Endemic Burkitt lymphoma (eBL) is the leading pediatric cancer in sub-Saharan Africa and is associated with Epstein-Barr virus (EBV) and Plasmodium falciparum malaria co-infections. Current treatment options in Africa are combination chemotherapy with a survival rate hovering around 50%. Relapsed or refractory eBL patients have failed to receive any targeted treatments in the clinic. Our focus was to delineate immune responses in eBL, interrogate the tumor variation in responses to targeted treatments and develop mouse models that can be used to target essential mediators of tumor pathogenesis. Immune-based treatments including immune checkpoint inhibition have recently become an effective therapeutic modality in oncology. However, some B cell lymphomas such as Hodgkin Lymphoma (HL), are more receptive to checkpoint inhibition than others suggesting a need to understand the efficacy of checkpoint inhibition on different lymphoma subtypes. Checkpoint inhibitors act by blocking inhibitory receptors on T cells and improving anti-tumor responses. One of the goals of this thesis was to characterize checkpoint inhibitors on Tumor-infiltrating lymphocytes (TILs) in eBL tumors and to identify T cell subsets that exhibit increased expression of inhibitory receptors, poor cytokine production, poor proliferation and express transcription factors associated with exhaustion. Using scRNA seq, we identified T cell clusters that co-expressed inhibitory receptors, poor proliferative markers but also sustained costimulatory signals, as well as cytokine expression suggesting a pre dysfunctional state and not terminally exhausted state. Furthermore, we quantified the dominant co-inhibitory receptors PD1 and TIGIT that are upregulated in the tumor microenvironment via immunohistochemistry (IHC) and in peripheral blood of eBL patients via flow cytometry. We compared eBL patients with healthy pediatric cohorts with a history of persistent malaria exposure to those who had little to no malaria infections, to understand uniquely T cell mediated responses in BL children. Tumors had high co-expression of PD1 and TIGIT but fewer PD1 only populations, suggesting that both ligands may play a role in restraining immune activation via IHC. Next, we investigated if PD1 ligands or TIGIT ligands were overexpressed in eBL tumors. Nectin-2, TIGIT ligand was highly expressed in eBL tumors but was not highly correlated with TIGIT expression. These studies provide insights for PD1/ TIGIT blockade in Burkitt lymphoma patients. Additionally, we established new patient-derived cell lines from eBL tumors to study tumor variation and to study targeted treatments. We established five new patient-derived eBL lines BL717, BL 719, BL720, BL725, and BL740 that were interrogated for their inter-patient variation by studying their gene expression profiles. Further, we developed a patient cell-line derived xenograft (CDX) mouse model by injecting newly patient-derived BL cell lines in immunodeficient mice (NSG BL) and studying BL tumorigenesis. Having successfully established NSG BL tumors, we observed differences in tumor growth sensitivity and survival. We tested rituximab efficacy, one of the most established treatments for B cell lymphomas in our mouse model. We also identified pathways associated with unfolded protein response (UPR) and the mammalian target of rapamycin (mTOR) signaling, as well as apoptosis in one of the cell line xenografts, BL740, in response to rituximab. BL717, BL720 cell line xenograft failed to control tumor growth and was enriched in IFN-ɑ signature genes. This mouse model will prove to be useful to study combination therapy against eBL tumors as well as mechanisms of resistance to drug targets. Collectively, these studies provide insights into intratumoral variation including subtypes during tumor progression and expression profiles of TILs in eBL tumors. This will be important in designing new therapeutic strategies as well as help pose novel therapeutic targets.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20260号 / 医博第4219号 / 新制||医||1020(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 前川 平, 教授 木原 正博, 教授 河本 宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21021号 / 医博第4367号 / 新制||医||1030(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 小柳 義夫, 教授 竹内 理, 教授 小川 誠司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The Notch signalling pathway is an evolutionary conserved pathway, named after the Notch receptors, Notch1-4 in mammals, which upon cell-cell contact and ligand binding releases the intracellular domain (NICD). NICD translocates into the nucleus where it binds the transcriptional repressor RBP-Jk, which together with co-activators belonging to the Mastermind-like family of proteins form a transcriptional activation complex. This complex activates genes controlling cell fate decision, embryonic development, proliferation, differentiation, adult homeostasis and stem cell maintenance. On the other hand, disrupted Notch signalling may result in pathological conditions like cancer, although the mechanisms behind the disruption are often complex and in many cases largely unknown. Notch1 drives the lymphocyte differentiation towards a T-cell fate and activating mutations in the gene have been suggested to be involved in T-cell lymphoma. In paper I, genetic alterations in Notch1 and the Notch1 regulating gene CDC4 were investigated in tumours from murine T-cell lymphoma induced with phenolphthalein, 1,3-butadiene or 2’,3’-dideoxycytidine. We identified activating Notch1 mutations in 39% of the lymphomas, suggesting that Notch1 is an important target gene for mutations in chemically induced lymphomas. While it is known that constitutively activated Notch signalling has a clear oncogenic function in several solid malignancies as well, the molecular mechanisms are less known in this context. Unpublished data of our lab, together with other recent studies, suggest that mutations of Notch and Notch-related genes per se are uncommon in solid malignancies including colorectal cancer, while a growing body of evidence indicates that aberrant Wnt/b-catenin signalling may result in pro-tumoural Notch activation in these contexts. In paper II, we therefore investigated potential transcriptional interactions between the Notch and Wnt signalling pathways in colorectal cancer cell lines. The proximal Notch and Wnt pathway gene promoters were bioinformatically identified and screened for putative TCF/LEF1 and RBP-Jk sites. In canonical Wnt signalling, Apc negatively regulates b-catenin leading to repression of TCF/LEF1 target genes. Upon repression of the Wnt pathway we observed that several genes in the Notch pathway, including Notch2, were transcriptionally downregulated. We also confirmed binding of Lef1 to Notch2 as well as other Notch pathway gene promoters and luciferase assays showed an increased activity for at least one LEF1/TCF-site in the Notch2 promoter upon co-transfection of HT29 or HCT116 cells with mutated b-catenin. HT29 cell lines were also treated with the g-secretase inhibitor DAPT, leading to inactivation of the Notch pathway by preventing release of NICD. However, results showed no effects on Apc, b-catenin or their target cyclin D1. Taken together, these results indicate that the Wnt pathway may function as a regulator of the Notch pathway through the TCF/LEF1 target gene program in colon cancer cell lines. In summary, Notch pathway deregulation is of importance in both murine T-cell lymphoma and human colorectal cancer, although the mechanisms differ. The current results give new insights in Notch pathway alterations as well as the signalling networks in which the Notch pathway interacts, and thus increase the understanding of Notch’s involvement in malignant diseases. / Studies on molecular genetic alterations in colorectal cancer

Title Complex preoperative brain tumor imaging Abstract The differentiation of glioblastoma, metastases and brain lymphoma using modern diagnostic imaging methods has a major impact on the strategy of further diagnostic examinations and treatment. In a group of 67 patients with glioblastoma and 31 with cerebral metastasis, the ability to differentiate them according to the evaluation of perfusion parameters changes in peritumoral white matter by T1 dynamic post-contrast magnetic resonance imaging was verified, with the positive predictive value in glioblastoma detection up to 91%. In a group of 36 brain lymphoma patients the importance of imaging submodalities and contribution of a complex magnetic resonance imaging protocol to detect lymphoma up to 80% were evaluated. Key words brain, glioblastoma, lymphoma, magnetic resonance imaging, neoplasm metastasis

Primary gastrointestinal lymphoma is the most common type of extra-nodal lymphoma, representing about 30-50% of all extra-nodal involvement. The stomach is the most common site, with the colon and rectum accounting for a minority of occurrences. Primary colorectal lymphoma is uncommon, representing only 0.3% of all large intestinal malignancies and approximately 3% of GI lymphomas with the majority of these being B-cell non-Hodgkin lymphoma and diffuse large B-cell lymphoma (DLBCL) being the most common subtype. We present a case of an 85-year-old male who presented with symptoms suggestive of bowel obstruction, who after further evaluation was diagnosed with primary non-Hodgkin lymphoma of the colon, DLBCL subtype.

No description available.

Cytokiner eller interleukiner är signalpeptider med låg molekylär vikt somreglerar många viktiga funktioner. De kan delas in i två grupper beroende på deraseffekt på celler. Interleukin 4 (IL-4) till exempel kan tillhöra gruppentillväxtfaktorer medan interleukin 6 (IL-6) kan tillhöra gruppen aktiverings- ellerdifferentieringsfaktorer. IgM-receptorn eller B-cellsreceptorn, BCR, finns på Bcelleroch är membranbundna immunoglobuliner (mIg) som har tvåhuvuduppgifter; att förmedla signaler som styr B-cellens utveckling samt att bindain antigen som sedan ska presenteras för T-celler. I studien aktiverades B-cellermed antikropp mot IgM (anti-IgM) samt rekombinant IL-4. Efter stimuleringanalyserades IL-6 produktionen med enzyme-linked immunosorbent assay(ELISA). Syftet med studien var att karakterisera uttrycket av IL-4 receptorn pålymfom- och leukemiceller med flödescytometri och polymeras chain reaction(PCR) samt produktion av IL-6 med ELISA. ELISA-analysen visade att tvåcellinjer, stimulerade Sp53 och stimulerade samt kontroller av WaC3CD5+, gaven ökning av IL-6 produktionen. Vid jämförelse med ELISA-resultaten ochflödescytometri-analyserna ser man att WaC3CD5+ som producerat storamängder IL-6, har en liten andel IL-4 receptorer på cellytan. Resultatet av PCRanalysenvisar att det var framförallt Sp53 som fick höga mängder IL-4R mRNA,men även I83, U2932 och WaC3CD5+ fick positiva resultat. Resultaten i dennastudie är preliminära och för att få mer säkerställda resultat krävs att alla analysergörs om ett antal gånger för att få ett mer tillförlitligt resultat. / Cytokines or interleukins are signal peptides of low molecular weight, whichregulates many important functions. They can roughly be divided into two groupsor divisions due to their effect on cells. Interleukin 4 (IL-4), for example, belongsto the group growth factors while interleukin-6 (IL-6) belongs to the groupactivation or differentiation factors. IgM receptors or B cell receptors, BCR, areexpressed on B cells and are membrane-bound immunoglobulins (mIg) and havetwo main functions: to convey signals that control B cell activation and to bindantigen which will then be presented to T cells. In the study B cells were activatedwith antibodies against IgM (anti-IgM) and recombinant IL-4. After stimulationthe IL-6 production was analysed by enzyme-linked immunosorbent assay(ELISA). The purpose of this study was to characterize the expression of IL-4receptor in lymphoma and leukemia cells by flow cytometry and polymerasechain reaction (PCR) and furthermore the production of IL-6 by ELISA. ELISAanalysis showed that two cell lines, stimulated Sp53 and stimulated and control ofWaC3CD5+, resulted in an increased IL-6 production. When comparing ELISAresults and flow cytometry assays, it can be seen that WaC3CD5+, whichproduced large amounts of IL-6, has a small percentage of IL-4 receptors on thecell surface. The results of the PCR analysis shows that particularly Sp53displayed high amounts of IL-4 mRNA, but also I83, U2932 and WaC3CD5+were positive for IL-4 mRNA. The results of this study are preliminary, and to getmore trustworthy results, all analyses have to be repeated to get more reliableresults.

Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous disease with multiple distinct molecular subtypes. Increased NF-κB activity and expression of the microRNA miR-155 (product of the BIC gene) are associated with one subtype, called the activated B-cell (ABC) subtype. It is shown here that induction of NF-κB activity leads to increased miR-155 expression, the levels of miR-155 in a panel of B-lymphoma cell lines correlate with increased NF-κB activity, and the NF-κB p50/p65 heterodimer binds to a specific DNA site in the BIC promoter. Also described is a regulatory network wherein NF-κB-dependent up-regulation of miR-155 leads to reduced PU.1 transcription factor expression and consequently reduced PU.1-driven expression of B-lymphoma marker CD10 in the human B-lymphoma cell line BJAB. Genetic variation in DLBCL can be used to explain the response of individual patients to chemotherapy. One cancer therapeutic approach currently in clinical trials uses histone deacetylase inhibitors (HDACi's) as a monotherapy or in combination with other vi agents. It is shown here that two pan-HDACi's, trichostatin A and vorinostat, induce apoptosis in seven of eight human DLBCL cell lines. Ectopic over-expression of antiapoptotic proteins BCL-2 and BCL-XL or the pro-apoptotic protein BIM in select DLBCL cell lines can confer further resistance or sensitivity, respectively, to HDACi treatment. Additionally, the BCL-2 family antagonist ABT-737 can increase the sensitivity of several DLBCL cell lines to vorinostat-induced apoptosis, including the HDACi-resistant SUDHL6 cell line. Moreover, one vorinostat-resistant variant of the HDACi-sensitive cell line SUDHL4 has increased expression of anti-apoptotic proteins BCL-XL and MCL-1 and decreased sensitivity to ABT-737, and a second such variant cell line has increased expression of anti-apoptotic protein MCL-1. These results suggest that the balance of anti- to pro-apoptotic BCL-2 family protein expression is important in determining the sensitivity of DLBCL cell lines to HDACi-induced apoptosis. Thus, the sensitivity of DLBCL cell lines to treatment with HDACi's appears to depend on the complex regulation of BCL-2 family members, suggesting that the response of a subset of DLBCL patients to HDACi treatment may benefit from co-treatment with BCL-2 antagonists.

<p dir="ltr">Lymphoma is a prevalent malignancy in dogs. Diffuse large B-cell Lymphoma (DLBCL) is the common subtype that represents about 50% of the clinically seen lymphoma cases. DLBCL diagnosis relies on cytological examination of a fine needle aspirate and histological evaluation by immunohistochemistry (IHC) in most common practices. This workflow is sufficient to confirm the diagnosis; however, it may be challenging to differentiate reactive and neoplastic forms in some controversial cases. In such cases, PCR-based clonality assays and flow cytometry (FC) can help with more conclusive diagnoses. So, finding more biomarkers that can detect and track DLBCL early and over time is a must for a final diagnosis and helps us learn more about how DLBCL starts at the molecular level. MicroRNAs (miRNAs), the small non-coding RNAs, regulate gene expression by binding to the 3'-untranslated region of protein-coding RNAs, leading to either RNA degradation or translational repression. They can switch on and off genes to regulate physiological and pathological processes. MicroRNA stability features and tissue availability make them promising biomarkers for identifying and sub-classifying patients and sequentially evaluating the disease status or the response toward a specific medicine. This dissertation investigates the small RNA sequence analysis, the differentially expressed miRNAs between healthy and DLBCL-affected lymph nodes, and the miRNA profile in DLBCL cases with different outcomes.</p>