L'environnement tumoral reste mal défini et cliniquement sous-utilisé, les agents tumoricides agissant directement sur les cellules tumorales. Cependant, il est admis que les cellules tumorales dépendent de leur environnement pour se développer au maximum. Ce projet de thèse s’est concentré sur la molécule « a proliferation inducing ligand » (APRIL) qui est impliquée dans la survie, la prolifération et la différenciation des cellules B. Son rôle pathologique a été investigué dans les lymphomes diffus à grandes cellules B (DLBCL) et les myélomes multiples (MM). Grâce à un modèle murin nous avons pu démontrer le rôle pro-tumoral direct d’APRIL dans les DLBCL. Chez les patients, APRIL est d’origine paracrine, produit par les cellules myéloïdes et ciblant les cellules tumorales. Son expression intra-tumorale est variable et un niveau élevé corrèle avec un mauvais pronostic. L’expression d’APRIL est dictée par des mécanismes chémotactiques. L’expression différentielle de certaines chémokines par les cellules tumorales influence le recrutement des cellules productrices d’APRIL. Nous avons également démontré qu’APRIL possède un mode de signalisation atypique dans les DLBCL. Les cellules ont besoin d’internaliser APRIL afin d’activer BCMA, un récepteur de signalisation qui est principalement exprimé de manière intracellulaire. Dans les MM, APRIL possède également un rôle pro-tumoral direct tel que démontré dans un modèle murin. Chez les patients, APRIL est d’origine paracrine et principalement exprimé par les cellules myéloïdes immatures. Malgré l’infiltration tumorale de la moelle osseuse, l’expression d’APRIL reste stable grâce au remodelage du microenvironnement. Un modèle murin nous a permis d’identifier une boucle autocrine basée sur l’interkeuline-6 qui permet la persistance des cellules myéloïdes immatures pendant le développement tumoral, fournissant une expression stable du facteur pro-tumoral APRIL. Globalement, nos travaux ont permis d’identifier APRIL comme un biomarqueur prédictif de la survie avec une valeur thérapeutique dans le DLBCL. La caractérisation des mécanismes moléculaires contrôlant l’expression différentielle d’APRIL ouvre des perspectives thérapeutiques pour son antagonisme. APRIL possède également une valeur thérapeutique dans le MM. La caractérisation de la source cellulaire d’APRIL et de l’interleukine-6, deux facteurs pro-tumorales pour le MM, nous a conduit à de nouvelles perspectives thérapeutiques. / Tumor microenvironment remains largely ill-defined and clinically underused, as all currently used tumoricidal agents act directly on tumor cells. However it is well known that tumor cells depend on their environment to fully develop. This thesis project focused on the molecule “a proliferation inducing ligand” (APRIL) which is involved in B-cell survival, proliferation and differentiation. Its pathological role was investigated in diffuse large B-cell lymphoma (DLBCL) and in multiple myeloma (MM). Thanks to a murine model we have been able to demonstrate the direct pro-tumoral role of APRIL in DLBCL development. In patients APRIL is of paracrine origin, being produced by myeloid cells and acting on tumor cells. Intra-tumoral expression is variable and a high level of APRIL expression correlates with a poor prognosis. APRIL expression is controlled by chemotactic mechanisms. Differential expression of certain chemokines by tumor cells influence APRIL-producing cells recruitment. We also showed that APRIL has an atypical way of signaling in DLBCL. Tumor cells need to internalize APRIL in order to activate BCMA, a signaling receptor which is mainly expressed intracellularly. In MM, APRIL also possesses a direct pro-tumoral effect as demonstrated in a mouse model. In patients, APRIL is of paracrine origin and mainly expressed by immature myeloid cells. Despite tumor-cell infiltration into the bone marrow APRIL expression remains stable thanks to microenvironment remodeling. A mouse model allowed us to identify an autocrine loop based on interleukin-6 which allows persistence of immature myeloid cells during tumor development, providing a stable expression of the pro-tumoral factor APRIL. Globally, our work identified APRIL as a predictive biomarker with a therapeutic value in DLBCL. Characterization of molecular mechanisms controlling differential expression of APRIL opens new therapeutic perspectives by its antagonism. APRIL also possesses a therapeutic value in MM; characterization of the cellular source for APRIL and interleukin-6, two pro-tumoral factors in MM, has led us to new therapeutic perspectives.

Abstract Diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL) are two of the most common lymphomas in the Western world. DLBCL is an aggressive disease with a good response to treatment; about 75% of patients achieve permanent remission after first-line treatment. In patients with relapses or primary refractory disease, prognosis is dismal; only 10–20% of them can be cured, even with aggressive treatments. FL is an indolent lymphoma with a very good response to treatment and slow progression. Median survival with modern treatments is over 15 years. Nevertheless, some patients have short remissions and succumb to disease. Oxidative stress, TP53 mutations, and translocations of MYC, Bcl-2 and Bcl-6 have been linked in many neoplasms to aetiology and poor prognosis. This thesis concerns oxidative stress and redox-state-regulating enzymes in DLBCL and FL, and TP53 mutations and translocations of MYC, Bcl-2 and Bcl-6 in DLBCL. High expression levels of the antioxidant enzyme thioredoxin and a marker of oxidative stress, nitrotyrosine, were related to poor prognosis in DLBCL. In FL, high-level expression of peroxiredoxin was associated with good prognosis. TP53 mutations in specific regions LSH and L3 and concurrent translocation of Bcl-2 were associated with poor prognosis in DLBCL. Not all TP53 mutations predicted survival. High expression levels of Bcl-2 and MYC were associated with poor prognosis in DLBCL. Based on the results presented here, antioxidant function may have protective roles, but also may cause resistance to treatment. TP53 mutations have prognostic roles in DLBCL, but should be further defined. Novel therapies could be developed in connection with these mechanisms. / Tiivistelmä Diffuusi suurisoluinen B-solulymfooma (DLBCL) ja follikulaarinen lymfooma (FL) ovat kaksi yleisintä lymfoomaa länsimaissa. DLBCL on aggressiivinen syöpä, joka reagoi hyvin hoitoihin, jopa 75 % paranee. Kuitenkin potilailla, joilla syöpä uusiutuu hoitojen jälkeen tai etenee hoidon aikana, on erittäin huono ennuste, noin 10-20 % näistä potilaista voidaan parantaa. FL on hyväennusteinen lymfooma, joka yleensä reagoi hyvin hoitoihin. Mediaani elossaoloaika kaikilla FL potilailla on yli 15 vuotta taudin toteamisesta. Osalla potilaista FL kuitenkin on aggressiivisempo. Oksidatiivinen stressin, TP53- mutaatioiden, MYC, Bcl-2 ja Bcl-6 -translokaatioiden on todettu olevan huonoon ennusteeseen yhteydessä olevia tekijöitä monissa syövissä, kuten lymfoomissa. Tämä väitöskirja tutki oksidatiivisen stressin ja hapetus-pelkistys reaktioon liittyvien entsyymien osuutta R-CHOP-hoidetuissa DLBCL:ssa ja FL:ssa immunohistokemian (IHC) avulla. DLBCL:ssa tutkittiin lisäksi TP53 mutaatioita, MYC, Bcl-2 ja Bcl-6 translokaatioiden roolia taudin kulussa. Korkea ekspressio oksidatiivisen stressin merkkiainetta nitrotyrosiinia ja antioksidantti thioredoksiinia olivat yhteydessä huonoon ennusteeseen DLBCL:ssa. FL:ssa runsas ilmentyminen antioksidativiisiin entsyymeihin kuuluvia peroksiredoksiineja olivat yhteydessä hyvään ennusteeseen. TP53 mutaatiot LSH ja L3 alueella ja Bcl-2 -translokaatiot yhdessä olivat yhteydessä huonoon ennusteeseen DLBCL:ssa. Kaikki TP53-mutaatiot eivät olleet assosioituneet huonoon ennusteeseen. DLBCL:ssa Bcl-2 ja MYC –proteiinien runsas ilmentyminen IHC:llä arvioituna liittyi huonoon ennusteeseen. Tulosten perusteella solujen hapetus-pelkistystilaa säätelevillä entsyymeillä voi olla dualistinen rooli, osittain suojeleva ja osittain vahingoittava lymfoomissa. TP53 -mutaatioilla voi olla ennusteellista merkitystä, mutta tämä vaatii lisää tutkimuksia.

Para avaliar a influência dos polimorfismos de nucleotídeo único (SNPs) do CYP2B6, CYP3A5, GSTM1, GSTP1, GSTT1, PON1, NQO1 e MDR1 na resposta ao tratamento com R-CHOP e CHOP, 82 pacientes com Linfoma Difuso de Grandes Células B, sem evidências de infecção por HIV, foram selecionados nesse estudo. Amostras de sangue periférico foram coletadas para extração de DNA. Os SNPs foram analisados por PCR-RFLP. Em relação aos pacientes que apresentaram resposta completa (RC) ao tratamento (70%), 51% foram tratados com R-CHOP. Sobre o tratamento, 50% dos pacientes com RC apresentaram classificação de ECOG 0-1 (p=0,0193) e a maioria desses pacientes (41%) não apresentaram envolvimento extranodal (p=0,0377). Não houve associação entre os SNPs do CYP2B6, CYP3A5, GSTT1, NQO1 e MDR1 (C3435T) e as variáveis estudadas. Apenas CYP3A5 (sexo p=0,0519), GSTM1 (idade p=0,016; tratamento p=0,0372), GSTP1 (envolvimento extranodal p=0,0307), PON1 (sintomas B p=0,0201; Bulky p=0,0148) e MDR1 C1236T (sexo p=0,0316) mostraram associação. Em relação à sobrevida global, apenas tratamento (p=0,0129), IPI (p=0,000342), idade (p=0,0155), estadiamento (p=0,00281) e ECOG (p=0,00869) apresentaram resultados significantes. Quanto à sobrevida livre de doença (SLD), apenas idade (p=0,0292), estadiamento (p=0,0402) e ECOG (p=0,0142) apresentaram resultados significantes / To evaluated the influence of single nucleotide polymorphisms (SNPs) of CYP2B6, CYP3A5, GSTM1, GSTP1, GSTT1, PON1, NQO1 and MDR1 in the treatment response with R-CHOP and CHOP, 82 patients with Diffuse Large B-cell Lymphoma, without evidence of HIV infection, were enrolled in this study. Peripheral blood samples were collected for DNA extraction. The SNPs were analyzed by PCR-RFLP. In relation the patients that showed complete response (CR) to the treatment (70%), 51% were treated with R-CHOP. About the treatment, 50% of the patients with CR showed ECOG classification of 0-1 and the most of these patients (41%) did not showed extranodal involvement (p=0,0377). There was no association between CYP2B6, CYP3A5, GSTT1, NQO1 and MDR1 (C3435T) SNPs and the variables studied. Only CYP3A5 (gender p=0,0519), GSTM1 (age p=0,016; treatment p=0,0372), GSTP1 (extranodal involvement p=0,0307), PON1 (B symptoms p=0,0201; Bulky p=0,0148) e MDR1 C1236T (gender p=0,0316) showed association. In relation to overall survival, only treatment (p=0,0129), IPI (p=0,000342), age (p=0,0155), stadiament (p=0,00281) and ECOG (p=0,00869) showed significant results. To disease-free survival, only age (p=0,0292), stadiament (p=0,0402) e ECOG (p=0,0142) showed significant results

Abstract Lymphomas are a group of more than 70 different malignancies arising from lymphoid tissues and diffuse large B-cell lymphoma (DLBCL) is the most common subtype of lymphoma. More than 70% of DLBCL patients can be cured with modern therapy, but some patients still die of the disease. The recognition of patients with adverse prognosis, justifying deviation from standard treatment and risking severe side effects, is problematic. The aim of this study was to identify potential biological factors for the prediction of poor treatment response and central nervous system (CNS) relapse in DLBCL patients. The study included 263 lymphoma patients. 205 patients had a DLBCL, and 37 of these represented primary CNS lymphoma (PCNSL). Immunohistochemistry was used to determine the expression of oxidative stress markers 8-hydroxydeoxyguanosine (8-OHdG) and nitrotyrosine, as well as antioxidant enzymes manganese superoxide dismutase (MnSOD), thioredoxin (Trx) and gamma cysteine ligase (GCL) from samples representing reactive lymphoid tissue and B-cell derived lymphomas. From DLBCL samples staining was also conducted for cell cycle regulating proteins p16, p53, p21 and p27 and chemokine receptors CXCR4, CXCR5 and CCR7. Immunoelectron microscopy (IEM) for CXCR4 and CXCR5, and their ligands CXCL12 and CXCL13 was performed on additional samples from reactive lymphoid tissue, nodal DLBCL, secondary CNS lymphoma and PCNSL. Factors associated with adverse prognosis included expression of nitrotyrosine, Trx and GCL. A prognostic score reflecting the degree of cell cycle dysregulation within each patient’s tumour identified 3 distinct prognostic groups among DLBCL patients. High cytoplasmic CXCR5 expression was associated with CNS involvement, whereas nuclear CXCR4 expression correlated with nodal disease. These results demonstrate the considerable biological heterogeneity seen within DLBCL, but further research is needed to confirm them. High antioxidant activity and the accumulation of damage to cell cycle regulating pathways separated patient groups with a poor prognosis that might benefit from new types of treatment. Chemokine receptor expression seems to play a role in the CNS tropism of DLBCL, and, if confirmed, could in the future contribute to more effective targeting of CNS prophylactic therapies. / Tiivistelmä Lymfoomat ovat yli 70 erilaisen maligniteetin muodostama ryhmä imukudoksen syöpiä, ja diffuusi suurisoluinen B-solulymfooma (engl. DLBCL) on yleisin lymfoomatyyppi. Yli 70 prosenttia DLBCL-potilaista pystytään parantamaan nykyaikaisilla hoidoilla, mutta osa potilaista kuolee edelleen tautiin. Nämä potilaat tarvitsisivat tehokkaampia hoitoja vakavien haittavaikutusten riskistä huolimatta, mutta huonon ennusteen potilaiden tunnistaminen etukäteen on vaikeaa. Tutkimuksen tavoitteena oli löytää biologisia tekijöitä DLBCL-potilaiden hoitovasteen ja taudin keskushermostossa (engl. CNS) uusiutumisen ennustamiseen. Aineisto sisältää 263 lymfoomapotilasta. 205 potilaalla on DLBCL, ja 37:llä näistä primaari aivolymfooma (PCNSL). Immunohistokemiallisilla värjäyksillä määritettiin oksidatiivisen stressin markkereiden 8-hydroksideoksiguanosiinin (8-OHdG) ja nitrotyrosiinin, sekä antioksidanttientsyymien mangaanisuperoksidi-dismutaasin (MnSOD), tioredoksiinin (Trx) ja gammakysteiiniligaasin (GCL) ilmentyminen reaktiivista imukudosta sekä B-soluperäisiä lymfoomia edustavissa näytteissä. DLBCL-näytteistä määritettiin lisäksi solusykliä säätelevien proteiinien p16, p53, p21 ja p27 sekä kemokiinireseptorien CXCR4, CXCR5 ja CCR7 ilmentyminen. Lisäksi reaktiivista imukudosta, imusolmuke-DLBCL:aa, sekundaarista CNS-lymfoomaa ja PCNSL:aa edustavista näytteistä määritettiin immunoelektronimikroskooppisesti reseptorien CXCR4 ja CXCR5 sekä ligandien CXCL12 ja CXCL13 ilmentyminen. Tulosten mukaan voimakas nitrotyrosiini-, Trx- ja GCL-positiivisuus ovat yhteydessä huonoon ennusteeseen. Solusyklin säätelyhäiriön vaikeusastetta kuvaava ennusteellinen pisteytys jaotteli DLBCL-potilaat kolmeen ennusteelliseen ryhmään. Runsas sytoplasminen CXCR5-positiivisuus oli yhteydessä CNS-tautiin, kun taas tumapositiivisuus CXCR4:lle korreloi imusolmuketautiin. Tutkimustulokset kuvaavat DLBCL:n merkittävää biologista heterogeenisyyttä, mutta tulosten varmistamiseksi tarvitaan lisää tutkimuksia. Korkea antioksidanttiaktiivisuus ja solusyklin säätelyhäiriöiden kasautuminen erottivat huonoennusteisia potilasryhmiä, jotka voisivat hyötyä uudenlaisista hoidoista. Kemokiinireseptorien ilmentyminen vaikuttaisi olevan yhteydessä DLBCL:n CNS-hakuisuuteen, ja tulosten varmistuessa ekspressioprofiilien analysointia voitaisiin tulevaisuudessa hyödyntää ennaltaehkäisevien hoitojen tehokkaammassa kohdentamisessa.

Abstract Lymphomas are a heterogeneous group of malignancies that arise from lymphatic tissues. Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma sub-type. It is an aggressive malignancy with an increasing incidence. The prognosis of DLBCL has improved significantly, but problems also remain. The clinical significance of central nervous system (CNS) relapses has become increasingly important. As secondary CNSL (sCNSL) and primary CNS lymphoma (PCNSL) are known to have poor prognoses; the prevention of sCNSL is of crucial importance. Peripheral T-cell lymphomas (PTCL) are rare neoplasms and include several lymphoma subtypes that possess complex and also overlapping morphological and immunophenotypic characteristics. The identification of different entities has improved, but the biological knowledge remains scarce when compared to DLBCL. The optimal treatment schemas for PTCLs are still lacking and they have long been treated with the same therapies as B-cell lymphomas, mainly with suboptimal treatment results. The aim of this study was to identify poor prognostic markers in DLBCL and PTCLs and potential biological markers for the prediction of DLBCL CNS relapse. The study material included patients with systemic DLBCL without CNS affision (sDLBCL), sCNSL, PCNSL and PTCLs. The expression of epithelial-mesenchymal transition (EMT) transcription factors (TFs), chemokines and their receptors and adhesion-, migration- and inflammatory responses-associated molecules were studied by means of immunohistochemistry. IEM was used to verify the specific subcellular location of the studied molecules. GEP was performed on 12 PTCL samples in order to compare the poor prognosis group with the good prognosis group and on one sDLBCL and one sCNSL sample from the time of primary diagnosis. The EMT TFs were found to be expressed in both DLBCL and PTCLs, where they ultimately proved to have prognostic relevance as well. In PTCLs, these TFs were able to delineate a disease group with a specific gene-expression profile. CXCR4, CXCR5, ITGA10, PTEN and CD44 were found to be differently expressed between DLBCL cases with CNS affision when compared to those without CNS disease. These molecules seem to play a role in the development of CNS relapse and hopefully, if further verified, will lead towards the identification of biological markers for CNS relapse prediction. / Tiivistelmä Lymfoomat ovat heterogeeninen ryhmä imukudossyöpiä, joista diffuusi suurisoluinen B-solulymfooma (DLBCL) on yleisin alatyyppi. Se on aggressiivinen maligniteetti, jonka insidenssi on noussut viime vuosina. DLBCL potilaiden ennuste on parantunut merkittävästi, mutta yhä osa potilaista menehtyy tautiinsa. DLBCL:n keskushermostorelapsin kliininen merkitys on tänä päivänä aiempaa suurempi. Sekundaarisen keskushermostolymfooman (sCNSL) ja primaarin aivolymfooman (PCNSL) ennusteet ovat nykyhoidoilla huonoja, joten keskushermostorelapsin ennaltaehkäiseminen on tärkeää. Perifeeriset T-solulymfoomat (PTCLs) ovat ryhmä harvinaisia neoplasioita, joka sisältää useita eri alatyyppejä, joiden morfologiset ja immunofenotyyppiset ominaisuudet ovat monimuotoisia ja osin päällekkäisiä. Eri entiteettien indentifiointi on parantunut, mutta PTCL:ien biologinen tietämys on yhä DLBCL:aa heikompaa. PTCL:ien optimaalinen hoito ei ole selvillä ja tätä tautiryhmää on pitkään hoidettu samoilla hoidoilla kuin DLBCL:aa, mutta huonommilla hoitotuloksilla. Tutkimuksen tavoitteena oli löytää huonon ennusteen markkereita, joilla myös pystyttäisiin ennustamaan DLBCL:n keskushermostorelapsia. Aineisto koostui DLBCL, sCNSL, PCNSL ja PTCL näytteistä. Immunohistokemiallisilla värjäyksillä tutkittiin epiteliaalis-mesenkymaalisen transition (EMT) transkriptiotekijöitä (TF), kemokiinireseptoreita sekä adheesioon-, migraatioon ja inflammaatioon assosioituja molekyylejä. Immunoelektronimikroskopialla varmennettiin molekyylien lokalisaatio soluissa. Geeniekspressioprofiloinnilla (GEP) verrattiin kahdentoista hyvän ja huonon ennusteen ryhmään kuuluvan PTCL näytteen välisiä geeniekspressioeroja sekä kahden DLBCL potilaan näytteitä, joista toiselle kehittyi keskushermostorelapsi. EMT TF:ien ekspressiota nähtiin DLBCL ja PTCL näytteissä, joissa niillä myös todettiin olevan ennusteellista merkitystä. PTCL:ssa TF:t pystyivät erottelemaan tautiryhmän, jolla oli oma spesifinen geeniekspressioprofiilinsa. CXCR4, CXCR5, ITGA10, PTEN ja CD44 ekspressio oli erilaista systeemisissä DLBCL tapauksissa verrattuna sCNSL tapauksiin. Edellä mainituilla molekyyleillä näyttää olevan oma roolinsa keskushermostotaudin kehittymisessä ja jos nämä tulokset pystytään vahvistamaan tulevissa tutkimuksissa, johtavat ne toivottavasti kohti keskushermostorelapsiriskin tarkempaa tunnistamista.

High grade aggressive lymphomas have high mortality. By their nature, more than 40% of patients die from these diseases even with the improved treatment strategies currently available for oncology patients. The characteristic feature is that they are functionally heterogeneous and therefore have different biological and molecular signatures which make it difficult for all groups to respond to same line of treatment. Based on the above, I set out to look at the impact of viral and cellular gene products on these groups of diseases: In chapter 3 I developed monoclonal antibodies against HERV‐K10. I subsequently investigated their expressions in aggressive lymphomas including Diffuse Large B‐cell lymphoma, Hodgkin’s lymphoma and Primary CNS lymphomas. I showed HERV‐K10 is expressed in cell lines of aggressive lymphomas, but not in paraffin‐embedded tissues. In chapter 4 I showed that the expression of ATM using immune‐histochemistry techniques in aggressive lymphomas does offer a guide to prognosis and treatment. Nearly 30% of Diffuse Large B‐cell lymphomas express ATM, 55% of Hodgkin’s lymphomas and more than 80% of Primary CNS lymphomas. I also showed there is a correlation of ATM expression and EBV‐driven aggressive lymphomas and that this has a poor prognostic significance. Chapter 5 analysed the results obtained by generating, validating and evaluating data base of DLBCL and PCNSL from a retrospective cohort over a 17‐year period. The results confirmed that prognostic indicators including ATM, S1PR2, Autotaxin and EBV using immuno‐histochemistry techniques help with categorising aggressive lymphomas into different prognostic groups and does influence future management. In summary, my results showed there is a critical place for immuno‐histochemistry techniques in convincingly helping understand the expressions of viral and cellular gene products in aggressive lymphomas and in contributing positively to their management.

Purpose: To investigate if apparent diffusion coefficient (ADC) values within primary central nervous system lymphoma correlate with cellularity and proliferative activity in corresponding histological samples. Materials and Methods: Echo-planar diffusion-weighted magnetic resonance images obtained from 21 patients with primary central nervous system lymphoma were reviewed retrospectively. Regions of interest were drawn on ADC maps corresponding to the contrast enhancing parts of the tumors. Biopsies from all 21 patients were histologically analyzed. Nuclei count, total nuclei area and average nuclei area were measured. The proliferation index was estimated as Ki-67 positive nuclei divided by total number of nuclei. Correlations of ADC values and histopathologic parameters were determined statistically. Results: Ki-67 staining revealed a statistically significant correlation with ADCmin (r = -0.454, p = 0.038), ADCmean (r = -0.546, p = 0.010) and ADCmax (r = -0.515, p = 0.017). Furthermore, ADCmean correlated in a statistically significant manner with total nucleic area (r = -0.500, p = 0.021). Conclusion: Low ADCmin, ADCmean and ADCmax values reflect a high proliferative activity of primary cental nervous system lymphoma. Low ADCmean values—in concordance with several previously published studies—indicate an increased cellularity within the tumor.

Diffuse large B cell lymphoma (DLBCL) is the most common non-Hodgkin’s lymphoma. Patients who fail conventional therapy (~50%) have a poor prognosis and few treatment options. It is essential to understand the underlying biological processes, the progression of the disease, and utilize this information to develop new therapeutics. DLBCL patients with high C-MYC expression have a poor prognosis and new therapeutics for these patients are needed. This thesis describes work testing the hypothesis that JQ1, which can indirectly inhibit C-MYC in some tumors, can be used as an effective treatment for DLBCL. Some tumors have an unknown mechanism causing high C-MYC expression, leading me to investigate the underlying mechanisms. YY1 is a transcriptional regulator of c- Myc and has been implicated in DLBCL and as a potential regulator of the germinal center (GC) reaction. DLBCL arises from GC cells or post-GC cells. I tested the hypothesis that YY1 regulates the GC reaction. SMURF2 is an E3-ubiquitin ligase for YY1 and a tumor suppressor for DLBCL. I was interested in examining the mechanism underlying the suppression of DLBCL by SMURF2 leading to the hypothesis that SMURF2 regulates the GC. This thesis shows JQ1 leads to cell death and cellular senescence in human DLBCL cells. I conclude that BRD4 inhibition by JQ1 or derivatives could provide a new therapeutic avenue for DLBCL patients. I also show loss of YY1 perturbs the GC by decreasing the dark zone and increasing apoptosis. Finally I show modulation of SMURF2 does not affect the GC, suggesting SMURF2 utilizes a different mechanism to act as a tumor suppressor and may not modulate YY1 in the context of the GC.

Bakgrund: Diffust storcelligt B-cellslymfom (DLBCL) är en högmalign cancersjukdom som drabbar B-lymfocyterna, en typ av vita blodkroppar. Incidensen i Sverige är omkring 600 fall per år. Utan effektiv behandling sker sjukdomsprogressionen med ett snabbt förlopp. Under de senaste två årtiondena har behandlingarna som använts i första och andra linjen kunnat bota 60–70% av patienterna med DLBCL. För patienterna som inte svarat på standardbehandlingarna är prognosen mycket allvarlig. YESCARTA är ett nytt genterapiläkemedel som består av patientens egna T-celler modifierade med en chimär antigen receptor (CAR). När CAR T-cellen binder till B-lymfocyter frisätts inflammatoriska mediatorer som orsakar celldöd av både normala och tumöromvandlade B-lymfocyter. Syfte: Hur påverkar behandling med YESCARTA den totala överlevnaden (OS), responsfrekvensen (ORR), progressionsfri överlevnad (PFS), händelsefri överlevnad (EFS), responsduration (DOR), samt komplett och partiell respons (CR & PR) hos patienter med diffust storcelligt B-cellslymfom (DLBCL)? Detta arbete syftar till att systematiskt sammanställa och utvärdera befintlig vetenskaplig litteratur om YESCARTA för att besvara denna frågeställning. Metod: Litteratursökningen utfördes i PubMed. Samtliga MeSH-termer för läkemedlet YESCARTA konsoliderades och filter för observation- och kliniska studier applicerades. Sökningen genererade 34 artiklar, varav 10 kunde inkluderas. Exklusion skedde huvudsakligen av studier som undersökt annat än den terapeutiska effekten av YESCARTA. Resultat: Majoriteten av studierna var av typen fas II. En fas III-studie med varianter i uppföljningstid och undergruppsanalys inkluderades. YESCARTA förefaller vara överlägsen standardbehandling i alla utfallsmått. Resultatet är mest robust för ORR, EFS och PFS. Cirka fyra av fem patienter kan förväntas uppnå remission efter behandling med YESCARTA. Effektfördelen av YESCARTA i OS och DOR är osäker med avseende på statistisk signifikans. Slutsats: För särskilt utvalda patienter med DLBCL är YESCARTA ett effektivt behandlingsalternativ.

INTRODUÇÃO: O linfoma difuso de grandes células B, sem outras especificaçoes (LDGCB, SOE) é uma neoplasia agressiva caracterizada pela heterogeneidade morfológica, imunofenotípica e molecular, porém o atual tratamento padrão utilizando imunoquimioterapia (R-CHOP) não considera tal diversidade. Há percentual significativo de pacientes que são refratários à terapia de primeira linha e alguns que apresentam recidiva precoce ou tardia, os quais representam as vítimas desta doença. O estudo imuno-histoquímico (IHQ), que é um método simples e universalmente disponível, vem sendo utilizado para reconhecer a diversidade biológica do LDGCB, SOE, identificando biomarcadores e subgrupos distintos da doença, que poderiam predizer a resposta terapêutica ao tratamento padrão e apontar possíveis candidatos a novas estratégias terapêuticas. OBJETIVOS: Este estudo avalia o valor prognóstico de cinco algoritmos para classificação do LDGCB segundo a célula de origem (COO) e da expressão de três biomarcadores (BCL2, CD30 e MYC) tendo como endpoint a sobrevida global. MÉTODOS: Foi realizado estudo retrospectivo com setenta e nove pacientes com LDGCB,SOE de novo tratados com imunoquimioterapia padrão, estadiados e acompanhados protocolarmente. Os casos foram classificados como subgrupo célula B centrogerminativa símile (GCB) ou como subgrupo célula B não-centrogerminativa símile (NGCB), de acordo com três algoritmos IHQ (Hans, Choi, e Visco-Young) pareados com estudo do perfil de expressão gênica (PEG) e dois algoritmos IHQ não-PEG pareados (Muris e Nyman). Foi estimado o valor prognóstico destes algoritmos e também avaliado a concordância entre eles. O valor prognóstico da expressão do BCL2, CD30 e MYC utilizando IHQ também foi analisado. RESULTADOS: Os algoritmos IHQ PEG pareados revelaram maior concordância entre si, porém nenhum deles revelou força prognóstica. A expressão do CD30 mostrou tendência a melhor prognóstico, porém a expressão de BCL2 e MYC avaliados isoladamente não revelaram impacto prognóstico. Contudo, a coexpressão do BCL2 e MYC, denominado como fenótipo linfoma duplo-expressor (LDE), revelou-se importante marcador prognóstico desfavorável. Foram identificados três subgrupos de risco baseado no fenótipo LDE e o Índice Prognóstico Internacional (IPI). CONCLUSÃO: Em pacientes com LDGCB, SOE de novo tratados com esquema terapêutico padrão, a pesquisa da expressão do fenótipo LDE é mais relevante do ponto vista prognóstico que a classificação em subgrupo GCB ou NGCB. Além disso, a expressão do CD30 pode ser relevante tanto para identificar subgrupo com tendência a melhor prognóstico como para identificar possíveis candidatos a nova terapia alvo / BACKGROUND: Diffuse large B-cell lymphoma, not otherwise specified (DLBCL, NOS) is an aggressive neoplasm characterized by morphological, phenotypic and molecular heterogeneity, but the current standard therapy using immunochemotherapy (R-CHOP) does not consider such diversity. There is a significant percentage of patients who are refractory to first-line therapy and those with early or late recurrence, whose represent the victims of this disease. Immunohistochemistry (IHC), a simple and universally available method, has been used to recognize the biological diversity of DLBCL, NOS, to identify biomarkers and distinct subgroups of the disease, which would predict the therapeutic response to standard treatment and point possible candidates for novel therapeutic strategies. OBJECTIVES: The current study was conducted to evaluate the prognostic value from five algorithms for classification of DLBCL based on cell of origin (COO) and the expression of three biomarkers (BCL2, CD30 and MYC) with overall survival (OS) as an endpoint. METHODS: We retrospectively evaluated seventy nine patients with de novo DLBCL, NOS treated with R-CHOP-like immunochemotherapy. The cases were assigned as germinal center B-cell like (GCB) or non-GCB subgroup (NGCB) according to five different IHC algorithms, including three algorithms based on gene expressing profile study (GEP), proposed by Hans, Choi, and Visco-Young, and two non-GEP based algoritms proposed by Muris, and Nyman. We evaluated their prognostic relevance and the concordance between these algorithms. The prognostic power of BCL2, CD30 and MYC expression were also assessed by IHC. RESULTS: None of the profiles assessed by IHC algorithms was able to predict overall survival (OS). The positive expression of CD30 showed a trend toward a better outcome. Neither the positive expression of BCL2 nor the positive expression of MYC were associated with outcome. However, the double-expressor lymphoma phenotype (DEL), represented by the concurrent expression of MYC and BCL2, exhibited a negative prognostic impact. Three different risk subgroups were identified based on the DEL phenotype and the International Prognostic Index (IPI) score. CONCLUSIONS: These data suggest that the DEL, rather than the cell of origin classification based on IHC, is a better predictor of OS in patients with DLBCL treated with R-CHOP-like immunochemotherapy. Besides, the CD30 expression may be a useful prognostic marker and a possible therapeutic target