Purpose: The five point Deauville (D) scale is widely used to assess interim PET metabolic response to chemotherapy in Hodgkin lymphoma (HL) patients. An International Validation Study reported good concordance among reviewers in ABVD treated advanced stage HL patients for the binary discrimination between score D1,2,3 and score D4,5. Inter-reader reliability of the whole scale is not well characterised. Methods: Five international expert readers scored 100 interim PET/CT scans from paediatric HL patients. Scans were acquired in 51 European hospitals after two courses of OEPA chemotherapy (according to the EuroNet-PHL-C1 study). Images were interpreted in direct comparison with staging PET/CTs. Results: The probability that two random readers concord on the five point D score of a random case is only 42% (global kappa = 0.24). Aggregating to a three point scale D1,2 vs. D3 vs. D4,5 improves concordance to 60% (kappa = 0.34). Concordance if one of two readers assigns a given score is 70% for score D1,2 only 36% for score D3 and 64% for D4,5. Concordance for the binary decisions D1,2 vs. D3,4,5 is 67% and 86% for D1,2,3 vs D4,5 (kappa = 0.36 resp. 0.56). If one reader assigns D1,2,3 concordance probability is 92%, but only 64% if D4,5 is called. Discrepancies occur mainly in mediastinum, neck and skeleton. Conclusion: Inter-reader reliability of the five point D-scale is poor in this interobserver analysis of paediatric patients who underwent OEPA. Inter-reader variability is maximal in cases assigned to D2 or D3. The binary distinction D1,2,3 versus D4,5 is the most reliable criterion for clinical decision making.

The fundamental subject of this thesis is the development of tools for the analysis of DNA methylation data as well as their application on bisulfite sequencing data comprising a large number of samples. DNA methylation is one of the major epigenetic modifications. It affects the cytosines of the DNA and is essential for the normal development of cells and tissues. Unusual alterations are associated with a variety of diseases and, specially, in cancergeneous tissues global changes in the DNA methylation level have been detected. To sequence DNA methylation on single nucleotide resolution, the sequences are treated with sodium bisulfite before sequencing, whereby unmethylated cytosines are represented as thymines. Thus, specialized techniques are required to process and analyze these kind of data. Here, the bisulfite analysis toolkit BAT is introduced, that is designed to facilitate an quick analysis of bisulfite treated DNA methylation sequencing data. It covers all steps of processing raw sequencing data up to calling of differential DNA methylation. At the begin of analysis, sodium bisulfite treated sequence data are aligned and DNA methylation rates for each covered cytosine in the reference genome are called. Subsequently, BAT integrates annotation data and performs basic analysis, i. e., methylation rate distribution plots and hierarchical clustering of the samples. In addition, calling of differentially methylated regions is performed and statistics of called regions are automatically created. Finally, DNA methylation and gene expression data integration is covered by the calculation of correlating regions. Secondly, a novel algorithm, metilene, for the calculation of differentially methylated regions (DMRs) between two groups of samples is introduced. Existing methods are limited in terms of detection sensitivity as well as time and memory consumption. Our approach is based on a circular binary segmentation, using a scoring function to detect sub-regions that show a stronger difference between the mean methylation levels of two groups than the surrounding background. These sub-regions are tested using a two-dimensional Kolmogorov Smirnov test (2D-KS test) [Fasano 1987] for significant differences taking all samples of each group into account. The use of the non-parametric 2D-KS test allows to avoid assumptions about a background distribution. Furthermore, the two dimensions of the problem, i. e., (i) the detection of a region, such that (ii) the methylation rates of the samples in the groups are significantly different, are taken into account in a single test. The algorithm calls DMRs in sufficiently short time on single sample comparisons as well as on about 50 samples per group. Furthermore, it works on whole-genome bisulfite sequencing (WGBS) and reduced representation bisulfite sequencing (RRBS) data and is able so estimate missing data points from the methylation rates of other samples in the group. Benchmarks on simulated and real data sets show that metilene outperforms other existing methods and is especially suitable for noisy datasets often found for example in cancer analysis. In the framework of this thesis, the previously introduced methods and algorithms are used to analyze a WGBS dataset of two different subtypes of germinal-center derived B-cell lymphomas and healthy controls. In both lymphoma subgroups genome-wide hypomethylation was found, with an exception for a specific type of promoter regions, i. e., poised promoters, that were frequently found to be hypermethylated. Using the previously presented algorithm, DMRs were called between the three entities. A strong enrichment of DMRs immediately downstream of the transcription start site was observed, indicating the regulatory relevance of this regions. The integration of gene expression data of the same samples, revealed that a considerable amount of the DMRs showed significant correlation between gene expression and DNA methylation. Finally, transcription factor binding sites and mutation data were combined with the methylation and expression data analysis. This identified strongly altered signaling pathways and cancer subtype specific genes. Furthermore, the data integration indicates that mutations and DNA methylation changes may act complementary to another. Finally, findings from the lymphoma study regarding the hypermethylation of poised promoters in cancer were extended to a huge data set comprising a variety of cancers. We could show that the relation of DNA methylation at a small set of frequently poised regions with respect to the background methylation level is sufficient to classify almost all samples based on DNA methylation data from 450k BeadChips into cancer or non-cancer probes. In addition, we found that the increase in methylation co-occurs with upregulated gene expression of several poised promoter regulated genes in almost all fresh cancer samples, implying a de-poising of poised regions. This upregulated gene expression is in contrast to the silencing of those genes in cancer cell lines, indicating that the upregulated gene expression might be a temporary status and possibly contributes to cancerogenesis.

<p>E and Id proteins are members of the basic helix-loop-helix (bHLH) transcription regulator family. These proteins control a broad range of lymphocyte biology, from the development of multiple lineages to execution of their effector functions. With the development of new experiment models, novel functions of E and Id proteins continued to be discovered. In this thesis, I focused my study on the role of Id2 in gamma delta T cells and CD4<super>+</super> alpha beta T cells, as well as the role of Id3 in B cells.</p><p> Id proteins have been shown to control gamma delta T cell development. Id3 knockout mice demonstrate a dramatic expansion of innate-like Vgamma1.1<super>+</super> Vdelta6.3<super>+</super> T cells in the neonatal stage, suggesting that Id3 is an inhibitor of their development. Interestingly, Id3 knockout mice with a B6/129 mix background have much less expansion of the Vgamma1.1<super>+</super> Vdelta6.3<super>+</super> T cells compared to mice with pure B6 background. Genetic studies showed that this difference is strongly influences by a chromosome region very close to the Id2 locus. Using the Id2<super>f/f</super> CD4Cre<super>+</super> mice, I found that Id2 is also an inhibitor of gamma delta T cell development. Deletion of Id2 alone is sufficient to enhance the maturation of these cells in the thymus and induce a moderate expansion of gamma delta T cells in the periphery. This study demonstrated the delicate balance of transcription control in cells of the immune system.</p><p> The Id2<super>f/f</super> CD4Cre<super>+</super> mice also enabled me to study the role of Id2 in peripheral CD4<super>+</super> alpha beta T cell functions, which was difficult in the past because Id2 knockout mice lack lymph node development. I found that CD4 T cells in these mice have a profound defect in mounting immune responses, demonstrated by a complete resistance to induction of experimental autoimmune encephalomyelitis (EAE). I found that Id2-deficient CD4 T cells fail to infiltrate the central nervous system, and the effector CD4 T cell population is smaller compared to that in control mice. Id2 is important for the survival and proliferation of effector CD4 T cells, and this phenotype was correlated with an increased expression of <italic>Bim</italic> and <italic>SOCS3</italic>. This study revealed a novel role of Id2 in the functioning of CD4<super>+ </super>alpha beta T cells.</p><p> Switching my focus to B cells, recent next generation sequencing of human Burkitt lymphoma samples revealed that a significant proportion of them have mutations of Id3. This finding suggests that Id3 may be a tumor suppressor gene in the lymphoid system. Utilizing various Id3 knockout and conditional knockout mouse models, I showed that Id3 deficiency can accelerate lymphoid tumor genesis driven by the over-expression of oncogene c-Myc. This work may lead to development of a more realistic mouse model of human Burkitt lymphoma, allowing more mechanistic studies and perhaps preclinical tests of new therapies.</p> / Dissertation

<p>Diss. (sammanfattning) Umeå : Umeå universitet, 1980 härtill 4 uppsatser</p> / digitalisering@umu

Treatment decision making (TDM) in oncology is complex. Understanding treatment information is essential for shared TDM. Research suggests many patients have low numeracy. This mixed methods study explored numeracy and experience with numbers in a sample of individuals diagnosed with follicular lymphoma. Participants completed questionnaires (N = 32) and interviews (N = 20) assessing numeracy, decisional conflict and regret, and number preference. Results suggest that mean objective numeracy was relatively high, and most reported high confidence in numerical ability. Most participants preferred to receive numbers during the TDM process. There was no relationship between numeracy and decision outcomes. Future research should investigate the use of numeracy measures in practice and the impact of patient preferences and beliefs on shared TDM.

No description available.

Follicular lymphoma (FL) is the second most common non-Hodgkin lymphoma (NHL). FL is clinically designated as an indolent disease with a long median survival of 8-10 years. However, the clinical and biological behavior of FL shows considerable variability, with some patients showing aggressive disease progression and very short survival. Because defects in the regulation of apoptotic cell death are fundamental in FL pathogenesis, we hypothesized that deregulated expression of components of the pRb signaling pathway may promote cell proliferation, thereby complementing antecedent anti-apoptotic mutations and producing more aggressive disease. In the present study we undertook an immunohistochemical (IHC) evaluation of expression of key cell-cycle regulatory proteins in diagnostic biopsies from 127 cases of FL using formalin-fixed, paraffin-embedded tissues (FFPE) in tissue microarray (TMA) sections immunostained for p53, pRb, p16INK4A and cyclin D3. Data analysis revealed that increased abundance of p53 or p16INK4A is associated with reduced overall survival (OS) (p=0.005 and p=0.014 respectively), and with conventional pathological markers of tumour aggressiveness including high histologic grade. Encouraged by this remarkable finding of a counterintuitive association between p16INK4A expression and clinical outcome, we analyzed CDKN2A gene deletion and methylation, as these are the most frequent mechanisms of the CDKN2A gene inactivation in NHL including FL. We determined the deletion and methylation status of CDKN2A in 105 FL cases. Laser-capture microdissection was used to enrich the samples for lymphoma cells. CDKN2A was deleted in 9 cases and methylated in 22 cases. The 29 cases (28%) with CDKN2A deletion or methylation had decreased overall survival (OS) (p=0.046) in all cases and in cases treated with rituximab (p<0.001). Our findings indicate that deleterious alterations of CDKN2A are relatively prevalent in FL at diagnosis and can predict poor clinical outcome. In summary, our data reveal novel insights into the pathogenesis of FL and suggest a relationship between increased p16INK4A expression and CDKN2A deletion or methylation and unfavorable clinical outcome in FL. We hope that the work presented herein will provide a useful prognostic tool for predicting the prognosis and choosing optimal treatment approaches to help patients suffering from FL. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2013-03-12 23:49:44.541

Thesis (PhD)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: BACKGROUND Lymphomas arise in cells of the immune system. They vary widely in cytomorphology, immunophenotype and clinical course as well as response to treatment - all of which are factors that determine prognosis. The substantial geographical differences that exist for Hodgkin and other lymphoproliferative disorders have been previously reported from the Lymphoma Reclassification Project. OBJECTIVE This investigation was in two parts. In the first we tested the hypothesis that, using comparable treatment regimens, outcome from a private academic centre in the Western Cape region of South Africa would be similar to that from the first world. To this end a series of 512 individuals were analysed. In the second tissue samples from these patients where the most common subtype is diffuse large B-cell lymphoma (n=93) randomly receiving either standard combination chemotherapy in the form of the CHOP regimen or the identical program with rituximab which is an anti-CD20 monoclonal antibody were further investigated in cooperation with the University of Nebraska Medical Centre using an immunohistochemistry based method, also known as tissue microarray. PATIENTS AND METHODS In the first cohort consecutive and comprehensive records of patients diagnosed with lymphoma aged 14 years and older seen between October 1998 in July 2006, were scrutinized. After exclusion for a variety of reasons 398 cases suitable for further definitive study remained. In the second group tissue samples from a total of 149 biopsy proven de novo diffuse large Bcell variants could be further evaluated. After additional refinement a total of 93 remained that met all the entry criteria for the study in which 48 received chemotherapy alone and the remaining 45 chemo-immunotherapy. Demographic features were well matched. The initial stratification of these 93 cases, based on phenotyping with immunohistochemistry employing a tissue microarray method, yielded two populations depending on the criteria used. Each of these primary subdivisions was further evaluated for expression of BCL2 and LMO2 both of which are recognised to predict response. Finally, for each variable, clinical characteristics and survival outcome were compared between chemotherapy as a single modality or the same regimen combined with rituximab. STATISTICAL ANALYSIS Overall and event-free survival were calculated as the years from diagnosis to death, loss to follow-up, first progression or relapse respectively. The Kaplan Meier method was used to estimate distribution and the log rank test to compare survival between groups. Patient characteristics for each cohort specified were tested with Chi-squared or Fisher's exact test for small samples. RESULTS In the first part of this study all 398 cases were retrospectively analysed and showed a similar treatment outcome with regards to overall and event-free survival at 36 months when compared to first world reference centers. Adverse factors identified were similar to published experience comprising constitutional symptoms, prior treatment with chemotherapy, intermediate or high-risk scores as defined by the International Prognostic Index, histologic grading and certain anatomical sites of primary tumour. In contrast gender, staging by Rye or Rai classification, retroviral infection and prior treatment with radiotherapy were without effect. In the second part tissue samples from 93 de novo DLBCL, subtyped using an investigational immunohistochemical based Tissue Micro Array (TMA) were contrasted to the approximately corresponding categories as defined either by Hans and associates using a three marker panel into germinal or non-germinal centre subtypes or by Choi and colleagues with two additional antibodies into germinal centre or activated B-cells. Not surprisingly when compared to DNA-based gene expression profiling, as a reference point, concordance was different being 86 as opposed to 93% for the respective algorithms. The rationale for this exercise was to determine relative cost-effectiveness in an affordable but accurate technology that may be applicable to under resourced areas of the globe. Additionally the prognostic marker expression for BCL2 and LMO2 was investigated with regards to treatment outcome. Using the investigational tissue microarray approach the addition of rituximab to standard chemotherapy group did not show any significant improvement on 5 years overall (63% vs 59%, p 0.68) or event-free survival (42% vs 39%, p 0.94). Similarly no differences were evident in subtype analysis. Interestingly however, when segregated on the Choi criteria, cytotoxic drugs alone showed a non-significant trend in improved survival (74% vs 55%, p 0.32) as well as event-free survival (44% vs 40%, p 0.42) for the germinal centre as opposed to the activated B-cell subtype. Nevertheless not even a small difference could be demonstrated in the presence of rituximab. According to Choi, both regimens (chemotherapy with or without the addition of rituximab) revealed similar results to the Hans algorithm on 5 years OS as well as 3 year EFS when comparing GCB versus non-GCB subgroups. BCL2 and LMO2 marker expression of the respective immunohistochemical (IHC) subtype, despite small sample size, revealed the following. Analysis by Choi criteria on survival for BCL2, no matter for which subsets (GCB or ABC) or treatment modality (chemotherapy with or without the addition of rituximab) showed no difference in 5 years OS or EFS. However, in contrast, a significant difference for better EFS (p=0.0015) in the BCL2 positive group of the ABC subgroups subtypes treated with rituximab containing chemotherapy. These results must be interpreted with care due to the very low sample size and the follow up time of only 9 months. For LMO2 similar results on survival outcome were seen thus showing no difference in 5 years OS or EFS – regardless of subtype or treatment modality. Also here, this was contrasted by better EFS (p=0.039) in the LMO2 positive group of ABC subtypes when treated with the rituximab containing regimen. Again the reservation about small numbers and the 14 months observation period apply. DISCUSSION AND CONCLUSION Most of the studies examining the proportions of subtypes in large series of DLBCL patients have been predominantly carried out on Western populations. While 50% of patients in North America or Europe express GCB phenotypes, this is only 31% in their Asian counterparts. Thus far, no study has been published on lymphoma subtypes in South African populations using a Tissue Micro Array (TMA) method. Despite certain limitations of this study, due to a variety of reasons such as loss of analysable data, variability of representation of the South African population as a whole or low sample size leading to a potential source of error, these results confirm that lymphoproliferative disorders are heterogeneous. Neverless this group can be treated successfully if exact staging, classification and risk assessment defines the holistic management plan – no matter if in a developed or under resourced setting. With the introduction of sophisticated methods such as gene expression profiling (GEP), diffuse large B-cell lymphoma (DLBCL) can be classified into the prognostically favourable germinal center B-cell–like (GCB) and the more aggressive activated B-cell–like (ABC) subtypes. Against the background of resource restriction we therefore used an immunohistochemical (IHC) stain method to analyse formalin-fixed, paraffin-embedded tissue samples. Here the algorithm by Choi et al., originally described by Hans et al., showing a high concordance rate, was comparable to the GEP classification. The use of the IHC based TMA methodology was shown to be a simple, cost effective and a robust alternative to GEP which is currently regarded as the gold standard for the classification in lymphomas. It provides a useful prognostic tool in stratifying DLBCL or other entities in future, even when frozen tissue samples are not available for GEP analysis. With the current budgetary limitations in public hospitals chemotherapy protocols for lymphoproliferative disorders exclude agents such as rituximab. Local therapeutic drug committees consider the approximately 15% overall survival benefit seen at 5 years for DLBCL when rituximab is added to combination chemotherapy as too marginal for justifying the arising additional expenses. Accordingly, demonstration that a specific molecular subtype accounts for superior outcome when using these regimens is needed and would provide convincing evidence for the use of this monoclonal antibody in a resource constrained setting. / AFRIKAANSE OPSOMMING: AGTERGROND Limfome ontstaan vanuit selle van die immuunsisteem. Hulle toon ‘n groot verskil met betrekking tot sitomorfologie, immunofenotipe, kliniese verloop, asook respons op behandeling – almal faktore wat prognose bepaal. Die Limfoom Herklassifikasieprojek het getoon dat daar substansiële geografiese verskille bestaan vir Hodgkin se limfoom en ander limfoproliferatiewe toestande. Huidige navorsing oor hierdie maligniteite skep die indruk dat hulle skaars is in Afrika, met die inligting wat wel beskikbaar is hoofsaaklik gebaseer op gevallestudies deur klinici en patoloë. DOEL Hierdie studie bestaan uit twee dele. In die eerste toets ons die hipotese dat die uitkomste vir die behandeling van limfoom in ‘n privaat akademiese instansie geleë in die Wes-Kaap, Suid- Afrika, dieselfde is as wat in die res van die wêreld gesien word indien soortgelyke behandeling toedgedien word.`n Reeks van 512 pasiënte is in die analise gebruik. Vir die tweede hipotese het`n kohort van die mees algemene subtipe, naamlik diffuse groot B-sellimfoom (DLBCL) (n = 93), lukraak òf die standaardkombinasie-chemoterapie in die vorm van CHOP òf `n identiese program met rituximab, `n anti-CD20 monoklonale teenliggaam, gekry. Die teenliggaam is in samewerking met die Universiteit van Nebraska se Mediese Sentrum getoets met behulp van `n immunohistochemies-gebaseerde metode, ook bekend as weefsel mikro-skikking (tissue microarray or TMA). PASIËNTE EN METODE Vir die eerste kohort is die opeenvolgende en volledige rekords van pasiënte wat ouer as 14 was en tussen Oktober 1998 en Julie 2006 in `n privaat-gebaseerde akademiese instansie gediagnoseer is, noukeurig ondersoek. Na sekere uitsluitingskriteria toegepas is, was daar 398 gevalle wat geskik was vir verdere analise. In die tweede deel van die navorsing kon 149 pasiënte wat nuut met diffuse groot B-sel-limfoom (DLBCL) gediagnoseer is, verder geëvalueer word. Drie-en-neëntig van hierdie 149 pasiënte het aan die kriteria vir insluiting voldoen; 48 het slegs die standaard chemoterapie (CHOP) ontvang en die oorblywende 45 het chemo-immunoterapie (chemoterapie plus rituximab (R-CHOP) ontvang. Die demografiese eienskappe van beide groepe het goed vergelyk. Vanweë die kriteria wat gebruik is, het die aanvanklike stratifikasie van 93 gevalle, wat deur middel van die TMA-metode op fenotipering met immunohistochemie gebaseer was, twee populasies gelewer. Hierdie primêre subdivisies is verder geëvalueer vir uitdrukking van BCL2 en LMO2, beide erkende voorspellers van respons. Laastens is kliniese eienskappe en oorlewingsuitkoms ná behandeling met chemoterapie as `n enkel modaliteit of dieselfde chemoterapie met rituximab, vir elke veranderlike met mekaar vergelyk. STATISTIESE ANALISE Algehele en insident-vrye oorlewing is bereken as die jare vanaf diagnose tot sterfte, verdwyning van pasiënte tydens opvolg, eerste progressie of terugval van die toestand. Die Kaplan Meier-metode is gebruik om distribusie te bepaal en die log rank-toets om oorlewing tussen die groepe te vergelyk. Pasiëntkenmerke vir elke gespesifiseerde kohort is met die Chikwadraattoets of Fisher se eksakte toets in die geval van kleiner groepe getoets. RESULTATE In die eerste gedeelte van die studie is al 398 gevalle terugwerkend geanaliseer en daar is gevind dat die behandelingsuitkoms met betrekking tot totale oorlewing en insident-vrye oorlewing teen 36 maande vergelykbaar was met uitkomste in eerstewêreldsentrums. Nadelige faktore met betrekking tot oorlewing was soortgelyk aan reeds gepubliseerde data en het die volgende ingesluit: gestelsimptome, voorafgaande behandeling met chemoterapie, intermediêre of hoë-risiko tellings soos deur die Internasionale Prognostiese Indeks bepaal, histologiese gradering en sekere anatomiese setels van primêre tumor. In teenstelling met internasionale ondervinding het geslag, steiering volgens Rye- of Rai-klassifikasie, retrovirale status en vorige behandeling met radioterapie geen invloed gehad nie. In die tweede gedeelte is weefsel van 93 nuut-gediagnoseerde DLBCL-pasiënte wat deur middel van die TMA-metode subtipeer is, vergelyk met naastenby ooreenstemmende kategorieë soos deur Hans et al. met `n drie-merkerpaneel in kiem- en nie-kiemsentrumsubtipes, of deur Choi et al. met twee bykomende teenliggame in kiemsentrum of geaktiveerde B-selle gedefinieer. Met die DNA-gebaseerde geen-uitdrukkingsprofiel as `n verwysingspunt, was die ooreenstemming tussen die onderskeie algoritmes na verwagting verskillend, met 86% teenoor 93%. Die onderliggende rasionaal was om die relatiewe lonendheid te bepaal van `n bekostigbare maar akkurate tegnologie, wat moontlik in gebiede met onvoldoende hulpbronne toegepas kon word Verder is die prognostiese merkeruitdrukking vir BCL2 en LMO2 ook met die oog op die uitkomste van behandeling ondersoek. In vergelyking met standaard chemoterapie, het die gebruik van die TMA-tegniek met toevoeging van rituximab geen noemenswaardige verbetering in die algehele 5-jaar oorlewing (63% vs 59%, p = 0.68) óf insident-vrye oorlewing (42% vs 39%, p = 0.94) getoon nie. Insgelyks was daar geen duidelike verskille in subtipe-analise nie. Dit is egter interessant dat, met die toepassing van die Choi-kriteria, sitotoksiese middels op hul eie, in teenstelling met die geaktiveerde B-sel-subtipe, nie ‘n statisties belangrike neiging tot beter oorlewing (74% vs 55%, p = 0.32) of insident-vrye oorlewing (44% vs 40%, p = 0.42) vir die kiemsentrumsubtipe (GCB) getoon het nie. Dit was selfs nie eers moontlik om ‘n klein verskil in die teenwoordigheid van rituximab te demonstreer nie. Volgens Choi, het chemoterapie (met óf sonder die toevoeging van rituximab) by vergelyking van kiemsentrumsubtipes met nie-kiemsentrumsubtipes soortgelyke resultate gelewer as die Hans algoritme na 5-jaar oorlewing, sowel as 3-jaar insident-vrye oorlewing. Ten spyte van ’n klein steekproef het verdere subtipe-analise van BCL2- en LMO2- merkeruitdrukking van die onderskeie immunohistochemiese (IHC) subtipes die volgende aan die lig gebring: Analise met gebruik van die Choi-kriteria vir subtipe-analise vir BCL2- uitdrukking het, ongeag die subtipe (GCB of ABC) en die behandelingsmodaliteit (chemoterapie met of sonder toevoeging van rituximab), geen verskil getoon in 5-jaar oorlewing en insident-vrye oorlewing nie. Daar was egter `n noemenswaardige verskil ten opsigte van beter insident-vrye oorlewing (p = 0.0015) in die BCL2-positiewe groep van die ABC-subtipes wat met rituximab-bevattende chemoterapie behandel is. Hierdie resultate moet egter met sorg geïnterpreteer word weens die klein steekproef en kort opvolgperiode van slegs nege maande. Soortgelyke resultate met betrekking tot die uitkoms vir oorlewing is vir LMO2 gelewer, naamlik geen verskil ten opsigte van 5-jaar oorlewing of insident-vrye oorlewing ongeag die subtipe of behandelingsmodaliteit. Hier ook was daar egter beter insident-vrye oorlewing (p = 0.039) in die LMO2-positiewe groep van die ABC-subtipe wanneer rituximab-bevattende chemoterapie toegedien is. Weereens moet die resultate met sorg interpreteer word weens die klein steekproef en die kort opvolgperiode van 14 maande. BESPREKING EN GEVOLGTREKKING Die meeste studies wat reeds die verhoudings van subtipes in groot getalle DLBCL-pasiënte ondersoek het, is onder westerse populasies uitgevoer en daar bestaan onsekerheid oor of dieselfde verhoudings in Afrika van toepassing is. Terwyl 50% van alle DLBCL-pasiënte in Noord-Amerika of Europa die GCB-fenotipe uitdruk, kom dit in slegs 31% van hierdie pasiënte in Asië tot uitdrukking. Geen studie is tot dusver met behulp van die TMA-metode onder limfoomsubtipes in populasies in Afrika onderneem nie, veral nie onder pasiënte met verswakte immuunstelsels nie. Ten spyte van sekere beperkings van hierdie studie, waarvoor daar verskeie redes is, soos die verlies van analiseerbare data, die wisselende verteenwoordiging van die Suid-Afrikaanse bevolking as ‘n geheel en die klein steekproef, wat vergissing in die hand kan werk, bevestig die resultate dat limfoproliferatiewe toestande heterogeen is. Desnieteenstaande kan hierdie groep met sukses behandel word indien ‘n holistiese bestuurplan presies volgens stadiums, klassifikasie en risiko-assessering uitgevoer word – ongeag of dit in `n ontwikkelde gebied of een met min hulpbronne plaasvind. Met die beskikbaarheid van ingewikkelde metodes soos geen-ekspressie profielskepping (GEP), kan DLBCL in prognosties-voordelige kiemsentrum B-sel-agtige (GCB) en die meer aggressiewe geaktiveerde B-sel-subtipes geklassifiseer word. Teen die agtergrond van beperkte hulpbronne, is immunohistochemiese (IHC) kleuringsmetodes gebruik om - weefselmonsters wat in formalien gefikseer en in paraffien ingebed was, te analiseer. Hier was die algoritme wat oorspronklik deur Hans et al. beskryf is en deur Choi et al. verder ontwikkel is, en hoë ooreenstemming toon, vergelykbaar met die GEP-klassifikasie. Die gebruik van die IHC-gebaseerde metodologie is as ‘n eenvoudige, effektief lonende en kragtige alternatief tot GEP, wat tans as die goudstandaard vir klassifikasie van limfoom beskou word, bewys. Dit verskaf ‘n nuttige prognose-hulpmiddel vir die stratifisering van DLBCL of ander entiteite in die toekoms, selfs wanneer gevriesde weefselmonsters nie vir GEP-analise beskikbaar is nie. Onder die huidige begrotingsbeperkings in staatshospitale is middels soos rituximab by die protokolle vir die behandeling van limfoproliferatiewe toestande uitgesluit. Plaaslike terapeutiese middel-komitees beskou die nagenoeg 15% algehele oorlewingsvoordeel teen vyf jaar, wat vir DLBCL moontlik is met die toevoeging van rituximab by kombinasiechemoterapie, as te randstandig om die bykomende onkoste te regverdig. Daarvolgens is dit noodsaaklik om te demonstreer dat `n spesifieke molekulêre subtipe tot ’n beter uitkoms lei wanneer hierdie metode gebruik word en dat dit oortuigende bewyse sal lewer vir die gebruik van hierdie monoklonale teenliggaam in `n omgewing met beperkte hulpbronne.

<p>Calcium (Ca2+) is a known important second messenger. Calcium/Calmodulin (CaM) dependent protein kinase kinase 2 (CaMKK2) is a crucial kinase in the calcium signaling cascade. Activated by Ca2+/CaM, CaMKK2 can phosphorylate other CaM kinases and AMP-activated protein kinase (AMPK) to regulate cell differentiation, energy balance, metabolism and inflammation. Outside of the brain, CaMKK2 can only be detected in hematopoietic stem cells and progenitors, and in the subsets of mature myeloid cells. CaMKK2 has been noted to facilitate tumor cell proliferation in prostate cancer, breast cancer, and hepatic cancer. However, whethter CaMKK2 impacts the tumor microenvironment especially in hematopoietic malignancies remains unknown. Due to the relevance of myeloid cells in tumor growth, we hypothesized that CaMKK2 has a critical role in the tumor microenvironment, and tested this hyopothesis in murine models of hematological and solid cancer malignancies. </p><p>We found that CaMKK2 ablation in the host suppressed the growth of E.G7 murine lymphoma, Vk*Myc myeloma and E0771 mammary cancer. The selective ablation of CaMKK2 in myeloid cells was sufficient to restrain tumor growth, of which could be reversed by CD8 cell depletion. In the lymphoma microenvironment, ablating CaMKK2 generated less myeloid-derived suppressor cells (MDSCs) in vitro and in vivo. Mechanistically, CaMKK2 deficient dendritic cells showed higher Major Histocompatibility Class II (MHC II) and costimulatory factor expression, higher chemokine and IL-12 secretion when stimulated by LPS, and have higher potent in stimulating T-cell activation. AMPK, an anti-inflammatory kinase, was found as the relevant downstream target of CaMKK2 in dendritic cells. Treatment with CaMKK2 selective inhibitor STO-609 efficiently suppressed E.G7 and E0771 tumor growth, and reshaped the tumor microenvironment by attracting more immunogenic myeloid cells and infiltrated T cells.</p><p>In conclusion, we demonstrate that CaMKK2 expressed in myeloid cells is an important checkpoint in tumor microenvironment. Ablating CaMKK2 suppresses lymphoma growth by promoting myeloid cells development thereby decreasing MDSCs while enhancing the anti-tumor immune response. CaMKK2 inhibition is an innovative strategy for cancer therapy through reprogramming the tumor microenvironment.</p> / Dissertation

Patients with primary Sjögren’s syndrome (pSS) are at increased risk of developing malignant lymphoma. The studies in this thesis aim at broadening our understanding of the association between these two conditions. Germinal centre (GC)-like structures were found in minor salivary gland biopsies taken at the time of pSS diagnosis in 25% of 175 studied patients. Lymphoma development was observed in 86% of the GC-positive pSS patients and 14% of the GC-negative patients. GC-like structures in salivary gland biopsies at pSS diagnosis might identify pSS patients at high risk for later lymphoma development. We used the National Patient Register and the Cancer Register to identify pSS patients with lymphoid malignancy for the following studies. The lymphoma tissues were reviewed and classified according to the WHO classification. In a study of 79 patients with available lymphoma tissues, we identified histopathological and clinical features compatible with IgG4-related disease (IgG4-RD) in one patient (1.3%). Histological features of IgG4-RD in lymphoma tissue in patients with an initial pSS diagnosis seem to be rare but, if present, may indicate underlying IgG4-RD. We identified and compared pSS patients with (n=18/17%) and without (n=87) pre-existing lymphoma at pSS diagnosis and found similar pSS characteristics in both groups. Mucosa-associated lymphoid tissue (MALT) lymphoma in salivary glands was more common in patients with pre-existing lymphoma. The findings support the removal of pre-existing lymphoma as a general exclusion criterion for a pSS diagnosis in classification criteria. Further, the findings suggest an investigation for pSS in patients presenting with MALT lymphoma in salivary glands. We compared the distribution of lymphoma subtypes with a general population reference. Both diffuse large B-cell lymphoma (DLBCL) (32%) and marginal zone lymphoma (MZL) (31%) were common, but only MZL (MALT lymphomas) occurred at an increased relative frequency compared to the general population. Men constituted 15% of 105 pSS patients with lymphoma. Men had a shorter time between the pSS and lymphoma diagnoses and more often had lymphoma in the salivary glands compared with women. Increased awareness of signs of lymphoma in salivary glands already during the first years after pSS diagnosis is justified in men with pSS.