treatment. Eight per cent of the patients with adequate CRT to achieve complete response also had a complete histological response of the luminal tumor in the resected bowel. Sixteen per cent of that group had remaining lymph node metastases in the operative specimen. Chemotherapy together with radiotherapy doubled the chance of complete response in the luminal tumour. Patients with remaining lymph node metastases had a lower survival rate compared to those without. We concluded that residual nodal involvement after neoadjuvant treatment was an important factor for reduced survival after complete response in the luminal tumour. Study IV followed up the results from the previous study by re-evaluating magnetic resonance imaging (MRI)- images in patients with complete tumour response. Two experienced MRI radiologists performed blinded re-staging of post CRT MR- images from patients with complete response in the luminal tumour. One group with lymph node metastases and another one without were studied and the results compared with the pathology reports. The sensitivity, specificity, and positive and negative predicted values for correct staging of positive lymph nodes was 37%, 84%, 70% and 57%. The size of the largest lymph node (4.5 mm, p=0.04) seemed to indicate presence of a tumour positive lymph node. We concluded that MRI couldn’t correctly stage patients for lymph node metastases in patients with complete response to CRT in the luminal tumour.

In recent years, the genetic landscape of B-cell derived lymphoid malignancies, including chronic lymphocytic leukemia (CLL), has been rapidly unraveled, identifying recurrent genetic mutations with potential clinical impact. Interestingly, ~30% of all CLL patients can be assigned to more homogeneous subsets based on the expression of a similar or “stereotyped” B-cell receptor (BcR). Considering that biased distribution of genetic mutations was recently indicated in specific stereotyped subsets, in paper I, we screened 565 subset cases, preferentially assigned to clinically aggressive subsets, and confirm the SF3B1 mutational bias in subset #2 (45%), but also report on similarly marked enrichment in subset #3 (46%). In contrast, NOTCH1 mutations were predominantly detected in subsets #1, #8, #59 and #99 (22-34%). This data further highlights a subset-biased acquisition of genetic mutations in the pathogenesis of at least certain subsets. Aberrant NF-κB signaling due to a deletion within the NFKBIE gene previously reported in CLL warranted extended investigation in other lymphoid malignancies. Therefore, in paper II, we screened 1460 patients with various lymphoid malignancies for NFKBIE deletions and reported enrichment in classical Hodgkin lymphoma (27%) and primary mediastinal B-cell lymphoma (PMBL) (23%). NFKBIE-deleted PMBL cases had higher rates of chemorefractoriness and inferior overall survival (OS). NFKBIE-deletion status remained an independent prognostic marker in multivariate analysis. EGR2 mutations were recently reported in advanced stage CLL patients; thus, in paper III we screened 2403 CLL patients for mutations in EGR2. An overall mutational frequency of 3.8% was reported and EGR2 mutations were associated with younger age, advanced stage and del(11q). EGR2 mutational status remained an independent marker of poor outcome in multivariate analysis, both in the screening and validation cohorts. Whole-genome sequencing (WGS) of 70 CLL cases, assigned to poor-prognostic subsets #1 and #2 and indolent subset #4, were investigated in Paper IV and revealed a similar skewing of SF3B1 mutations in subset #2 and NOTCH1 mutations in subset #1 to that reported in Paper I. Additionally, an increased frequency of the recently proposed CLL driver gene RPS15 was observed in subset #1. Finally, novel non-coding mutational biases were detected in both subset #1 and #2 that warrant further investigation.

Hodgkin lymphoma (HL) is a heterogeneous condition varying from engaging one single lymph node site to a widespread condition. The prognosis with contemporary treatment is excellent for the vast majority. However, the treatment might cause severe late adverse effects in a proportion of the affected individuals. We evaluated all children and adolescents diagnosed in Sweden and registered in the Swedish Childhood Cancer Register over a period of 25 years. The incidence has been stable and the overall survival (OS) is very good, comparable to the best results in the world. Approximately ten percent encountered a relapse, but even after relapse the chances of survival were good. During the study period there were no detectable changes in survival estimates. The use of radiotherapy has decreased. Epstein Barr virus (EBV) and numbers of eosinophils, mast cells and macrophages in the tumors were investigated in 98 cases. Young children were more likely to express EBV. In patients with advanced disease the mast cell and macrophage counts were higher and they also had more affected laboratory parameters. Patients with Nodular Lymphocyte Predominant Hodgkin Lymphoma did not express EBV in the tumor, had significantly lower numbers of eosinophils, mast cells and macrophages and less affected laboratory parameters compared to classical HL. Outcome and clinical presentation were investigated in a cohort of children, adolescents and young adults in Sweden and Denmark and treatment in pediatric and adult departments was compared. OS and event-free survival (EFS) did not differ between the three age groups nor between pediatric and adult treatment. However, the Danish pediatric patients had lower EFS, which corresponded to less use of radiotherapy. Adolescents and young adults shared similar characteristics, while children presented differently with less advanced disease and male preponderance. Hospitalization rates and outpatient visits after end of treatment were evaluated to see whether the excess need of resources described in the literature is evenly distributed among the survivors or whether it is limited to a smaller group. Most of the patients had a low burden of health care use and the relapsing patients were the main drivers of the excess need.

Bakgrund: Lungcancer och malignt melanom är exempel på två sjukdomar som undersöks med dual-modaliteten positron emission tomography/computed tomography (PET/CT). Vid undersökning med PET/CT erhåller patienten både en stråldos från Flourine-18 (18F) märkt med 2-[18F] fluoro-2-deoxy-D-glucose (18FDG) och från CT-modaliteten. Det finns strålningsrisker med undersökningen som kan uttrycka sig i form av stokastiska skador som exempelvis cancer. Syftet med studien var att jämföra stråldoserna mellan lungcancergruppen (misstänkt eller verifierad) och malignt melanomgruppen genom att beräkna den totala effektiva stråldosen samt redovisa riskerna med PET/CT-undersökningen. Material och metod: Materialet omfattades av parametrar gällande undersökningen och urvalet bestod av 20 patienter från lungcancergruppen respektive malignt melanomgruppen som hämtades från Nuklearmedicin, Länssjukhuset Ryhov, Jönköping. En retrospektiv metod med kvantitativ ansats användes för genomförandet av studien. Resultat: En signifikant skillnad (p <0,001) mellan sjukdomsgrupperna förekom där lungcancergruppen erhöll 11,95 milliSievert (mSv) och malignt melanomgruppen 6,03 mSv och den procentuella riskökningen av letal cancer var 0,06 % respektive 0,03 %. Slutsatser: Lungcancergruppen erhöll en dubbelt så hög effektiv dos som malignt melanomgruppen. Den effektiva dosen är dock så låg att riskökningen av letal cancer är marginell och nyttan med undersökningen överväger riskerna. / Background: Lung cancer and malignant melanoma are diseases investigated by the dual-modality positron emission tomography/computed tomography (PET/CT). There are radiation risks with the examination that can appear as stochastic effects such as cancer. The aim of this study was to compare the radiation doses between the lung cancer group (suspected or verified) and the malignant melanoma group by calculating the total effective radiation dose and to declare the risk with the PET/CT examination. Material and method: The material contained parameters regarding the examination and the sample contained 20 patients from the two groups. The method was retrospective with a quantitative approach. Results: There was a significant difference (p <0,001) between these two groups, were the lung cancer group received 11,95 milliSievert (mSv) and the malignant melanoma group 6,03 mSv and the percentage risk for lethal cancer increased by 0,06% and 0,03%, respectively. Conclusions: The lung cancer group received twice as much effective dose than the malignant melanoma group. However, the effective dose is so low that the risk increase of the lethal cancer is marginal, and the benefit of the examination outweighs the risks.

Radiotherapy is the use of ionising radiation to induce localised DNA damage to cancerous tissues, leading to cell death and disease control. In order to maximise tumour growth control and to limit damage of the healthy surrounding tissues and the consequent side effects for the patient, molecular determinants of tumour radioresistance are investigated as potential clinical targets. A high-throughput siRNA colony formation assay screen in HeLa cervical carcinoma cells previously published by our laboratory identified modulators of radiosensitivity. From the list CSF1R, EPHB2, GAK and TOPK, were selected and validated. TOPK (T-LAK cell-originated protein kinase, also known as PDZ-binding kinase, PBK) was selected for further investigation because it is overexpressed in most malignancies but not in normal tissues, apart from testis and placenta. Knockdown of TOPK was shown to induce radiosensitisation in a panel of cancer cell lines with no significant effects on normal cells. A role for TOPK in the cell cycle response to ionising radiation (IR) was discovered in HCT116 colorectal cancer cells, with alterations in the G₁/S and G₂/M checkpoints. Furthermore, immunoprecipitation experiments identified a physical interaction between TOPK and CDKN1A (p21) at 8 hours after IR. Apoptosis and the number of multinucleated cells were significantly increased in TOPK depleted cells exposed to IR, suggesting the possibility of aberrant mitosis and mitotic catastrophe in these cells. High TOPK expression in early breast cancer patients was shown to be associated with poor recurrence-free survival. In addition, immunohistochemistry (IHC) analysis on samples from prostate cancer patients identified a strong correlation between high levels of TOPK and poor clinical response to radiotherapy. In order to facilitate future in vivo experiments, an HCT116 shRNA stable knockdown cell line was developed and two commercially available TOPK inhibitors were tested and optimised. Taken together, these data suggest that TOPK is a molecular determinant of radiosensitivity with a great potential for future clinical applications.

Cancer is a well-known disease that many people encounter in their lifetime. There is constantly research being performed on cancer to find treatments for those types where none has been found, or even find better or more efficient treatments for those cancer types where there already is treatment available. Two types of cancer that have been studied in this thesis are neuroblastoma, which is a form of cancer that affects children and infants, and breast cancer. The 13-cis retinoic acid is presently used as treatment for neuroblastoma post-surgery and post-chemo therapy, but the treatment is quite invasive. It has been shown that 1,25-dihydroxyvitamin D3 is a good candidate for cancer treatment, and the aim of this study was to investigate whether a combination of 1,25-dihydroxyvitamin D3 and two forms of retinoic acid, all-trans and 13-cis, could cause synergistic effects on cell viability, invasion, and differentiation of the cells. The two vitamins were combined at different concentrations and ratios to make the different treatments. A proliferation assay with absorbance measurement was performed to determine cell viability, and a migration assay was performed to observe the migratory properties of the cells after treatment. The results concluded that the combined treatments had greater effect than the single treatments on cell viability in both neuroblastoma and breast cancer cells. The results showed that single treatment of 13-cis retinoic acid and combined treatments had the highest effect on invasion and differentiation on neuroblastoma cells.

Pyelonefrit är en inflammation i njurarna och undersökningen som främst används vid utredning är dimerkaptosuccinat (DMSA)-scintigrafi. Pyelonefrit drabbar framförallt barn och risk finns för njurparenkymskador. Syftet med studien var att utreda om DMSA-scintigrafi efter pyelonefrit hos barn är berättigad. Detta granskades genom att beräkna den generella risken för cancer, specifika riskökningen för njurparenkymcancer, antal upptäckta njurparenkymskador och eventuella könsskillnader. Vetenskapliga artiklar söktes upp via sökmotorn PRIMO. Metoden var retrospektiv med kvantitativ ansats där materialet bestod av svarsutlåtanden från DMSA-scintigrafier på barn 0-2 år med frågeställning njurparenkymskador efter pyelonefrit. Urvalet bestod av 91 barn; 52 flickor och 39 pojkar varav 16 stycken exkluderades. Av de studerade 75 barnen hade sex (8 %) njurparenkymskador, med medelålder på 9,2 månader, och det fanns ingen signifikant skillnad mellan kön och njurparenkymskada (p=0,246). Medelvärdet på given aktivitet gav en effektiv medeldos på 0,69 mSv. Den generella riskökningen vid en DMSA-scintigrafi blev 0,01-0,014 och 0,00019 för njurparenkymcancer. Trots att relativt få barn drabbas av njurparenkymskador, finns ändå risk att drabbas av komplikationer från skadan. Skadorna är därför viktiga att upptäcka. Riskökningen för cancerutveckling och njurparenkymcancer efter DMSA-scintigrafi är mycket låg. Nyttan (att upptäcka njurparenkymskadorna) överväger risken (strålningen), vilket gör DMSA-scintigrafin till en berättigad undersökningsmetod. / A dimercaptosuccinic acid (DMSA) scintigraphy is used to test for pyelonephritis, an inflammation of the kidneys with risk of renal scarring. Aiming to investigate if DMSA scan after pyelonephritis in children is justified, we calculated the general cancer risk, the specific increased renal cancer risk, the number of discovered renal scarring and potential differences between the sexes. The method was retrospective and quantitative and data was based on results from DMSA scans of children aged 0-2 years. From the original set of 91 children (52 girls, 39 boys), 16 were excluded. Of the remaining 75, six (8 %) had renal scarring; with an average age of 9,2 months, and there was no significant difference between sex and renal parenchymal damage (p=0,0246). The mean activity from a DMSA scan equaled an effective dose of 0.69 mSv, with general cancer versus renal cancer risk being 0.01-0.014 and 0.00019, respectively. Even though only a few children develop renal scarring, there is still a risk of complications. Renal scarring is therefore important to discover. The increased risk for cancer and renal cancer after a DMSA scan is low. The benefits (discovering renal scarring) are greater than the risk (radiation), making the DMSA scan justified.

Deregulation of the epigenome is associated with the initiation and progression of various types of human cancers. Here we investigated the level of 5-hydroxymethylcytosine (5hmC), expression and function of TET1 and TET2, and DNA methylation in parathyroid tumors and small intestinal neuroendocrine tumors (SI-NETs). In Paper I, an undetectable/very low level of 5hmC in parathyroid carcinomas (PCs) compared to parathyroid adenomas with positive staining, suggested that 5hmC may represent a novel biomarker for parathyroid malignancy. Immunohistochemistry revealed that increased tumor weight in adenomas was associated with a more aberrant staining pattern of 5hmC and TET1. A growth regulatory role of TET1 was demonstrated in parathyroid tumor cells. Paper II revealed that the expression of TET2 was also deregulated in PCs, and promoter hypermethylation was detected in PCs when compared to normal parathyroid tissues. 5-aza-2′-deoxycytidine treatment of a primary PC cell culture induced TET2 expression and further supported involvement of promoter hypermethylation in TET2 gene repression. TET2 knockout demonstrated a role for TET2 in cell growth and migration, and as a candidate tumor suppressor gene. In Paper III, variable levels of 5hmC, and aberrant expression of TET1 and TET2 were observed in SI-NETs. We demonstrated a growth regulatory role for TET1, and cytoplasmic expression with absent nuclear localization for TET2 in SI-NETs. In vitro experiments supported the involvement of exportin-1 in TET2 mislocalization, and suggested that KPT-330/selinexor, an orally bioavailable selective inhibitor of exportin-1 and nuclear export, with anti-cancer effects, could be further investigated as a therapeutic option in patients with SI-NETs. In Paper IV, DNA methylation was compared between SI-NET primary tumors and metastases by reduced representation bisulfite sequencing. Three differentially methylated regions (DMR) on chromosome 18 were detected and chosen for further analyses. The PTPRM gene, at 18p11, displayed low expression in SI-NETs with high levels of methylation in the presumed CpG island shores, and in the DMR rather than the promoter region or exon 1/intron 1 boundary. PTPRM overexpression resulted in inhibition of cell growth, proliferation, and induction of apoptosis in SI-NET cells, suggesting a role for PTPRM as an epigenetically deregulated candidate tumor suppressor gene in SI-NETs.

Malignant melanoma (MM) is the deadliest skin cancer with an ever-increasing incidence. New treatments have improved the prognosis for patients with advanced MM. Still, most patients do not respond, and the side effects can be severe, underlining the need for better therapies. The overall aim of this thesis was to evaluate new means to improve the treatment for patients with advanced MM. Immunostimulatory gene therapy (AdCD40L) was evaluated in a clinical study and BRAF-inhibitory treatment in rare cases of BRAF-mutated MM. Due to its immunogenicity, MM is an attractive target for immunostimulatory gene therapy. AdCD40L is an adenovirus carrying the human gene for CD40 ligand, which in different ways can stimulate the immune system to combat cancer. We conducted a Phase I/IIa study with AdCD40L in patients with metastatic MM having received established treatments. In cohort 1 (n=6), four weekly, intratumoural AdCD40L injections were given. In cohort 2 (n=9), low dose cyclophosphamide was added to increase the immune response. Since irradiation may act synergistically with immunotherapy, patients in cohort 3 (n=9) also received a single fraction of radiotherapy (8 Gy). This fraction was given towards the lesion selected for injections. The primary objectives were to assess the feasibility and safety of AdCD40L-treatment and secondarily its anti-tumour effects. Patients were thoroughly assessed for toxicity. The anti-tumour response was evaluated by imaging techniques (FDG-PET/CT, DW-MRI scans), tumour biopsies and blood tests. Plasma protein markers were measured with a multiplex platform. Another objective was to evaluate the potential of DW-MRI and FDG-PET/CT for prediction of AdCD40L treatment response, in terms of overall survival (OS). AdCD40L was well tolerated with mild transient reactions. Local and distant responses in PET/CT scans along with a significantly better 6-month survival in the cohorts that received cyclophosphamide conditioning were observed. Effector lymphocyte responses were elicited. All patients had an increased T effector/T regulatory-cell ratio and death receptors were significantly up-regulated post therapy. Inflammatory cytokines and other plasma proteins were altered in favourable ways by the AdCD40L treatment. The analyses support that the functional DWI parameters may be better early predictors of OS than the established metabolic and morphologic criteria of FDG-PET/CT and CT/MRI, respectively. In conclusion, the stimulation of the CD40 pathway to initiate anti-tumour immunity is a promising treatment alternative for MM patients. However, further studies with developed treatment schemes are warranted. In the first report ever on treatment of a pregnant patient with a BRAF-inhibitor, the therapy was initiated in the second trimester. The treatment with vemurafenib enabled prolonged gestation, hence reducing the risk of immaturity-related complications. Further, we report the first case worldwide of a patient with metastatic conjunctival melanoma who benefitted from treatment with vemurafenib. Additional studies are needed to assess the efficacy of BRAF -inhibitors in the different subtypes of ocular melanoma.

Acute lymphoblastic leukemia (ALL) has poor prognosis in older/elderly adults and in high-risk/relapsed disease. Recommended treatment of ALL was evaluated (study I-IV). Data was obtained from the Swedish Acute Leukemia registries and from patient records. I. We assessed ALL relapse treatment and outcome in 76 patients aged 15-65 years (y). Complete remission (CR) was achieved in 50/71 patients (70%). Of them, 29 underwent allogeneic hematopoietic stem cell transplantation (hSCT). Five year overall survival (OS) was 15%, but close to 50% in 19 patients <35y after hSCT. II. We studied outcome of treatment with the Hyper-CVAD protocol in 19 of 24 patients with T-ALL aged 18-72y. CR was reached in 89%, but 5y leukemia-free survival was only 29%, and 20% in 15 patients not transplanted in CR1. Six patients received hSCT in CR2. Finally, 5y OS in all 19 patients was 47%. The only negative prognostic factor found was age ≥35y. III. We evaluated minimal residual disease (MRD) monitoring in 35 patients with Philadelphia (Ph) negative B-ALL aged 46-79y and treated with the ABCDV protocol. The CR rate was 91%. MRD was measured by flow cytometry in 73% in CR1 (MRD1) and omitted in those >70y or with high-risk ALL. Five patients received hSCT (only one due to MRD). Five year OS in the whole cohort was 47%. Continuous CR but not OS was improved in patients with MRD1 <0.1 %. IV. We studied 155 patients with ALL (Ph+ in 35%) aged 55-85y and treated with remission induction/palliation (124/31). Both, intensive, and palliative treatment resulted in the CR rates of 70/83/16% and 3y OS of 26/32/3%. OS was negatively influenced by age and platelet count ≤35×109/L (but not Ph+). OS was not enhanced by introduction of an age-adapted protocol. We concluded that intensive treatment with subsequent allogeneic hSCT is the most reasonable option in younger patients with ALL recurrence (I). Hyper-CVAD has low relapse-preventing efficacy (II). MRD guided intensification is probably feasible in only a minority of older patients (III). Prognosis in elderly ALL is poor, but no longer impaired by Ph+ (IV).