Orientador: Maura Moscardi Bacchi / Resumo: O linfoma de Hodgkin (LH) tem características clínicas e anátomo-patológicas distintas dos linfomas não-Hodgkin. Seu componente neoplásico, as células células de Hodgkin/Reed-Sternberg (H-RS), corresponde a cerca de 2% do tumor. As células H-RS apresentam imunofenótipo peculiar e sua origem ainda é objeto de estudo. O LH é classificado em LH clássico (que inclui os subtipos celularidade mista, esclerose nodular, depleção linfocítica e LH rico em linfócitos) e LH predominância linfocítica nodular. A associação do vírus de Epstein-Barr (EBV) com LH é conhecida e acredita-se que este vírus desempenha importante papel no desenvolvimento de parcela significativa dos casos de LH. No LH, o acúmulo de células reativas como linfócitos, plasmócitos, eosinófilos, histiócitos e fibroblastos ocorre em resposta a citocinas secretadas pelas células H-RS. A interleucina 10 (IL-10), importante citocina antiinflamatória da resposta imunitária, tem sido encontrada em grande quantidade em indivíduos com LH. É possível que essa elevada expressão de IL-10 esteja vinculada a determinados polimorfismos de nucleotídeos simples (SNP) na seqüência promotora do gene da IL-10, que podem se associar a características anátomoclínicas do LH. O presente trabalho avaliou a freqüência dos polimorfismos da IL-10 nas posições promotoras -1082, -819/-592 e estudou a correlação destes polimorfismos em LH com subtipos histológicos, infecção pelo EBV, faixa etária e, em alguns casos, estadiamento clínico da doença. Os resultados demonstram que os diferentes fenótipos para produção de IL-10, genótipos na posição -1082 e genótipos nas posições -592/-819 não têm relação com subtipos do LH e idade dos pacientes. Por outro lado, verificou-se que o genótipo GG na posição -1082 e a combinação de haplótipos GCC/GCC para alta... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Clinical and pathologic features of Hodgkin's lymphoma (HL) reflect an abnormal immune response, which is in part due to the elaboration of a variety of cytokines. It was reported that interleukin 10 (IL -10), which may be produced by the malignant Hodgkin/Reed-Sternberg (H-RS) cells, is an important prognostic factor in HL, and it could play a role in the pathogenesis of this neoplasm. It is well established that genetic factors affect protein expression and function. In this regard, polymorphisms in the promoter region of IL-10 gene may cause different phenotypes for IL- 10 synthesis and activity. Three dimorphic single nucleotide polymorphisms (SNPs) have been identified at positions -1082, -819 and -519 within the IL-10 promoter region (SNPs/IL-10). These polymorphisms are in close proximity to several transcription factors binding-sites, and may interfere with IL-10 gene transcription. The aim of this study was to evaluate whether is there a particular distribution of SNPs at positions -1082, -819 and -519 in the IL-10 gene promoter in patients with HL, as well as to access the differences in the SNPs/IL10 frequency regarding HL subtype, patient age, EBV infection status, and clinical staging of the disease. For these purposes, sixty-five cases of HL and fifty cases of reactive follicular lymphoid hyperplasia (RFLH) were evaluated for SNPs/IL-10 by polymerase chain reaction amplification plus restriction fragment length polymorphism analysis (PCR-RFLP). Compared to HL EBV-negative cases, in HL EBV-positive cases it was observed a significant increase in the frequency of GG genotype at position -1082 of IL-10 gene promoter region, which is associated to high level of IL -10 production. No differences were observed in the frequency of different SNPs/IL-10 concerning patient age, HL subtype, and clinical stage of the disease. These results... (Complete abstract, click electronic address below) / Mestre

No description available.

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / FundaÃÃo de Amparo à Pesquisa do Estado do Cearà / O Linfoma de Hodgkin à uma hemopatia linfÃide pode ocorrer em qualquer faixa etÃria; no entanto, à mais comum na idade adulta jovem, dos 15 aos 40 anos. O TP53 à um gene de 20kb de comprimento que possui 11 Ãxons situado no cromossomo 17 e codifica a proteÃna p53, uma proteÃna cuja principal funÃÃo està relacionada à preservaÃÃo da integridade do cÃdigo genÃtico, e, durante o ciclo celular faz verificaÃÃo quanto à eventual ocorrÃncia de uma mutaÃÃo na seqÃÃncia do cÃdigo genÃtico. Na presenÃa dessas mutaÃÃes, impede que esta cÃlula entre em processo de mitose e complete a divisÃo celular. A maioria dos cÃnceres apresenta mutaÃÃes pontuais na seqÃÃncia do TP53. A anÃlise dos padrÃes dessas mutaÃÃes à de grande valia uma vez que o conhecimento dessas mutaÃÃes leva a um melhor entendimento das funÃÃes dos vÃrios domÃnios da proteÃna p53 e seu envolvimento com o mecanismo de supressÃo tumoral, alÃm de permitir que essas mutaÃÃes sejam utilizadas como biomarcadores para desvendar a oncogÃnese humana. Na literatura vigente, poucos trabalhos abordam a identificaÃÃo dessas mutaÃÃes em relaÃÃo ao linfoma de Hodgkin â forma clÃssica. Dessa forma, este trabalho se propÃe a investigar quantitativa e qualitativamente os padrÃes de polimorfismos existentes nesta forma do linfoma de Hodgkin. A primeira etapa do nosso experimento constou da seleÃÃo de 42 casos de linfonodos diagnosticados como linfoma de Hodgkin entre os anos de 2000 e 2006, arquivados em blocos de parafina. Os casos foram selecionados de pacientes com diagnÃstico de linfoma de Hodgkin, sem predileÃÃo por idade, sexo ou raÃa. Em seguida, o DNA foi extraÃdo das amostras selecionadas para reaÃÃo de PCR, realizada para isolar e amplificar, o fragmento de 137pb correspondente ao Ãxon 8 do gene TP53, atravÃs de primers exclusivos desenhados para o experimento: PFw8 e PRv8. ApÃs a reaÃÃo, os produtos de PCR foram purificados para reaÃÃo de SeqÃenciamento de DNA, utilizando o seqÃenciador automÃtico de DNA da marca ABI Prism 3100 de 16 capilares (Applied Biosystems). A Ãltima etapa aconteceu em laboratÃrio de bioinformÃtica â dry lab, onde as seqÃÃncias de DNA obtidas foram analisadas qualitativa- e quantitativamente. Os processos de extraÃÃo do DNA genÃmico, amplificaÃÃo do Ãxon 8, purificaÃÃo do produto de PCR foram realizadas com sucesso em todas as amostras obtidas de material parafinizado. No seqÃenciamento do DNA parafinizado foi possÃvel determinar, com seguranÃa e confiabilidades previstas em parÃmetros convencionais, a seqÃÃncia de pelo menos 32 amostras; contudo, nÃo se obteve distinÃÃo suficiente dos picos de eletroferogramas para a determinaÃÃo de eventuais polimorfismos na regiÃo analisada. NÃo foi possÃvel portanto, verificar com margem de seguranÃa razoÃvel, a presenÃa ou ausÃncia de SNPs nas amostras seqÃenciadas. Houve, contudo, uma qualificaÃÃo e competÃncia laboratorial instalada localmente (em Fortaleza), a partir da experiÃncia desenvolvida com o esforÃo deste trabalho, para continuar a investigaÃÃo molecular em prol da determinaÃÃo, em futuro breve, da ocorrÃncia ou nÃo de SNPs no exon 8 do TP53 em linfoma de Hodgkin. / Hodgkin lymphoma is a hematololgic B neoplasm occurring at patients within any age, however more likely to affect those from 15 to 40 years-old. The TP53 gene is 20kb length gene with 11 exons that encodes the p53 protein, which main function is related to the conservation and integrity of the genetic code. Most cancers show point mutations in the TP53 sequence. The analysis of these mutations allows a better understanding of the function of the diverse domains of the protein and its relationship to tumor suppression. There is only a few data about TP53 polymorphisms and Hodgkin lymphoma. In this manner, in our study we try to detect polymorphisms within the codons 272, 273, 278, 282, 306 of the exon 8 of the TP53 gene in Hodgkin lymphoma. In our survey we analyzed 42 paraffin-embedded tissues from 2000 to 2006. These samples were prepared for DNA extraction and PCR isolation and amplification of the 137bp fragment of the exon 8 of the TP53 gene, using exclusive primers specially designed to our experiment: PFw8 e PRv8. After PCR amplification, the products of the reaction were purified to the Sequencing reaction. The last part of the experiment encoded the bioinformatics analysis of sequences. DNA extraction and PCR amplification were successfully obtained in our study in all the samples. However, the DNA sequencing was only obtained in 32 samples, but there was no characteristic electropherogram of the analyzed region of the gene. Therefore, it was not possible to determine the presence or absence of SNPs in the TP53 exon 8.

The efficacy of therapeutic modalities for non-Hodgkin’s lymphoma have been tested and improved throughout the 19th century through various series of drug trials aimed at eliminating cellular malignancies, first through chemotherapy treatment, and more recently through immunotherapy. While to an extent successful in eliminating cancerous lesions and affected cells, chemotherapy treatments have shown to influence the induction of new malignancies, through genetic mutation, as well as unwanted toxic effects of systemic poisoning. The purpose of this thesis is to compare treatment methods in terms of their biomolecular activity, precision of intended results, and possible drawbacks, as well as their application to specific populations of Non-Hodgkin lymphoma diagnoses, including Follicular and Diffuse Large B-Cell lymphomas. In the following sections on contributing factors specific to Diffuse Large B-Cell lymphomas and Follicular lymphoma, elements of disease prognosis will be analyzed from a molecular and clinical point of view. This includes a focus on the impact of genetic mutation, the immunohistochemical evidence these changes present, as well as the variances in immune cell functionality, and finally a description of symptoms with direction to specific underlying causes. An analysis of standard of care chemotherapy, and monoclonal antibody treatments will then be provided for each occurrence. The second segment will discuss novel techniques being developed for the treatment of lymphoma including but not limited to new monoclonal antibodies, synthetic lethality modulation, inhibition of selected chemokine receptors, DNA vector immunization for production of internal host antibodies, concepts of cell mediated bispecific antibody induced destruction, and new generations of Immunomodulatory drugs. With the recent development of cost effective sequencing technology, included is a discussion of the shift towards personalized medicine treatments, targeting appropriate phenotypic specific populations for optimal results, as it relates to therapies for Diffuse Large B-Cell lymphoma and Follicular lymphoma.

Le lymphome de Hodgkin est caractérisé, d’un point de vue histologique, par la présence de rares cellules tumorales nommées cellules de Reed et Sternberg, au sein d’un infiltrat cellulaire polymorphe, inflammatoire et réactionnel. Cette dernière résulte de la transformation tumorale de cellules lymphocytaires B qui acquièrent des propriétés d’échappement au système immun, de prolifération, de résistance à l’apoptose et une instabilité chromosomique. Néanmoins, la rareté des cellules tumorales, impliquant des problèmes techniques mais aussi de caractérisation des évènements primaires dans l’initiation de cette instabilité chromosomique, a été bien débattue dans la littérature. Mais les mécanismes impliqués dans l’instabilité chromosomique dans le lymphome de Hodgkin demeurent obscurs.La première partie de cette thèse a été consacrée à l’étude des mécanismes impliqués dans l’instabilité génomique du lymphome de Hodgkin via l’instabilité des microsatellites et l’instabilité chromosomique en utilisant 7 lignées de lymphome de Hodgkin. Nous avons montré pour la première fois l’implication des microsatellites dans l’instabilité génomique des lymphomes de Hodgkin (MSI-H (microsatellite instability-high) dans 3/7 lignées). De plus, nous avons montré que deux mécanismes favorisent l’émergence d’une instabilité chromosomique : le premier implique une instabilité télomérique qui est présente essentiellement dans les petites cellules tumorales induisant la formation des chromosomes dicentriques, des amplifications des gènes (Jak2 comme exemple) et des arrangements chromosomiques complexes. Le deuxième mécanisme est lié essentiellement à un défaut de réparation des cassures double-brin avec l’apparition des chromosomes dicentriques sporadiques et une fréquence importante des micronoyaux avec la formation des ponts anaphasiques.La deuxième partie de cette thèse a été consacrée à l’étude des mécanismes de maintenance des télomères dans les ganglions tumoraux du lymphome de Hodgkin (50 patients) comme dans les lignées tumorales. Nous avons montré qu’il existe une cohabitation entre les deux mécanismes importants de maintenance des télomères, l’activation de la télomérase d’une part et le mécanisme ALT (alternative lengthening of telomeres) d’autre part. Nous avons identifié la présence de petites cellules dans les ganglions hodgkiniens comme dans les lignées tumorales avec une forte activité de la télomérase par contre la cellule de Reed Sternberg est caractérisée par un profil ALT avec la présence des corps PML et une très faible activité de télomérase. La fréquence des cellules télomérase ou ALT varie d’un ganglion à un autre et d’une lignée à une autre. Un drastique raccourcissement télomérique a été observé dans les cellules exprimant la télomérase. Pour les cellules ALT, une grande hétérogénéité de la taille des télomères ainsi que la présence des chromosomes dicentriques sporadiques ont été détectées. Le suivi des patients à long terme pendant 10 ans, nous a permis d’établir une corrélation entre le profil ALT et la survenue de mortalités et de morbidités. De plus, l’étude de la radiosensibilité des lignées tumorales a montré que les lignées ALT sont plus résistantes que les lignées télomérases.La troisième partie de cette thèse a été consacrée à la validation de ces deux concepts d’instabilité chromosomique via l’instabilité télomérique et à celle des mécanismes de maintenance des télomères, en utilisant un modèle de lymphome de Hodgkin établi dans le laboratoire à partir de la lignée L428.Ces données auront une retombée clinique importante non seulement dans la compréhension et le traitement des lymphomes de Hodgkin mais aussi dans d’autres pathologies malignes. / The study of Hodgkin lymphoma (HL), with its unique microenvironment and long clinical outcomes, has provided exceptional insights into several areas of tumour biology. Findings in HL have not only improved our understanding of human carcinogenesis, but have also pioneered its translation into the clinic.Tumoral cells in HL, called Hodgkin and reed Sternberg cells (HRS), are characterized by a highly altered genomic landscape with a wide spectrum of genomic alterations, including somatic mutations, copy number alterations, complex chromosomal rearrangements, and aneuploidy. Moreover, the scarcity of HRS cells and the resulting technical problems of their in situ characterization, the primary cytogenetic events and the clonality of these possible aberrations has been a matter of debate in the past. As a consequence, a few accepted and established HL cell lines are widely used in the majority of research projects conducted worldwide.In this thesis, first we have first investigated the possible mechanisms underlying genomic instability including microsatellite and chromosomal instability in HL cell lines. We provide the first evidence that the genomic instability observed in HL is related to microsatellite instability and chromosomal instability related to two major mechanisms: first, telomere fusion leading to dicentric chromosomes formation and breakage/fusion/bridge (B/F/B) cycles involving the repeated fusion and breakage of chromosomes following the loss of telomeres in small cells associated with the lower expression of TRF2, as well as an elevated copy number of the Jak2 gene and the presence of nucleoplasmic bridges containing telomere and centromere sequences. The second mechanism is related to defective DNA repair via non homologous end-joining (NHEJ) repair with the presence of nucleoplasmic bridges without telomere or centromere sequences, accompanied by the micronucleus without centromere sequences and a higher frequency of sporadic dicentric chromosomes.The second part of this thesis has focused on investigating telomere maintenance mechanisms (TMMs) not only in HL cell lines but also in lymph nodes of HL patients. A telomerase-independent mechanism for TMM in HL has been proposed in the absence of detectable telomerase activity (TA) in some cases. The major finding of this work has been the demonstration of the presence of both telomerase and ALT mechanism in lymph nodes of HL patients as well as in HL cell lines. We have identified a subset of tumors with some small cells expressing telomerase and Reed Sternberg cells containing ALT-associated PML bodies. A significant correlation was observed between telomere length and TMMs. Drastic telomere shortening was observed in cells with telomerase expression and elevated heterogeneity of telomere length was found in ALT profile cells. Interestingly, complex chromosomal rearrangements, included sporadic dicentric formation, were observed in ALT profile cell lines. Interestingly, the relationship between TMMs and all-cause mortality and morbidity during 10 years of follow-up of HL patients using cox proportion hazard models demonstrated a poor clinical outcome for HL patients exhibiting primarily ALT mechanisms. Similarly, higher radiation sensitivity was observed for cell lines with high telomerase activity compared to cell lines with the ALT profile.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2009. / Härtill 4 uppsatser.

Evidence that radiation-related cardiovascular disease and second primary cancers can occur in cancer survivors following radiation therapy (RT) has emerged from several independent sources. Cardiotoxicity and second cancers are of particular concern for patients with good prognosis, such as those with Hodgkin lymphoma (HL). HL patients are among the youngest to receive RT, which means that those who are cured of their cancer have decades-long natural life-expectancies during which treatment-related long-term toxicities may cause years of excess morbidity or premature mortality. A considerable amount of research has been conducted to investigate the risk of radiation-related cardiotoxicity and second cancers. However, there are still substantial gaps in knowledge. It is therefore important to improve our understanding regarding these risks and develop treatment approaches and survivorship care to minimise their impact on patients' quality of life. In this thesis, I have investigated the risk of congestive heart failure (CHF) in a cohort of 2619 HL survivors and presented, for the first time, dose-response relationships for risk of CHF versus cardiac radiation doses. I also validated the radiation dosimetry method used to estimate the cardiac doses in this study as well as for other reconstruction methods, versus a gold standard based on the patients' own computed tomography scans. Additionally, I investigated what effect the dose reconstruction errors had on the dose-response relationships. I then focused on modern RT methods and specifically on proton RT. Based on published dose-response relationships (including that developed in this thesis) I predicted cardiovascular and second cancer risks for patients treated with advanced RT. This thesis has provided new knowledge in the study of late effects in HL patients who were treated decades ago as well as for patients treated more recently with advanced RT methods. The results here can be used to facilitate progress towards personalised RT in terms of choosing the appropriate RT method by integrating individualised risk prediction in advanced RT treatment planning. The research here provides the basis for further work towards evidence-based case selection for HL patients for the first NHS proton therapy centres in the UK, opening in 2018-2021.

Το Depreotide είναι ένα νέο συνθετικό δεκαπεπτίδιο ανάλογο της σωματοστατίνης, που επισημαίνεται με 99m-Τεχνήτιο (Tc-99m), το οποίο έχει την ικανότητα να συνδέεται με τους υποτύπους 2,3 και 5 των υποδοχέων της σωματοστατίνης, όπως και το οκτρεοτίδιο, παρουσιάζει όμως μερικές διαφορές και πλεονεκτήματα: υψηλότερη συγγένεια για τον υποδοχέα 3 της σωματοστατίνης, ανώτερη ποιότητα απεικόνισης, δεδομένου ότι πρόκειται για τεχνητιο-παράγωγο, πρωτόκολλο μιας ημέρας, μικρότερη ακτινοβόληση του ασθενή και άμεση διαθεσι-μότητα του ρ/φ στο εργαστήριο σε καθημερινή βάση. Στην παρούσα διατριβή μελετάται η διαγνωστική και προγνωστική αξία της απεικόνισης με 99mTc-depreotide σε μια σειρά ασθενών με διάφορους υπότυπους λεμφώματος, πριν, κατά την διάρκεια και μετά την θεραπεία. Τα ευρήματα συγκρίθηκαν ευθέως με αυτά του Ga-67, ενώ για την σωστή εξαγωγή συμπερασμάτων, μελετήθηκαν και χρησιμποποιήθηκαν ως σημείο αναφοράς, τόσο οι συμβατικές μέθοδοι σταδιοποίησης, τα ιστολογικά ευρήματα, όσο και η παρακολούθηση της πορείας των ασθενών. Συνολικά μελετήθηκαν 106 ασθενείς, εκ των οποίων οι 47 έπασχαν από νόσο Hodgkin (HL) και οι 59 από διάφορους ιστολογικούς τύπους μη Hodgkin λεμφώματος (NHL). Οι ασθενείς αυτοί παραπέμφθηκαν στο Εργαστήριο της Πυρηνικής Ιατρικής για να απεικονιστούν με Ga-67 στα πλαίσια διερεύνησης λεμφώματος και υποβλήθηκαν επίσης σε απεικόνιση με 99mTc-depreotide. Αρχικά έλαβε χώρα ολόσωμη στατική σπινθηρογραφική απεικόνιση (WB acquisition) και στην συνέχεια τομογραφική μελέτη σε συνδιασμό με αξονική τομογραφία χαμηλής διακριτικής ικανότητας (SPECT/low resolution CT imaging). Έγιναν συνολικά 142 ραδιοϊσοτοπικές μελέτες σε διάφορες φάσεις της νόσου, ενώ η σύγκριση των ευρημάτων έγινε τόσο ποιοτικά (οπτικά) όσο και ημιποσοτικά (semi-quantitatively). Στα περισσότερα περιστατικά με νόσο Hodgkin, σε αυτά με μετρίου και χαμηλού βαθμού κακοήθειας Β-NHL όσο και στα Τ-NHL, το 99mTc-depreotide ανίχνευσε περισσότερες θέσεις νόσου ή/και εμφάνισε υψηλότερο βαθμό πρόσληψης (uptake) σε σχέση με το Ga-67. Δεν συνέβη όμως το ίδιο στα επιθετικά Β-NHL, στα οποία το Ga-67 ήταν ανώτερο. Υπήρχαν βεβαίως και περιπτώσεις στις οποίες παρατρήθηκαν διαφοροποίησεις σχετικά με τον παραπάνω κανόνα. Το 99mTc-depreotide κατά την αρχική σταδιοποίηση κατάφερε να ανιχνεύσει το 93.3% των προσβεβλημένων λεμφαδένων πάνω από το διάφραγμα, το 100% των βουβωνικών λεμφαδένων και όλες τις περιπτώσεις με σπληνική προσβολή από την νόσο. Βάσει των ευρημάτων του 99mTc-depreotide, οι ασθενείς που βρίσκονταν στα αρχικά στάδια της νόσου Hodgkin κατατάχθηκαν σε δυσμενέστερο στάδιο (upstaged) σε ποσοστό 32%. Όμως στην περίπτωση των ασθενών με προχωρημένο HL και NHL παρατηρήθηκε μετάπτωσή τους σε ευμενέστερο στάδιο(downstaged) εξαιτίας της μικρής ευαισθησίας του ρ/φ στην ανίχνευση παθολογικών κοιλιακών λεμφαδένων (22.7%), της προσβολής του ήπατος (45.5%) και της συμμετοχής του μυελού των οστών στην νόσο (36.4%). Στους ασθενείς που απεικονίστηκαν μετά την θεραπεία το 99mTc-depreotide εντόπισε το 94.7% των περιπτώσεων με ανθεκτική ή υπολειπόμενη νόσο. Η συνολική ειδικότητα του ρ/φ κυμάνθηκε στο 57.1%. Τα συχνότερα ψευδώς θετικά ευρήματα αφορούσαν σε περιστατικά με αντιδραστική υπερπλασία του θύμου αδένα, διάφορες φλεγμονώδεις εξεργασίες και περιπτώσεις με μη ειδική πρόσληψη του ρ/φ. Ο συνδιασμός των δύο ραδιοϊσοτοπικών εξετάσεων, του 99mTc-depreotide και του Ga-67, βελτίωσαν αισθητά την ειδικότητα (100%), ενώ βοήθησε να περιοριστεί ο αριθμός των ψευδώς θετικών περιστατικών. Συμπερασματικά, με εξαίρεση πιθανώς τις περιπτώσεις με νόσο Hodgkin σε πρώιμο στάδιο, to 99mTc-depreotide έχει περιορισμένη αξία στην αρχική σταδιοποίηση του λεμφώματος. Όμως σε ό,τι αφορά τα περιστατικά μετά θεραπεία φαίνεται ότι σαφώς μπορεί να προσφέρει στην ανίχνευση νόσου σε συγκεκριμένες ανατομικές περιοχές, ιδίως εάν πρόκειται για λεμφώματα μετρίου και χαμηλού βαθμού κακοήθειας. Ο συνδιασμός δε με Ga-67 δύναται να αυξήσει την ευαισθησία και την ειδικότητα της μελέτης. Τέλος, το 99mTc-depreotide θα μπορούσε να έχει ενεργό ρόλο στις περιοχές όπου το FDG-PET δεν είναι διαθέσιμο και ιδιαίτερα όταν πρόκειται για περιστατικά ορισμένων τύπων ηπίου βαθμού (indolent) λεμφώματος. / Previous studies have demonstrated the feasibility of targeting lymphoma lesions with somatostatin receptor binding agents, mainly with In-111-pentetreotide. However, Pentreotide has not been established as a useful radiopharmaceutical for lymphoma diagnosis, mainly due to non-uniform uptake in lymphoma lesions leading to variable sensitivity. In the present study, the potential value of the Tc-99m-depreotide scintigraphy in a series of patients with various lymphoma types, prior to and post-therapy is investigated. Depreotide was directly compared to Ga-67. We used contemporary hybrid imaging technology with both radioisotopic agents, namely single photon emission tomography coupled with low resolution computerized tomography (SPECT/CT). Methods: One-hundred and six patients, 47 with Hodgkin’s (HL) and 59 with various types of non-Hodgkin’s lymphoma (NHL) were imaged with both Tc-99m-depreotide and Ga-67 citrate. Planar whole-body and SPECT/CT images were obtained. A total of 142 examinations were undertaken at different phases of the disease. Depreotide and gallium findings were compared visually and semi-quantitatively, with reference to the results of conventional work-up and the patients’ follow-up data. Results: In most HL, intermediate- and low-grade B-cell, as well as in T-cell NHL, depreotide depicted more lesions than Ga-67 and/or exhibited higher tumor uptake. The opposite was true in aggressive B-cell NHL. However, there were notable exceptions in all lymphoma subtypes. During initial staging, 93.3% of affected lymph nodes above the diaphragm, 100% of inguinal nodes and all cases with splenic infiltration were detected by depreotide. On the basis of depreotide findings 32% of patients with early stage HL were upstaged. However, advanced HL and NHL cases were frequently downstaged, due to low sensitivity for abdominal lymph node (22.7%), liver (45.5%) and bone marrow involvement (36.4%). Post-therapy, depreotide detected 94.7% of cases with refractory disease or recurrence. Its overall specificity was moderate (57.1%). Rebound thymic hyperplasia, various inflammatory processes and sites of unspecific uptake were the commonest causes of false positive findings. Combination of depreotide and gallium enhanced sensitivity (100%), while various false positive results of either agent could be avoided. Conclusion: Except perhaps for early-stage HL, Tc-99m-depreotide as a stand-alone imaging modality has a limited value for the initial staging of lymphomas. Post-therapy, however, depreotide scintigraphy seems useful in the evaluation of certain anatomic areas, particularly in non-aggressive lymphoma types. Combination with Ga-67 potentially enhances sensitivity and specificity. If flurodeoxyglucose positron emission tomography (PET) is not available or in case of certain indolent lymphoma types, Tc-99m depreotide may have a role as an adjunct to conventional imaging procedures.

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.

Diffuse large B-cell lymphoma (DLBCL) is the most common form of non-Hodgkin lymphoma (NHL). The current standard of care is the combination of rituximab with cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP), but this only results in a 60% over-all 5-year survival rate, thus highlighting a need for new therapeutic approaches. Histone deacetylase inhibitors (HDACi) are novel therapeutics that is being clinically evaluated for combination therapy. Rational selection of companion therapeutics for HDACi is difficult due to their poorly understood, cell-type specific mechanisms of action. To understand these mechanisms better, we developed a pre-clinical model system of response to the HDACi belinostat. Using this model system, we identified two major responses. Resistance, consisting of a reversible G1 cell cycle arrest with little induction of apoptosis; or sensitivity, consisting of mitotic arrest and high levels of apoptosis. In this dissertation, we determine that the induction of G1 cell cycle arrest is due to the increased expression of cyclin dependent kinase inhibitors (CDKi) that bind to and inhibit the cyclin E/CDK2 complex thereby blocking the final repressive phosphorylation steps of Rb protein. Repression of transcriptional elongation blocked CDKi upregulation and prevented G1 cell cycle arrest in belinostat-resistant cells. Additionally, we identified that belinostat arrests sensitive cells prior to metaphase and belinostat-resistant cells slow-down in mitosis but complete the process prior to arresting in G1. The combination of belinostat with the microtubule-targeting agent, vincristine resulted in strong synergistic induction of apoptosis by targeting mitotic progression. Furthermore, this combination prevents polyploidy, a key mechanism of resistance to microtubule targeting agents. Finally, we utilized selective class one HDAC inhibitors to identify the individual contributions of HDACs in the eliciting the responses observed with belinostat treatment. HDAC1&2 inhibition recapitulated the belinostat-resistant phenotype of G1 cell cycle arrest with little apoptosis, in both belinostat-resistant and sensitive cell lines. HDAC3 inhibition resulted in the induction of DNA damage, increased S phase and the induction of apoptosis in belinostat-resistant cells. Belinostat-resistant cells did not have observable effects to HDAC3 inhibitor alone but when combined with vincristine had significantly increased G2/M population at early time points. This suggests that HDAC3 maintains roles in DNA replication and also in mitotic progression. HDAC3 inhibition combined with vincristine resulted in a significant increase in polyploidy, suggesting that HDAC3 might not regulate the expression of apoptotic regulating factors as belinostat does.