EH 3_2015

www.KUGEL-medical.de YOUR SPECIALIST FOR LABORATORY, PATHOLOGY & HISTOLOGY MADE IN GERMANY EUROPEAN HOSPITAL Vol 24 Issue 3/15 8 LABORATORY Genetic alterationsProfessor Christine Mannhalter highlights the impact changes have on the occurrence and severity of diseases and their influence on therapy response. Many different genetic alterations (mutations) influence the clinical phenotypes of monogenetic and polygenetic inherited but also somatic diseases. Germ line muta- tions that confer a severe impact on the phenotype are usually found in rare diseases (orphan diseas- es). In contrast, common germ line mutations (polymorphisms) with little negative selection pressure occur frequently in the gene pools of populations (e.g. the factor V Leiden mutation, associated with venous thrombosis, has a frequency of about 5% in Caucasians). It is important to be aware that muta- tion carriers do not always develop the disease. Furthermore, mutations do not necessarily lead to the same phenotype in every mutation carrier, which is due to endogenous and exogenous modulators. Up to now the influences of modulators are not well known, and it is often difficult to predict the consequences of a mutation for indi- vidual patients. Despite these limita- tions, genetic tests are very valuable because they allow the early detec- tion of mutations and are important for diagnosis, prognosis, prediction of relapses, selection of therapies, monitoring of therapy responses and family studies (identification of carrier relatives). This article discusses examples of applications of genetic analyses in coagulation disorders and haem- atological and oncological diseases. Molecular genetic analyses in coagulation disorders Most proteins involved in blood coagulation are well characterised, their functions are known and their genes have been cloned and sequenced around 30 years ago. Despite that, we still do not have international guidelines on molecu- lar genetic diagnostics of coagula- tion disorders. This is partly due to the heterogeneity of the disor- ders of the haemostatic system. For example, in haemophilia A, a disease caused by a deficiency of coagulation factor VIII, we can dif- ferentiate severe and mild forms. Most patients with factor VIII levels below 1% exhibit a severe bleeding phenotype. However, about 10-15% of patients with extremely low or undetectable factor VIII levels show a milder disease phenotype with reduced frequencies of spontane- ous bleeding. Furthermore, in some patients the same missense muta- tions lead to a severe phenotype, while they cause a mild form in other patients. The disease-modify- ing causes are unclear and are cur- rently under investigation. Surprisingly, even in this mono- genic disease no mutation can be found with conventional molecular methods in approximately 2% of severe patients. Novel technologies, such as next generation sequenc- ing, quantitative RNA analysis or methylation studies, offer options to identify the underlying causes for the disease in these patients. It has been shown that molecular genetic analyses allow the identification of patient subgroups and the selection of customised, personalised treat- ments in haemophilia A. Patients with a high risk to develop therapy- induced inhibitors, who need specif- ic treatment regimes, can be found by mutation analysis. As has been presented above, imbalances within the haemostatic system lead to bleeds but they may also cause thrombosis in arteries or veins. Despite successful research, our understanding of the disease pathogenesis of thrombotic disor- ders is still incomplete. While for venous thrombosis the role of mutations is well estab- lished, genetic risk factors for arte- rial thrombosis are less well defined. Although many single nucleotide polymorphisms (SNPs) in candidate genes have been associated with plasma concentrations of the respec- tive proteins and a risk of ischaemic stroke or myocardial infarction, pub- lished results for individual poly- morphisms and genes are inconclu- sive and often controversial. Risk genotypes and risk profiles are associated with these diseases in large patient collectives (>10,000 cases), but the relevance for indi- vidual patients is frequently unclear. Nevertheless, it can be expected that genetic, genomic, and epigenetic discoveries will enhance the diag- nostic capability and improve the treatment options for these common diseases. Molecular genetic analyses in haematology-oncology The finding that every gene has a specific position in the genome is of central importance. The loss of the appropriate position by e.g. translocations or inversions usu- ally leads to significant changes in gene expression and subsequently to severe disease. Chronic myeloid leukaemia (CML) is a stem cell neoplasm caused by the reciprocal translocation t(9;22). The transloca- tion generates the fusion gene BCR- ABL1, which is found in leukaemic cells of more than 95% of human CML patients and about 30% of adult patients suffering from ALL. BCR- ABL1 is a constitutively active tyros- ine kinase activating several signal- ling pathways. The constitutively active tyrosine kinase is responsible for impaired apoptosis and uncon- trolled proliferation of cells, and the initiation and manifestation of the disease. The identification and under- standing of the molecular lesion led to the development of a targeted therapy. The first BCR-ABL1 kinase inhibitor was imatinib, a small mol- ecule, which produces major and stable cytogenetic responses in a majority of patients in chronic phase of CML. Although a large percentage of patients show complete cytogenetic response to imatinib, drug resist- ance may occur, which is usually due to mutations in the BCR-ABL1 fusion gene. Monitoring of mini- mal residual disease with molecular genetic tests during imatinib-therapy is of high importance. If BCR-ABL1 is detectable, sequence analysis has to be performed to identify the mutation. The type of the mutation is relevant for treatment switch to other tyrosine kinase inhibitors In addition to translocations, point mutations can be disease rel- evant in hematologic leukaemias. Until about a decade ago, no molec- ular abnormalities were known for polycythaemia vera (PV), essential thrombocythemia (ET) or myelofi- brosis (PMF). The discovery of a point mutation in codon 617 (valin to phenylalanine) of the JAK2 gene in a large percentage of these mye- loproliferative neoplasms increased our understanding of these diseases and improved their diagnosis. The JAK2 V617F mutation results in the constitutive activation of the JAK- STAT pathway. In PV, approximately 95% of patients have a JAK2 muta- tion, in ET or PMF the frequency is approximately 50–60%. Point mutations are also found in solid tumours (e.g. mutations in the p53 gene, the retinoblastoma gene or the KRAS gene). Mutations in the KRAS gene are early events in tumo- rigenesis. The detection of the muta- tions supports diagnosis, prognosis and also therapy selection. New publications have shown that colon carcinoma patients with metastases can only be treated suc- cessfully with monoclonal antibod- ies if they have no KRAS muta- tions in the tumour cells. Therefore, genetic analysis of the KRAS gene is the state of the art diagnosis for colon carcinoma. Methodological aspects Even today, relatively few molecular tests are certified for in vitro diag- nostic use. Thus, every laboratory is responsible for the validation of the methods and reagents used for diagnoses. After gaining her doctorate at Vienna University Medical School, Dr Christine Mannhalter received a post-doctoral fellowship at the University of Southern California (USC) School of Medicine in Los Angeles. From 1985-2000 she was first Assistant Professor in the internal medicine clinic at Vienna School of Medicine and then Associate Professor in the Laboratory Medicine Department. Since 2000, she is full Professor of Molecular Diagnostics in Clinical Chemistry in the same unit. With major research interests in biochemistry, physiology and pathology of oncology etc., with awards and research funding bestowed for her work – currently on myeloproliferative neoplasms as well as cell communication in health and disease – and with her strong links to ethics groups, (she currently is President of the Austrian Agency of Scientific Integrity) Dr Mannhalter is a much welcomed lecturer. Continued from page 1 Endoscopy jumps the boundaries to be the “switching off” of the duo- denum. Its mucosa produces met- abolic hormones, incretins, which are believed to stimulate insulin secretion and to play a crucial role in the development of Type 2 dia- betes. Details of the pathogenesis are currently being investigated. If we relate this finding to Type 2 diabetics it seems likely that we can help these patients through a “reset” of the mucosa in the duodenum which re-regulates the production of metabolic substances,’ the professor explained. ‘This is where the endoscope comes into play – by helping the doctor to insert a special balloon in to the duodenum, heating it and ablating the mucosa. The duode- nal mucosa regenerates, but then appears to have different character- istics from the original one,’ he said, summarising the result of a pilot study in a Chilean hospital. In Europe this new procedure is currently being tested in sev- eral clinical centres including, in Germany, the Evangelical Hospital in Dusseldorf. Two other German hospitals will also participate. The basis for this advance in inter- vention is the continuous technology development. ‘It’s not only about the camera but also about the con- trol and mobility of the endoscope, which has to be thin enough to enter the smallest channels. In addi- tion, without a very good flushing function we cannot carry out any surgical interventions,’ Neuhaus said, mindful of the interest in coopera- tion with the industry. ‘The devices need to be tested and developed further, and engineers regularly visit to watch us at work. With them we discuss future technologies based on clinical need.’ To promote the internation- al exchange on procedures and technologies, many years ago Neuhaus founded the International Endoscopy Symposium, held annu- ally in Dusseldorf. What’s on the 2016 agenda? ‘Our work increasingly revolves around treatment and intervention, but also good diagnostics must not be neglected. The new possibili- ties offered by interventions under endoscopic ultrasound guidance are particularly exciting. In one endos- copy session they allow examining several organs and positioning stents to create anastomoses, for example between stomach and small intes- tine. Moreover, new opportunities to carry out endoscopies of the entire small intestine, or to position medi- cation in a targeted fashion in cer- tain organ sections or tumours cer- tainly will be on the 2016 agenda.’ Source:MedUniWien

EH 3_2015

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