Defects in tricarboxylic acid cycle enzymes Fumarate hydratase and Succinate dehydrogenase in cancer

Hereditary leiomyomatosis and renal cell cancer (HLRCC) is a recently characterized cancer syndrome which predisposes to cutaneous and uterine leiomyomas as well as renal cell carcinoma (RCC). Uterine leiomyosarcoma (ULMS) has also been observed in certain Finnish HLRCC families. The predisposing gene for this syndrome, fumarate hydratase (FH), was identified in 2002. The well-known function of FH is in the tricarboxylic acid cycle (TCAC) in the energy metabolism of cells. As FH is a novel cancer gene, the role of FH mutations in tumours is in general unknown. Similarly, the mechanisms through which defective FH is associated with tumourigenesis are unclear. The loss of a wild type allele has been observed in virtually all HLRCC patients tumours and the FH enzyme activities are either totally lost or remarkably reduced in the tissues of mutation carrier patients. Therefore, FH is assumed to function as a tumour suppressor. Mutations in genes encoding subunits of other TCAC enzyme SDH have also been reported recently in tumours: mutations in SDHB, SDHC, and SDHD genes predispose to paraganglioma and pheochromocytoma. In the present study, mutations in the SDHB gene were observed to predispose to RCC. This was the first time that mutations in SDHB have been detected in extra-paraganglial tumours. Two different SDHB mutations were observed in two unrelated families. In the first family, the index patient was diagnosed with RCC at the age of 24 years. Additionally, his mother with a paraganglioma (PGL) of the heart and his maternal uncle with lung cancer were both carriers of the mutation. The RCC of the index patient and the PGL of his mother showed LOH. In the other family, an SDHB mutation was detected in two siblings who were both diagnosed with RCC at the ages of 24 and 26 years. Both of the siblings also suffered PGL. All these tumours showed LOH. Therefore, we concluded that mutations in SDHB predispose also for RCC in certain families. Several tumour types were analysed for FH mutations to define the role of FH mutations in these tumour types. In addition, patients with a putative cancer phenotype were analysed to identify new HLRCC families. Three FH variants were detected, of which two were novel. One of the variants was observed in a patient diagnosed with ULMS at the age of 41 years. However, LOH was not detected in the tumour tissue. The FH enzyme activity of the mutated protein was clearly reduced, being 43% of the activity of the normal protein. Together with the results from an earlier study we calculated that the prevalence of FH mutations in Finnish non-syndromic ULMS is around 2.4%. Therefore, FH mutations seem to have a minor role in the pathogenesis on non-syndromic ULMS. Two other germline variants were detected in a novel tumour type, ovarian mucinous cystadenoma. However, tumour tissues of the patients were not available for LOH studies and therefore LOH status remained unclear. Therefore, it is possible that FH mutations predispose also for ovarian tumours but further studies are needed to verify this result. A novel variant form of the FH gene (FHv) was identified and characterized in more detail. FHv contains an alternative first exon (1b), which appeared to function as 5 UTR sequence. The translation of FHv is initiated in vitro from exons two and three. The localization of FHv is both cytosolic and nuclear, in contrast to the localization of FH in mitochondria. FHv is expressed at low levels in all human tissues. Interestingly, the expression was induced after heat shock treatment and in chronic hypoxia. Therefore, FHv might have a role e.g. in the adaptation to unfavourable growth conditions. However, this remains to be elucidated.

Microsurgical Aneurysm Model in Rats and Mice: Development of Endovascular Treatment and Optimization of Magnetic Resonance Imaging

The rupture of a cerebral artery aneurysm causes a devastating subarachnoid hemorrhage (SAH), with a mortality of almost 50% during the first month. Each year, 8-11/100 000 people suffer from aneurysmal SAH in Western countries, but the number is twice as high in Finland and Japan. The disease is most common among those of working age, the mean age at rupture being 50-55 years. Unruptured cerebral aneurysms are found in 2-6% of the population, but knowledge about the true risk of rupture is limited. The vast majority of aneurysms should be considered rupture-prone, and treatment for these patients is warranted. Both unruptured and ruptured aneurysms can be treated by either microsurgical clipping or endovascular embolization. In a standard microsurgical procedure, the neck of the aneurysm is closed by a metal clip, sealing off the aneurysm from the circulation. Endovascular embolization is performed by packing the aneurysm from the inside of the vessel lumen with detachable platinum coils. Coiling is associated with slightly lower morbidity and mortality than microsurgery, but the long-term results of microsurgically treated aneurysms are better. Endovascular treatment methods are constantly being developed further in order to achieve better long-term results. New coils and novel embolic agents need to be tested in a variety of animal models before they can be used in humans. In this study, we developed an experimental rat aneurysm model and showed its suitability for testing endovascular devices. We optimized noninvasive MRI sequences at 4.7 Tesla for follow-up of coiled experimental aneurysms and for volumetric measurement of aneurysm neck remnants. We used this model to compare platinum coils with polyglycolic-polylactic acid (PGLA) -coated coils, and showed the benefits of the latter in this model. The experimental aneurysm model and the imaging methods also gave insight into the mechanisms involved in aneurysm formation, and the model can be used in the development of novel imaging techniques. This model is affordable, easily reproducible, reliable, and suitable for MRI follow-up. It is also suitable for endovascular treatment, and it evades spontaneous occlusion.

Epidemiology and Treatment Options of Primary Biliary Cirrhosis

Primary biliary cirrhosis (PBC) is caused by an autoimmune inflammation of the small bile ducts. It results to destruction of bile ducts, accumulation of the bile in the liver, and cirrhosis. The prevalence and incidence of PBC is increasing in the Western world. The prevalence is highest in the USA (402 per million) and incidence in Scotland (49/million/year). Our aim was to assess the epidemiology of PBC in Finland. Patients for the epidemiological study were searched from the hospital discharge records from year 1988 to 1999.The prevalence rose from 103 to 180/million from 1988 to 1999, an annual increase of 5.1%. The incidence rose from 12 to 17 /million/year, an annual increase of 3.5%. The age at death increased markedly from 65 to 76 years. The risk of liver related deaths diminished over time. The treatment of PBC is based on Ursodeoxycholic acid (UDCA). During 20 years 50% of patients end up with cirrhosis. Our treatment option was to combine budesonide, a potent corticosteroid with a high first pass metabolism in the liver, to UDCA and evaluate the liver effects and systemic effects such as bone mass density (BMD) changes. Our aim was to find out if combination of laboratory tests would serve as a surrogate marker for PBC and help reducing the need for liver biopsy. Non-cirrhotic PBC patients were randomized to receive budesonide 6 mg/day combined to UDCA 15 mg /kg/day or UDCA alone for three years. The combination therapy with UDCA and budesonide was effective: stage improved 22%, fibrosis 25%, and inflammation 32%. In the UDCA group the changes were: 20% deterioriation in stage and 70% in fibrosis, but a 10% improvement in inflammation. BMD in femoral neck decreased by 3.6% in the combination group and by 1.9% in the UDCA group. The reductions in lumbar spine were 2.8% and 0.7%. Pharmacokinetics did not differ between the stages of PBC. HA, PIIINP, bile acids, and AST were significantly different within stages I-III and could differentiate the mild fibrosis (F0F1) from the moderate (F2F3). The combination of these individual markers (PBC-score) further improved the accuracy. The area under the ROC of the PBC score, using a cut of value 66, had a sensitivity of 81.4% and a specificity of 65.2% to classify the stage of PBC. The prevalence of PBC in Finland increases, which results from increasing incidence and improved survival. The combination of budesonide and UDCA improves liver histology compared to UDCA alone in non-cirrhotic stages of PBC. The treatment may reduce BMD. Hyaluronic acid, PIIINP, AST, and bile acids may serve as tools to monitor the treatment response in the early stages of PBC. The budesonide and UDCA combination therapy is an option for those patients who do not receive full response from UDCA and are still at the non-cirrhotic stage of PBC.

Non-small cell lung cancer : studies on pathogenesis, tumour targeting and treatment outcomes

Lung cancer accounts for more cancer-related deaths than any other cancer. In Finland, five-year survival ranges from 8% to 13%. The main risk factor for lung cancer is long-term cigarette smoking, but its carcinogenesis requires several other factors. The aim of the present study was to 1) evaluate post-operative quality of life, 2) compare clinical outcomes between minimally invasive and conventional open surgery, 3) evaluate the role of oxidative stress in the carcinogenesis of non-small lung cancer (NSCLC), and 4) to identify and characterise targeted agents for therapeutic and diagnostic use in surgery. For study I, pneumonectomy patients replied to 15D quality of life and baseline dyspnea questionnaires. Study III involved a prospective quality of life assessment using the 15D questionnaire after lobectomy or bi-lobectomy. Study IV was a retrospective comparison of clinical outcomes between 212 patients treated with open thoracotomy and 116 patients who underwent a minimally invasive technique. Study II measured parameters of oxidative metabolism (myeloperoxidase activity, glutathione content and NADPH oxidase activity) and DNA adducts. Study V employed the phage display method and identified a core motif for homing peptides. This method served in cell-binding, cell-localisation, and biodistribution studies. Following both pneumonectomy and lobectomy, NSCLC patients showed significantly decreased long-term quality of life. No significant correlation was noted between post-operative quality of life and pre-operative pulmonary function tests. Women suffered more from increased dyspnea after pneumonectomy which was absent after lobectomy or bi-lobectomy. Patients treated with video-assisted thoracoscopy showed significantly decreased morbidity and shorter periods of hospitalization than did open surgery patients. This improvement was achieved even though the VATS patients were older and suffered more comorbid conditions and poorer pulmonary function. No significant differences in survival were noted between these two groups. An increase in NADPH oxidase activity was noted in tumour samples of both adenocarcinoma and squamous cell carcinoma. This increase was independent from myeloperoxidase activity. Elevated glutathione content was noted in tumour tissue, especially in adenocarcinoma. After panning the clinical tumour samples with the phage display method, an amino acid sequence of ARRPKLD, the Thx, was chosen for further analysis. This method proved selective of tumour tissue in both in vitro and in vivo cell-binding assay, and biodistribution showed tumour accumulation. Because of the significantly reduced quality of life following pneumonectomy, other operative strategies should be implemented as an alternative (e.g. sleeve-lobectomy). To treat this disease, implementation of a minimally invasive surgical technique is safe, and the results showed decreased morbidity and a shorter period of hospitalisation than with thoracotomy. This technique may facilitate operative treatment of elderly patients with comorbid conditions who might otherwise be considered inoperable. Simultaneous exposure to oxidative stress and altered redox states indicates the important role of oxidative stress in the pathogenesis and malignant transformation of NSCLC. The studies showed with great specificity and with favourable biodistribution that Thx peptide is specific to NSCLC tumours. Thx thus shows promise in imaging, targeted therapy, and monitoring of treatment response.