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Myleom är en cancersjukdom som uppkommer i benmärgen. I Sverige insjuknar ungefär 500-600 personer i myelom per år. Syftet med litteraturstudien var att med hjälp av de sex dimensionerna av smärta belysa upplevelsen av att leva med sjukdomen myelom. Studien genomfördes utifrån en teoretisk referensram. För att få en helhetsbild av individernas upplevelser av sjukdomen redovisas resultatet utifrån sex dimensioner av smärta. Resultatet visar att individer med myelom får ett inskränkt socialt liv, förändrade vanor och både fysisk och psykisk ohälsa. Individerna påverkas av smärta, trötthet och upprepade infektioner som orsakas av ett nedsatt immunförsvar. Då kunskap om sjukdomen i samhället är begränsad får individer med myelom minskad förståelse när det gäller sjukdomsbilden. För att sjuksköterskan skall få förståelse för individerna och deras livskvalitet krävs en adekvat smärtbedömning, som bör beröra hur individen påverkas av sin sjukdom i det vardagliga livet. Mer forskning kring sjukdomen, behandlingar och mediciner behövs och det vore önskvärt om målet vore att sjukdomen skall kunna botas. / Multiple myeloma is a cancer illness that arises in the bone marrow. In Sweden 500-600 people per year get diagnosed with multiple myeloma. The purpose of the literature study was to highlight the experiences of individuals with multiple myeloma through the six dimension of pain. The study is based on a theoretical frame of reference. To get at overall picture of the individuals experiences of living with multiple myeloma, the result was described through the six dimensions of pain. The result shows that multiple myeloma patient´s social life becomes reduced, their habit changes and booth their physical and psychological health reduces. Pain, fatigue and infections caused by lower immune system affects the individuals. The knowledge of the illness in society is trivial and this makes the understanding for people with multiple myeloma and their illness slight. A proper pain assessment is necessary for the nurse to complete, to get a full understanding about the individuals and their quality of life. More research about the illness, treatments and medications is needed and the goal should be to cure the disease.

Analysis of M-components is an important part of the diagnosis of monoclonal gammopathies and for the evaluation of disease response during treatment. In this project, two widely used electrophoresis methods and their corresponding immunotyping method were compared to evaluate the sensitivity of each method for the detection of small M-components. The project included 30 plasma samples from patients with identified M-components; 10 samples containing each IgG, IgA and IgM, respectively. All samples were diluted with normal EDTA plasma to achieve M-components of 5,00g/L. The samples were then serially diluted to achieve M-component concentrations of; 5,00, 2,50, 1,25, 0,63, 0,31 and 0,16g/L. All 180 samples were analysed with agarose gel electrophoresis and capillary electrophoresis. The dilutions above and below the detection level of each method were then analysed with immunofixation and immunosubtraction. The results showed good agreement between agarose gel electrophoresis and capillary electrophoresis in the highest concentrations of IgG and IgM. With agarose gel electrophoresis, IgA was detected in the same location as transferrin and the lowest concentration detected were therefore 1,25g/L. Besides the samples containing IgG, immunofixation was the most sensitive method.

Šioje disertacijoje aprašyti tyrimai, atlikti siekiant įvertinti plazminių ląstelių imunotipavimo, taikant tėkmės citometriją, prognostinį potencialą. Patikrinome hipotezę, jog cirkuliuojančių plazminių ląstelių kinetika gydymo metu gali, anksčiau, nei standartiniai metodai įvertinti atsaką į gydymą. Tyrime naudojome iki tol neaprašytą plazminių ląstelių imunofenotipavimo metodiką. Mėginiai dažyti dviem skirtingais žymenų deriniais: CD56/CD138/CD45/CD19/CD38/CD20 ir cLambda/cKappa/CD138/CD19/CD38/CD56. Nustatėme, kad sveikų donorų kaulų čiulpuose aptinkama ~30% plazminių ląstelių, turinčių atipine CD56 ir/ar CD19 žymenų raiška. Optimizavome tėkmės citometrijos metodiką normalių ir piktybinų plazminų ląstelių aptikimui. Nustatėme, kad piktybinių cirkuliuojančių plazminių ląstelių proporcijos nesumažėjimas po pirmojo chemoterapijos kurso, su 91,7% jautrumu bei 93,3% specifiškumu prognozuoja ankstyvą progresiją. Pacientų, kuriems cirkuliuojančių plazminių ląstelių proporcija nesumažėjo, laikas iki progresijos ir bendras išgyvenamumas buvo statistiškai patikimai trumpesnis, nei pacientų, kuriems piktybinių cirkuliuojančių plazminių ląstelių proporcija sumažėjo ar šios ląstelės buvo neaptinkamos. Ištyrėme normalių plazminių ląstelių populiacijos klinikinę vertę pacientams, sergantiems mielomine liga. Apibendrinant, plazminių ląstelių imunofenotipavimas taikant tėkmės citometrijos metodą suteikia prognostiškai reikšmingos informacijos. Svarbiausias radinys, jog cirkuliuojančių... [toliau žr. visą tekstą] / The investigations presented in this dissertation were initiated with the intention to evaluate the prognostic value of plasma cells immunophenotypic analysis in multiple myeloma patients. We tested the hypothesis that kinetics of peripheral blood circulating plasma cells in response to first chemotherapy cycle could identify patients refractory to given treatment. We employed novel original methodology for plasma cells immunophenotyping: cells were stained in two tubes with antibody combinations CD56/CD138/CD45/CD19/CD38/CD20 and cLambda/cKappa/CD138/CD19/CD38/CD56. We found that ~30% of all plasma cells in bone marrow of healthy donors are immunophenotypically aberrant by CD56 and/or CD19 marker expression. We optimized immunophenotypic differentiation between malignant and normal plasma cells. Non reduction of malignant circulating plasma cells in response to first chemotherapy cycle predicted early progression with sensitivity and specificity of 91.7% and 93.3%, respectively. Time to progression and overall survival were significantly shorter in these patients as compared to patients with undetectable or reduced malignant circulating plasma cells. We also evaluated the clinical value of normal plasma cell subpopulation detection in peripheral blood and bone marrow of multiple myeloma patients. In summary, we demonstrated that immunophenotyping of plasma cells using multiparameter flow cytometry provides important prognostic information. The major finding was that the... [to full text]

The investigations presented in this dissertation were initiated with the intention to evaluate the prognostic value of plasma cells immunophenotypic analysis in multiple myeloma patients. We tested the hypothesis that kinetics of peripheral blood circulating plasma cells in response to first chemotherapy cycle could identify patients refractory to given treatment. We employed novel original methodology for plasma cells immunophenotyping: cells were stained in two tubes with antibody combinations CD56/CD138/CD45/CD19/CD38/CD20 and cLambda/cKappa/CD138/CD19/CD38/CD56. We found that ~30% of all plasma cells in bone marrow of healthy donors are immunophenotypically aberrant by CD56 and/or CD19 marker expression. We optimized immunophenotypic differentiation between malignant and normal plasma cells. Non reduction of malignant circulating plasma cells in response to first chemotherapy cycle predicted early progression with sensitivity and specificity of 91.7% and 93.3%, respectively. Time to progression and overall survival were significantly shorter in these patients as compared to patients with undetectable or reduced malignant circulating plasma cells. We also evaluated the clinical value of normal plasma cell subpopulation detection in peripheral blood and bone marrow of multiple myeloma patients. In summary, we demonstrated that immunophenotyping of plasma cells using multiparameter flow cytometry provides important prognostic information. The major finding was that the... [to full text] / Šioje disertacijoje aprašyti tyrimai, atlikti siekiant įvertinti plazminių ląstelių imunotipavimo, taikant tėkmės citometriją, prognostinį potencialą. Patikrinome hipotezę, jog cirkuliuojančių plazminių ląstelių kinetika gydymo metu gali, anksčiau, nei standartiniai metodai įvertinti atsaką į gydymą. Tyrime naudojome iki tol neaprašytą plazminių ląstelių imunofenotipavimo metodiką. Mėginiai dažyti dviem skirtingais žymenų deriniais: CD56/CD138/CD45/CD19/CD38/CD20 ir cLambda/cKappa/CD138/CD19/CD38/CD56. Nustatėme, kad sveikų donorų kaulų čiulpuose aptinkama ~30% plazminių ląstelių, turinčių atipine CD56 ir/ar CD19 žymenų raiška. Optimizavome tėkmės citometrijos metodiką normalių ir piktybinų plazminų ląstelių aptikimui. Nustatėme, kad piktybinių cirkuliuojančių plazminių ląstelių proporcijos nesumažėjimas po pirmojo chemoterapijos kurso, su 91,7% jautrumu bei 93,3% specifiškumu prognozuoja ankstyvą progresiją. Pacientų, kuriems cirkuliuojančių plazminių ląstelių proporcija nesumažėjo, laikas iki progresijos ir bendras išgyvenamumas buvo statistiškai patikimai trumpesnis, nei pacientų, kuriems piktybinių cirkuliuojančių plazminių ląstelių proporcija sumažėjo ar šios ląstelės buvo neaptinkamos. Ištyrėme normalių plazminių ląstelių populiacijos klinikinę vertę pacientams, sergantiems mielomine liga. Apibendrinant, plazminių ląstelių imunofenotipavimas taikant tėkmės citometrijos metodą suteikia prognostiškai reikšmingos informacijos. Svarbiausias radinys, jog cirkuliuojančių... [toliau žr. visą tekstą]

Multiple myeloma is a painful and uncurable malignant disease with an increasing incidence and mortality in several countries, e.g., Sweden. Some factors are suspected to be of aetiological significance, such as ionising radiation and chronic antigenic stimulation in certain inflammatory diseases. A familial factor has also been indicated. Furthermore, some studies have demonstrated farming as an occupation entailing an increased risk for the disease. The aim of this investigation was to further elucidate the impact of different aetiological factors in relation to multiple myeloma. The knowledge of aetiology is always a prerequisite for prevention. A case-control study on multiple myeloma was performed in a high-inddence area, the northern part of Sweden. One part of this study dealt with occupations and different exposures. The results supported farming as being an occupation with an increased risk. Within farming two kinds of pesticides, phenoxyacetic adds and DDT, and contact with certain domestic animals, i.e., cattle, horses and goats, were assodated with multiple myeloma. Farming as a risk factor was also confirmed by a register-based linkage study using the Swedish Cancer Environment Register. In this study a time trend was indicated, with increasing standardized inddence ratios over the different time periods studied. Another part of the case-control study showed that rheumatoid arthritis entailed an increased risk for multiple myeloma, a finding earlier suggested from register-based linkage studies, but not from any çase-control study. A third part of the case-control study indicated an increased risk for multiple myeloma if any first-degree relative had a history of haematological malignancy, or other malignant tumour, espedally prostatic cancer, brain tumour, and renal cancer. A case study encompassing 942 patients with haematological malignandes in the county of Jämtland, Sweden, during a 22-year period showed that about 5% of the patients had at least one relative who also suffered from such a disease. An espedally strong familial occurrence was found in the group of chronic lymphoprohferative diseases, including multiple myeloma. / <p>S. 1-69: sammanfattning, s. 71-129: 5 uppsatser</p> / digitalisering@umu

This thesis is focused on multiple myeloma (MM), a haematological malignancy that still remains incurable. The pathogenesis of MM is not fully understood and there is a large intra-tumour and interclonal genetic variation in MM patients. One of the most challenging areas in MM research is to find mechanisms for initiation and progression of MM, but also to overcome the arising resistance to therapy. In paper I, a signature of under-expressed genes in MM was found to significantly correlate with already defined Polycomb target genes. In selected genes from the profile we found an enrichment of H3K27me3, a repressive mark catalysed by Polycomb repressive complex 2 (PRC2), in MM patients and MM cell lines. Treatment with LBH589 (HDAC inhibitor) and DZNep (methyltransferase inhibitor) reactivated the H3K27me3 target genes and induced apoptosis in MM cell lines. LBH589 reduced tumour load and increased overall survival in the 5T33MM mice. These results suggest an important role for Polycomb complex in MM development and highlight PRC2 as a drug target in MM. In paper II, the insulin-like growth factor type 1 receptor tyrosine kinase (IGF-1RTK) inhibitor picropodophyllin (PPP) in combination with LBH589 synergistically inhibited cell proliferation and enhanced the apoptotic effect in MM. Since the bone marrow microenvironment has an important role in MM disease and also contributes to drug-resistance, we therefore evaluated the drug combination in the immunocompetent 5T33MM murine model. The drug combination significantly prolonged the survival of the 5T33MM mice compared to single drug treatment. We conclude that the combination of PPP and LBH589 has a therapeutic potential in MM. In paper III, the role of the cellular inhibitor of apoptosis protein 2 (cIAP2) was evaluated in MM cells harbouring TRAF3 deletion/mutation. By overexpressing cIAP2 in these cells we found an increased resistance to proteasome inhibitors. cIAP2 over-expression by lentiviral constructs led to decreased caspase activation, activation of the canonical NF-κB pathway, and down-regulation of tumour suppressor genes and genes that contribute to apoptosis. Supporting the role of cIAP2 mediated drug-resistance, we here demonstrate that inhibiting cIAP2 using an IAP antagonist, increased the sensitivity to the proteasome inhibitor, bortezomib.

A major contributor to mortality in patients with plasma cell dyscrasias (PCDs); i.e., multiple myeloma, light chain deposition disease and AL amyloidosis is the deposition as insoluble aggregates of monoclonal immunoglobulin light chain proteins (LC) in the kidneys and other organs. Currently anti-plasma cell chemotherapies are used to reduce LC synthesis, and slow deposition. While effective, these treatments are toxic, non-specific, expensive, and might not be appropriate in all cases, making the identification of an alternate means of reducing toxic LC species desirable. To this end, we have investigated whether RNA interference (RNAi) could achieve these goals. Human (RPMI 8226, Bur) and transfected mouse myeloma (SP2/O-lambda 6) cells which produce measureable quantities of human LC protein were used as model systems for testing the efficacy of both synthetic small interfering RNAs (siRNAs) and short hairpin RNA (shRNA) expression vectors in reducing LC synthesis. Sequencing of LC genes provided the basis for design of siRNA duplexes targeting either the variable (V) or joining (J) regions of individual LCs, or the constant (C) region of either kappa or lambda LC isotypes. Myeloma lines were transfected with siRNAs using lipid-based transfection media. Cells receiving non-silencing siRNAs served as controls. Exposure of myeloma lines to siRNAs was well tolerated and no cytotoxicity was observed. LC mRNA expression was shown to be reduced ≥40% in 8226 and SP2/O- lambda 6 cell lines receiving siRNA treatment as compared with untreated controls. Exposure to siRNAs was also effective in significantly reducing both intracellular and secreted LC protein levels in cell lines tested as evidenced by flow cytometry or enzyme-linked immunosorbent assays (ELISAs). Effective siRNA nucleotide sequences were used to generate shRNA cassettes which were ligated into lentiviral expression vectors under the control of the RNA polymerase III promoter, U6. These expression systems were used to generate replication incompetent lentiviral particles. Exposure of 8226 to lentiviral particles resulted in significant knockdown of LC mRNA and protein both in vitro and in xenograft tumor bearing immune compromised mice. These results provide positive evidence for the ability of RNAi based approaches to reduce LC secretion in models of PCD.

The Mater Medical Research Institute proposes to undertake a Phase I clinical trial using CMRF-56 immunoselected blood dendritic cells (BDC) loaded with control and myeloma-associated tumour peptide antigens for the treatment of multiple myeloma (MM) patients with minimal residual disease. This thesis describes some of the fundamental pre-clinical in vitro experiments undertaken in preparation for this trial so as to maximise the potential of this vaccine to induce myeloma-specific immune responses. These experiments involved determining the parameters for optimal activation of the CMRF-56 immunoselected cell preparation and exploring the potential of novel myeloma peptide antigens to induce anti-myeloma cytotoxic T lymphocyte (CTL) responses. CMRF-56 immunoselected cell preparations, containing predominately myeloid BDC, monocytes and B cells, were prepared from both healthy donors and myeloma patients. Activation of this preparation with granulocyte macrophage colony stimulating factor (GM-CSF) was found to increase co-stimulatory molecule expression by and survival of BDC, improve peptide- and lysate-specific CTL induction, and, in combination with prostaglandin E2 (PGE2), improve chemokine-specific migration of BDC. Following optimisation of in vitro CTL generation protocols, GM-CSF activated CMRF-56 immunoselected cells were examined for their ability to induce myeloma-specific immunity. Using lysate from myeloma cell line U266 as an antigen source, a polyclonal T cell pool was generated within which peptide specific CTL recognising myeloma antigens Muc1, HM1.24/BST2, DKK-1 and CT-7/MAGE-C1 could be identified. Furthermore, GM-CSF activated CMRF-56 immunoselected cells pulsed with HLA-A*201 restricted peptides derived from Muc1, HM1.24/BST2 and CT-7/MAGE-C1 could induce CTL capable of lysing both peptide- and myeloma cell line targets in both healthy donors and myeloma patients. These results provide the first evidence of immunogenic HLA-A*201 restricted epitopes of novel myeloma antigen CT-7/MAGE-C1. The data collected in this study supports the application of GM-CSF activated CMRF-56 immunoselected cells loaded with defined myeloma peptide antigens for the therapeutic vaccination of MM patients with minimal residual disease.

The Mater Medical Research Institute proposes to undertake a Phase I clinical trial using CMRF-56 immunoselected blood dendritic cells (BDC) loaded with control and myeloma-associated tumour peptide antigens for the treatment of multiple myeloma (MM) patients with minimal residual disease. This thesis describes some of the fundamental pre-clinical in vitro experiments undertaken in preparation for this trial so as to maximise the potential of this vaccine to induce myeloma-specific immune responses. These experiments involved determining the parameters for optimal activation of the CMRF-56 immunoselected cell preparation and exploring the potential of novel myeloma peptide antigens to induce anti-myeloma cytotoxic T lymphocyte (CTL) responses. CMRF-56 immunoselected cell preparations, containing predominately myeloid BDC, monocytes and B cells, were prepared from both healthy donors and myeloma patients. Activation of this preparation with granulocyte macrophage colony stimulating factor (GM-CSF) was found to increase co-stimulatory molecule expression by and survival of BDC, improve peptide- and lysate-specific CTL induction, and, in combination with prostaglandin E2 (PGE2), improve chemokine-specific migration of BDC. Following optimisation of in vitro CTL generation protocols, GM-CSF activated CMRF-56 immunoselected cells were examined for their ability to induce myeloma-specific immunity. Using lysate from myeloma cell line U266 as an antigen source, a polyclonal T cell pool was generated within which peptide specific CTL recognising myeloma antigens Muc1, HM1.24/BST2, DKK-1 and CT-7/MAGE-C1 could be identified. Furthermore, GM-CSF activated CMRF-56 immunoselected cells pulsed with HLA-A*201 restricted peptides derived from Muc1, HM1.24/BST2 and CT-7/MAGE-C1 could induce CTL capable of lysing both peptide- and myeloma cell line targets in both healthy donors and myeloma patients. These results provide the first evidence of immunogenic HLA-A*201 restricted epitopes of novel myeloma antigen CT-7/MAGE-C1. The data collected in this study supports the application of GM-CSF activated CMRF-56 immunoselected cells loaded with defined myeloma peptide antigens for the therapeutic vaccination of MM patients with minimal residual disease.