Diss. (sammanfattning) Uppsala : Univ., 2009. / Härtill 4 uppsatser.

Abstract Cutaneous T cell lymphoma (CTCL) is a rare, incurable, chronic disease accounting for approximately 3% of non-Hodgkin's lymphoma diagnoses every year. Patients with CTCL have skin lesions that can vary in severity putting patients at risk for developing symptoms that may impair their quality of life (QOL). The disease burden can lead to increased depressive symptoms, fatigue distress, and anxiety that the disease may be worsening. Seventy-five participants agreed to take part in an exploratory, prospective study to evaluate depressive symptoms, anxiety, fatigue distress, and spirituality as predictors of QOL in CTCL patients. Demographic variables including stage of disease, ethnicity, age, gender, marital status, level of education, and time since diagnosis, were also included in the analyses to assess for relationships. Bivariate correlations, t-tests, and regression analyses were conducted to assess for relationships among the predictor variables and QOL. The analyses revealed that the proposed model explained 64% of the variance, and depressive symptoms (t= -2.4, p= 0.020) and stage of disease (t= -3.0, p= 0.004) significantly predicted the QOL of CTCL patients. Evaluating for predictors that influence the QOL helps us to better understand the needs of the patients afflicted with CTCL. The importance of studying the QOL of the CTCL patients lies in the fact that nurses can assist in helping patients alleviate some of the symptoms they experience, thereby improving their QOL. Further study is warranted in developing interventions to assist in the preservation of QOL.

Burkitt lymphoma (BL) was the first tumor found to be strongly associated with Epstein-Barr virus (EBV). Almost 100% of the lymphoma cells are cycling, necessitating dose- and time-intense multi-agent chemotherapy regimens to achieve a cure of the disease. Whilst standard risk BL can be cured with this approach, high risk BL with leukaemic and CNS disease has significantly inferior survival. The intensive chemotherapy regimen causes considerable toxicity to the patients and relapse of BL is largely incurable. Thus, novel therapeutic approaches for high risk and relapsed BL are needed. Histone deacetylase inhibitors (HDACis) represent a novel class of drugs with potent anti-cancer effect in a wide range of malignancies. In the first part of this study, we tested HDACis of different classes for their ability to inhibit cell proliferation and activate the lytic cycle of EBV in a panel of EBV-positive BL cells of different latent viral gene expression patterns (type I, Wp-restricted and type III latency with highly restrictive, partial and full spectrum of EBV latent gene expression, respectively). Different HDACis could inhibit proliferation of EBV-positive BL cells in a time- and dose-dependent manner but only weakly activate the viral lytic cycle indicating that the drugs’ cytotoxic effect is independent of the EBV lytic cycle. Of note, BL cells of Wp-restricted or type III latency were more resistant to killing by HDACis than those of latency I, suggesting a possible link between relative resistance to the drug and expression of the latent viral genes. Bortezomib, a proteasome inhibitor, may have synergistic action with HDACis on lymphoid malignancies. We hypothesized that Bortezomib could potentiate the killing of EBV-positive BL cells by HDACis. In the second part, we tested the effect of combination of a FDA-approved HDACi, suberoylanilide hydroxamic acid (SAHA) and Bortezomib in the same panel of BL cells and also EBV-transformed lymphoblastoid cell lines (LCLs) which represent an in-vitro model of EBV-associated post-transplant lymphoproliferative disorder (PTLD). Interestingly, combination of SAHA and Bortezomib significantly enhanced the killing of BL cells of Wp-restricted or type III latency. Furthermore, the resistance to either SAHA or Bortezomib alone in contrast to synergistic killing by the combination of the two drugs could be observed in LCLs which also have the type III latency pattern. Compared with either drug alone, combination of SAHA and Bortezomib induced enhanced apoptosis in Wp-restricetd BL cells and LCLs as shown by the increase in the percentage of annexin V-positive cell, sub-G1 population and the proteolytic cleavage of apoptotic markers including PARP, caspase-3 and -9. The drug combination hyper-acetylated histone and induced cell cycle arrest. Combination of SAHA and Bortezomib was further shown to suppress the growth of BL xenograft in nude mice. In conclusion, our data indicated that expression of partial or full spectrum of viral latent genes in EBV-positive BL cells of Wp-restricted or type III latency confers resistance of the tumor cells to cytotoxic effect of HDACis. Bortezomib could potentiate SAHA-induced apoptosis of both BL cells and LCLs and might overcome mechanism of drug resistance. / published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

Autoimmune hepatitis (AIH) was identified as an entity by the Swedish professor Jan Waldenström in the 1950s. It was then denoted lupoid hepatitis, characterized by liver inflammation and most often affecting young women. During the years the diagnosis has become more defined (as the non A non B hepatitis has been identified as Hepatitis C) and now can be safely separated from other diseases with liver inflammation. Studies of epidemiological data and long term prognosis have been scarce in the literature. Within a collaboration between the university hospitals in Sweden, we collected what we believe is the largest cohort in the world of patients with AIH. Data from the medical records of 473 individuals was, after AIH-score calculations where the diagnosis was confirmed, collected in a data base, in which most of the analysis was done. Data from the Swedish national registers of cancer, death cause, and birth register was searched for these patients as well as controls. The aim of the thesis was to explore epidemiological and clinical outcome of AIH.The onset of AIH may be at any age, but the incidence seems to increase after 50 years of age; 75% are females, the overall incidence (0.85/ 100,000 inhabitants and year) and prevalence (11/100,000 inhabitants) are figures that are within the range of another but smaller Scandinavian study. Approximately 30 % had cirrhosis already at diagnosis and 87% displayed at some time positive auto-antibodies indicating AIH (Smooth muscle ab and or antinuclear ab).  Indications of future risk for liver transplantation or death is an advanced AIH at diagnosis with liver cirrhosis, decompensated liver disease, elevated PK INR as well as age. Acute hepatitis-like onset seems to carry a lower risk for later liver transplantation or death. Current Swedish national therapy traditions with immune suppression seem to be well tolerated. Five and ten years overall life expectancy does not differ from controls. Thirty-five women gave birth to 63 children, for 3 after liver transplantation of the mother. Thirteen of the women had liver cirrhosis. Current pharmacological treatment seems to be safe both for the patient and the foetus. Thirty percent of the patients experienced flair after delivery. It has been supposed that there is an overrisk for hepatocellular cancer (HCC) associated with AIH. Our figures are the first in the world to be presented that confirms a twenty-three fold overrisk (95% Confidence Interval 7.5-54.3) for hepatobiliar cancer. We found as well an overrisk of non-Hodgkin lymphomas of 13.09 (95% CI 4.2-30.6).Conclusion:  Our epidemiological results confirm that AIH is a fairly uncommon disease, and that many already at time of diagnosis have an advanced disease with liver cirrhosis. There is a clear overrisk for HCC and lymphoma. For those women with AIH who become pregnant the prognosis for the child as well as for the mother is good, even for those women who already have compensated cirrhosis. There is a risk for relapse after delivery. The overall survival for AIH patients with current therapy is good.

Cell proliferation is a process that is strictly regulated by a large number of proteins. An alteration in one of the encoding genes inserts an error into the regulative protein, which may result in uncontrolled cell growth and eventually tumor formation. Lymphoma is a cancer type originating in the lymphocytes, which are part of the body’s immune defence. In the present thesis, Znfn1a1, Notch1 and Bcl11b were studied; all involved in the differentiation of T lymphocytes. The three genes are located in chromosomal regions that have previously shown frequent loss of heterozygosity in tumor DNA. Ikaros is a protein involved in the early differentiation of T lymphocytes. In this thesis, mutation analysis of the Znfn1a1 gene in chemically induced murine lymphomas revealed point mutations and homozygous deletions in 13 % of the tumors. All of the detected deletions lead to amino acid substitutions or abrogation of the functional domains in the Ikaros protein. Our results support the role of Ikaros as a potential tumor suppressor in a subset of tumors. Notch1 is a protein involved in many differentiation processes in the body. In lymphocytes, Notch1 drives the differentiation towards a T-cell fate and activating alterations in the Notch1 gene have been suggested to be involved in T-cell lymphoma. We identified activating mutations in Notch1 in 39 % of the chemically induced murine lymphomas, supporting the involvement of activating Notch1 mutations in the development of T-cell lymphoma. Bcl11b has been suggested to be involved in the early T-cell specification, and mutations in the Bcl11b gene has been identified in T-cell lymphoma. In this thesis, point mutations and deletions were detected in the DNA-binding zinc finger regions of Bcl11b in 15 % of the chemically induced lymphomas in C57Bl/6×C3H/HeJ F1 mice. A mutational hotspot was identified, where four of the tumors carried the same mutation. Three of the identified alterations, including the hotspot mutation in Bcl11b, increased cell proliferation when introduced in a cell without endogenous Bcl11b, whereas cell proliferation was suppressed by wild-type Bcl11b in the same cell line. Mutations in Bcl11b may therefore be an important contributing factor to lymphomagenesis in a subset of tumors. A germ line point mutation was identified in BCL11B in one of 33 human B-cell lymphoma patients. Expression of BCL11B in infiltrating T cells was significantly lower in aggressive compared to indolent lymphomas, suggesting that the infiltrating T cells may affect the B-cell lymphomas.

As new therapeutic targets and drugs are discovered for B-cell lymphoma and other cancers, companion diagnostics are also needed to determine target engagement, therapeutic efficacy, and patient segmentation for clinical trials. We first employed synthetic chemistry to build a platform for modifying small molecule drugs into imaging probes, using the poly(ADP-ribose) polymerase 1 (PARP1) inhibitor AZD2281 (Olaparib) as a model for technology development. Our results showed that small-molecule companion imaging drugs can be used for fluorescence imaging in cells, as well as for pharmacokinetic studies and positron emission tomography (PET) imaging in vivo, without significantly perturbing their target binding properties or cellular uptake. To apply this approach to B-cell lymphoma drugs currently in clinical trials, we modified an irreversible inhibitor of Bruton's Tyrosine Kinase (BTK), PCI-32765 (Ibrutinib), with the fluorophore Bodipy FL (BFL), and used it for imaging in cells and in a mouse window-chamber xenograft model. The excellent co-localization of our probe (Ibrutinib-BFL) with BTK demonstrated its utility for studying additional BTK inhibitors and as a companion imaging probe. In parallel, we hypothesized that central nervous system (CNS) lymphoma diagnosis from paucicellular cerebrospinal fluid (CSF) samples could be improved with molecular profiling of putative lymphoma cells trapped in a customized microfluidic chip. Following fabrication and characterization of a polydimethylsiloxane (PDMS) diagnostic device containing an array of affinity-free single-cell capture sites, we were able to efficiently recover >90% of lymphocytes, perform immunostaining on chip, and apply an image-processing algorithm to group cells based on their molecular marker expression, such as kappa/lambda light chain restriction. Additionally, in combination with Ibrutinib-BFL or other imaging drugs, we demonstrated the potential for on-chip drug imaging for use in conjunction with drug development. Finally, we applied bioorthogonal conjugation chemistries on cellulose paper for potential applications in lowering the cost of drug screening. We anticipate that these approaches will enable direct, molecular information for personalized treatment decisions in B-cell lymphomas, as well as provide a roadmap for the development of companion diagnostic probes and devices for additional indications.

A possible treatment for patients with lymphoma and myeloma is stem cell transplantation (SCT). SCT is preceded with cytostatic treatment. There are several side effects related to this treatment, for example fatigue, nausea, constipation/diarrhoea, pain, mucositis and loss of appetite. Aim: Investigate which side effects related to the treatment where most troubling after SCT and if nurse assessment and patient assessment differ. Methods: A quantitative empirical study with repeated measuring. The two groups of nurses and patients answered a form independently. Results: Loss of appetite and fatigue are the most troubling side effects according to both nurse and patient. Older patients tended to be more affected by fatigue. The nurses estimated the side effects such as loss of appetite, fatigue, diarrhoea and nausea lower than the patients did. Conclusion: No definitive conclusion could be made because of the small patient/nurse sample. However, there is a tendency showing difficulty for nurses to estimate correctly the side effects suffered by the patients. The nurses tend to estimate the side effects lower than the patients do.

Die Behandlung von Lymphomen konnten durch zahlreiche Therapieoptimierungsstudien verbessert werden. Für das Erzielen weiterer Fortschritte ergibt sich die Forderung auch zukünftig möglichst viele Patienten in Therapieoptimierungsstudien einzuschließen. Ziel dieser Arbeit war es, einen Überblick über die Praxis des Studieneinschlusses an einem deutschen Studienzentrum zu geben und hieraus ggf. einen Verbesserungsbedarf für bestimmte Lymphomentitäten oder Patientensubgruppen aufzuzeigen. Weiterhin wurde untersucht inwieweit sich die Studienergebnisse im klinischen Alltag wiederspiegeln und reproduzieren lassen. Es wurden anhand der elektronisch hinterlegten Arztbriefe die Inzidenz, die Therapieergebnisse sowie die Häufigkeit des Studieneinschlusses in der Abteilung Hämatologie und Onkologie der Universitätsmedizin Göttingen ermittelt. Desweiteren wurden für das diffus großzellige B-Zell-Lymphom (DLBCL) die Therapieergebnisse mit denen wichtiger klinischer Studien verglichen. Hierzu wurden die Patienten analog der deutschen Studiengruppe Hochmaligne Non-Hodgkin-Lymphome (DSHNHL) in die Gruppen „junge Patienten mit guter Prognose“, „junge Patienten mit schlechter Prognose“, „ältere Patienten“ und „Rezidivpatienten“ eingeteilt. Bezüglich der Studieneinschlussrate zeigt sich ein Defizit für die Patienten, die älter als 60 Jahre sind und die bei den meisten Lymphomentitäten den Großteil der Patienten stellen. In der Erstlinientherapiegruppe der „jungen Patienten mit guter Prognose“ (alters-justierter Internationaler Prognostischer Index (aIPI) <2) zeigen sich die besten Remissionsraten. Die Ergebnisse dieser Gruppe sind mit den Beobachtungen der MabThera-International-Trial-Studie (MInT) vergleichbar. In der Gruppe der „jungen Patienten mit schlechter Prognose“ (aaIPI≥2) zeigen sich schlechtere Therapieergebnisse als bei den „jungen Patienten mit guter Prognose“. Dies bestätigt die prognostische Relevanz der IPI-Risikofaktoren in der Rituximab-Ära. Der Vergleich der Therapieergebnisse mit den Ergebnissen der Mega-CHOEP-Studie fiel hinsichtlich der Overall response-rate (ORR), der progessive disease (PD)-, und der complete remission/complete remission unconfirmed (CR/Cru)-Rate uneinheitlich aus. Lediglich 37,5% der Patienten wurden mit 8 Zyklen R-CHOEP-14 behandelt, das sich in der Mega-CHOEP-Studie als überlegen herausstellte. Die Gruppe der „älteren Patienten“ zeigte eine vergleichbare ORR und PD-Rate zu den Patienten im 6xR-CHOP-14-Arm der RICOVER-60-Studie. Jedoch konnte die dort erreichte hohe CR/Cru-Rate im Göttingern Kollektiv der „älteren Patienten“ nicht beobachtet werden. Für die Gruppe der Rezidivpatienten konnten die Beobachtungen der PARMA-Studie bestätigt werden. Die Zeit, die bis zum Auftreten eines Rezidivs vergeht, erwies sich auch bei den Göttinger Patienten als bedeutender Prognosefaktor. Innerhalb der drei Erstlinientherapiegruppen weisen die Göttinger Studienpatienten jeweils höhere Remissionsraten als die Nichtstudienpatienten auf. Zur weiteren Therapieoptimierung und zum Erreichen besserer Remissionsraten sollte somit auch weiterhin größte Aufmerksamkeit auf das Randomisieren der Patienten, insbesondere der älteren Patienten, in neue klinische Therapieoptimierungsstudien gelegt werden.

Human T-cell Leukemia Virus type 1 (HTLV-1) is the etiologic agent of the Adult T-cell Leukemia (ATL), an aggressive lymphoproliferation of activated CD4+ T cells. The lack of a reliable small animal model to reproduce in vivo the leukemogenic process associated with HTLV-1 infection has impaired the understanding of the early stages of this process as well as the discovery of effective therapeutic approaches. Recently, improvement in the models of humanized mouse models were achieved allowing the development of a human immune system in mice. Injection of human hematopoietic stem and progenitors cells purified from cord blood into Balb/c Rag2-/-γc-/- newborns allows the de novo production of human dendritic, B and T cells. We infected humanized mice with HTLV-1 producing cell lines resulting in infection of human cells within the mice and the development of lymphomas and leukemias. This infection also results in the alteration of the T-cell development within the thymus pushing the thymocytes toward a more mature phenotype. This small animal model recapitulating in vivo the HTLV-1 infection and its associated pathogenesis gave us the opportunity to study the evolution of the clonality of the virus among human cells in different lymphoid organs. Based on these observations, preliminary results on the use of a new therapeutic approach were obtained. We finally tried to adjust the humanization protocol in order to obtain better engraftment in this model.

Telomeres are nucleoprotein complexes located at the end of chromosomes. They have an essential role in protecting chromosome ends. Telomerase or ALT (alternative lengthening of telomeres) mechanisms maintain telomeres by compensating natural telomeric loss. We have set up a flow-FISH method and using mouse lymphoma cell lines we identified unexpectedly the presence of subpopulations of cells with different telomere lengths. Subpopulations of cells with different telomere lengths were also observed in a human ALT and non-ALT cell line. Differences in telomere length between subpopulations of cells were significant and we term this phenomenon TELEFLUCS (TElomere LEngth FLUctuations in Cell Subpopulations). By applying flow-FISH we could successfully measure telomere lengths during replicative senescence in human primary fibroblasts with different genetic defects that confer sensitivity to ionising radiation (IR). The results from this study, based on flow-FISH and Southern hybridisation measurements, revealed an accelerated rate of telomere shortening in radiosensitive fibroblasts. We also observed accelerated telomere shortening in murine BRCA1 deficient cells, another defect conferring radiosensitivity, in comparison with a BRCA1 proficient cell line. We transiently depleted BRCA1 by siRNAs in two human mammary epithelial cell lines but could not find changes in telomere length in comparison with control cells. Cytological evidence of telomere dysfunction was observed in all radiosensitive cell lines. These results suggest that mechanisms that confer sensitivity to IR may be linked with mechanisms that cause telomere dysfunction. Furthermore, we have been able to show that human ALT positive cell lines show dysfunctional telomeres as detected by either the presence of DSBs at their telomeres or cytogenetic analysis and usually cells with dysfunctional telomeres are sensitive to IR. Finally, we assessed hTERT mRNA splicing variants and telomerase activity in brain tumours, which exhibit considerable chromosome instability suggesting that DNA repair mechanisms may be impaired. We demonstrated that high levels of hTERT mRNAs and telomerase activity correlate with proliferation rate. The presence of hTERT splice variants did not strictly correlate with absence of telomerase activity but hTERT spliced transcripts were observed in some telomerase negative brain tumours suggesting that hTERT splicing may contribute to activation of ALT mechanisms.