Análise citogenética e investigação molecular do gene RASSF1A em indivíduos com síndrome mielodisplásica

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Oral chronic graft-versus-host disease: analysis of dendritic cells subpopulations

The graft-versus-host disease is the major cause of morbidity and mortality in patients who have undergone hematopoietic stem cell transplantation. Aiming at contributing to the understanding of the role of myeloid and plasmacytoid dendritic cells, and natural killer cells in chronic graft-versus-host disease, we examined biopsies of jugal mucosa of 26 patients with acute myeloid leukemia who had undergone allogenic hematopoietic stem cell transplantation. Half of these patients developed oral chronic graft-versus-host disease. Microscopic sections were immunohistochemically stained for anti-CD1a, anti-CD123 and anti-CD56. We calculated the number of immunostained cells in the corium per square millimeter and applied the Mann-Whitney test. Results showed a statistically significant increase of myeloid dendritic cells (CD1a+; p=0,02) and natural killer cells (CD56; p=0,04) in patients with oral chronic graft-versus-host disease. CD123 immunostaining showed no statistical difference between groups. It was concluded that myeloid dendritic cells and natural killer cells participate in the development of oral chronic graft-versus-host disease.

Simultaneous Occurrence of Biphenotypic T Cell/Myeloid Lesions Involving t(12;13)(p13;q14) in a Pediatric Patient

This paper chronicles a 2-year-old girl who presented with acute leukemia/lymphoma syndrome of the T cell immuno-phenotype. At this time, the cytogenetic analysis of her bone marrow cells showed a reciprocal translocation between the short arm of chromosome 12 and the long arm of chromosome 13, t(12;13)(p13;q14). The immunophenotyping of bone marrow blast cells by flow cytometry revealed a population of cells positive for CD56, CD117, CD45, partial CD33, partial HLA-DR, CD13, CD7, CD2 and CD5. Therefore, a diagnosis of acute leukemia with a mixed T cell/myeloid phenotype was made. The patient had a poor response to classic T cell acute lymphocytic leukemia/lymphoma therapy; thus, her treatment was changed to a myeloid leukemia protocol, which produced a good response. She underwent a successful cord blood transplantation from an unrelated HLA partially matched donor. The coexistence of these two phenotypes prompts questions about the existence of clonal instability, which might influence the choice of therapy. The rarity of the t(12;13)(p13;q14) and the coexistence of T cell/myeloid markers suggest a nonrandom association. To the best of our knowledge, this is the first reported case in which a cell clone bearing a t(12;13)(p13;q14) translocation in a mixed T cell/myeloid lesion was detected. Copyright (C) 2012 S. Karger AG, Basel

Human telomere disease due to disruption of the CCAAT box of the TERC promoter

Mutations in the coding region of telomerase complex genes can result in accelerated telomere attrition and human disease. Manifestations of telomere disease include the bone marrow failure syndromes dyskeratosis congenita and aplastic anemia, acute myeloid leukemia, liver cirrhosis, and pulmonary fibrosis. Here, we describe a mutation in the CCAAT box (GCAAT) of the TERC gene promoter in a family in which multiple members had typical features of telomeropathy. The genetic alteration in this critical regulatory sequence resulted in reduced reporter gene activity and absent binding of transcription factor NF-Y, likely responsible for reduced TERC levels, decreased telomerase activity, and short telomeres. This is the first description of a pathogenic mutation in the highly con-served CCAAT box and the first instance of a mutation in the promoter region of TERC producing a telomeropathy. We propose that current mutation-screening strategies should include gene promoter regions for the diagnosis of telomere diseases. This clinical trial was registered at www.clinicaltrials.gov as #NCT00071045. (Blood. 2012;119(13):3060-3063)

BCL2A1a Over-Expression in Murine Hematopoietic Stem and Progenitor Cells Decreases Apoptosis and Results in Hematopoietic Transformation

We previously reported the development of a lethal myeloid sarcoma in a non-human primate model utilizing retroviral vectors to genetically modify hematopoietic stem and progenitor cells. This leukemia was characterized by insertion of the vector provirus into the BCL2A1 gene, with resultant BCL2A1 over-expression. There is little information on the role of this anti-apoptotic member of the BCL2 family in hematopoiesis or leukemia induction. Therefore we studied the impact of Bcl2a1a lentiviral over-expression on murine hematopoietic stem and progenitor cells. We demonstrated the anti-apoptotic function of this protein in hematopoietic cells, but did not detect any impact of Bcl2a1a on in vitro cell growth or cell cycle kinetics. In vivo, we showed a higher propensity of HSCs over-expressing Bcl2a1a to engraft and contribute to hematopoiesis. Mice over-expressing Bcl2a1a in the hematologic compartment eventually developed an aggressive malignant disease characterized as a leukemia/lymphoma of B-cell origin. Secondary transplants carried out to investigate the primitive origin of the disease revealed the leukemia was transplantable. Thus, Bcl2a1 should be considered as a protooncogene with a potential role in both lymphoid and myeloid leukemogenesis, and a concerning site for insertional activation by integrating retroviral vectors utilized in hematopoietic stem cell gene therapy.

Impact of complex NOTCH1 mutations on survival in paediatric T-cell leukaemia

Background: Molecular alterations occur frequently in T-ALL and the potential impact of those abnormalities on outcome is still controversial. The current study aimed to test whether NOTCH1 mutations and additional molecular abnormalities would impact T-ALL outcome in a series of 138 T-ALL paediatric cases. Methods: T-ALL subtypes, status of SIL-TAL1 fusion, ectopic expression of TLX3, and mutations in FBXW7, KRAS, PTEN and NOTCH1 were assessed as overall survival (OS) and event-free survival (EFS) prognostic factors. OS and EFS were determined using the Kaplan-Meier method and compared using the log-rank test. Results: The frequencies of mutations were 43.5% for NOTCH1, while FBXW7, KRAS and PTEN exhibited frequencies of 19.1%, 9.5% and 9.4%, respectively. In 78.3% of cases, the coexistence of NOTCH1 mutations and other molecular alterations was observed. In multivariate analysis no statistical association was revealed between NOTCH1 mutations and any other variable analyzed. The mean length of the follow-up was 68.4 months and the OS was 50.7%. SIL-TAL1 was identified as an adverse prognostic factor. NOTCH1 mutation status was not associated with outcome, while the presence of NOTCH1 complex mutations (indels) were associated with a longer overall survival (p = 0.031) than point mutations. Conclusion: NOTCH1 mutations alone or in combination with FBXW7 did not impact T-ALL prognosis. Nevertheless, complex NOTCH1 mutations appear to have a positive impact on OS and the SIL-TAL1 fusion was validated as a negative prognostic marker in our series of T-ALL.

Cytotoxicity of cashew flavonoids towards malignant cell lines

The leaves of the Cashew plant (Anacardium occidentale L.) are used by the folk medicine in South America and West Africa. This plant is rich in flavonoids, which are polyphenolic compounds widespread in plants, and that have diverse physiological effects. In a sub-acute toxicity assay it was found that an ethanolic extract of Cashew leaves elicited lymphopenia in rats. The extract was also found to be cytotoxic and to induce apoptosis in Jurkat (acute lymphoblastic leukemia) cells. The crude ethanolic extract was fractionated and resolved by HPLC. One of the four fractions obtained led to the isolation of the biflavonoid agasthisflavone. [H-3]-thymidine incorporation assays and flow cytometry analysis showed that the isolated compound displayed a high anti-proliferative effect in Jurkat cells with an IC50 of 2.4 mu g/ml (4.45 mu M). The effect of agathisflavone on the acute promyelocytic leukemia cell line HL60, Burkitt lymphoma Raji cells and Hep-2 laryngeal carcinoma cells was also tested. The two latter ones were only mildly affected by agathisflavone. It is also shown that agathisflavone induces apoptosis in Jurkat cells and it this proposed that this is the likely mechanism of agathisflavone specific cytotoxicity. (C) 2010 Elsevier GmbH. All rights reserved.

Age-adjusted recipient pretransplantation telomere length and treatment-related mortality after hematopoietic stem cell transplantation

Telomere attrition induces cell senescence and apoptosis. We hypothesized that age-adjusted pretransplantation telomere length might predict treatment-related mortality (TRM) after hematopoietic stem cell transplantation (HSCT). Between 2000 and 2005, 178 consecutive patients underwent HSCT from HLA-identical sibling donors after myeloablative conditioning regimens, mainly for hematologic malignancies (n = 153). Blood lymphocytes' telomere length was measured by real-time quantitative PCR before HSCT. Age-adjusted pretransplantation telomere lengths were analyzed for correlation with clinical outcomes. After age adjustment, patients' telomere-length distribution was similar among all 4 quartiles except for disease stage. There was no correlation between telomere length and engraftment, GVHD, or relapse. The overall survival was 62% at 5 years (95% confidence interval [CI], 54-70). After a median follow-up of 51 months (range, 1-121 months), 43 patients died because of TRM. The TRM rate inversely correlated with telomere length. TRM in patients in the first (lowest telomere length) quartile was significantly higher than in patients with longer telomeres (P = .017). In multivariate analysis, recipients' age (hazard ratio, 1.1; 95% CI, .0-1.1; P = .0001) and age-adjusted telomere length (hazard ratio, 0.4; 95% CI; 0.2-0.8; P = .01) were independently associated with TRM. In conclusion, age-adjusted recipients' telomere length is an independent biologic marker of TRM after HSCT. (Blood. 2012;120(16):3353-3359)

Espressione dell'Enzima ad Azione Immunoregolatoria Indoleamina 2,3-Diossigenasi nella Leucemia Mieloide Acuta dell'Adulto: correlazioni Clinico-Biologiche

L’enzima IDO interviene nella via di degradazione del triptofano, essenziale per la vita cellulare; l’iperespressione di IDO favorisce la creazione di un microambiente immunotollerante. Nelle LAM IDO è funzionalmente attivo nelle cellule blastiche e determina l’acquisizione di un fenotipo regolatorio da parte delle cellule T alloreattive; l’espressione della proteina aumenta in modo consensuale con l’evoluzione clinica della patologia. Scopo della Tesi è indagare l’esistenza di una correlazione tra l’iperespressione di IDO da parte delle cellule leucemiche, le caratteristiche di rischio alla diagnosi e l’outcome dei pazienti. Sono stati esaminati 45 pazienti adulti affetti da LAM afferiti all’Istituto di Ematologia di Bologna. I pazienti sono stati stratificati a seconda di: età di insorgenza della leucemia, secondarietà a Mielodisplasia o radio chemioterapia, iperleucocitosi, citogenetica, biologia molecolare (sono state valutate le alterazioni a carico dei geni FLT3 ed NPM). I pazienti sono stati analizzati per l’espressione del gene IDO mediante RT-PCR, seguita da Western Blot, allo scopo di stabilire la presenza di una proteina attiva; successivamente si è proceduto a verificare l’esistenza di una correlazione tra l’espressione di IDO e le caratteristiche di rischio alla diagnosi per identificare una relazione tra l’espressione del gene ed un subset di pazienti a prognosi favorevole o sfavorevole. Dei 45 pazienti adulti affetti da LAM il 28,9% è risultato negativo per l’espressione di IDO, mentre il rimanente 71,1% è risultato positivo ed è stato suddiviso in tre ulteriori categorie, in base ai livelli di espressione. I dati non sembrano al momento suggerire l’esistenza di una correlazione tra l’espressione di IDO e le caratteristiche di rischio alla diagnosi. Nel gruppo di pazienti ad elevata espressione di IDO si riscontra un rate di resistenza alla chemioterapia di induzione più elevato, con una quota di pazienti resistenti pari al 71,4%, contro il 23,1% nel gruppo di pazienti IDO-negativi.

Evaluation der Leukämie-Reaktivität von PRAME- und p53-spezifischen T-Zellen bei lymphohämatopoetischen Neoplasien im Rahmen einer adoptiven Immuntherapie

Der Transplantat-gegen-Leukämie (GVL) Effekt als immuntherapeutisches Mittel bei der allogenen hämatopoetischen Stammzell Transplantation (HSZT) ist hauptsächlich durch Spender Lymphozyten vermittelt, welche hämatopoetische Minor-Histokompatibilitäts Antigene bzw. Leukämie-assoziierte Antigene (z. B.: PRAME, p53) erkennen. Der adoptive Transfer von Leukämie-spezifischen T-Zellen kann den GVL-Effekt, ohne ein Auftreten einer Transplantat-gegen-Wirt Erkrankung (GVHD), steigern. Unter Verwendung von HLA-A2 und human CD8 transgenen Mäusen (CD8yCyA2Kb) konnten in dieser Arbeit PRAME spezifische CD8+ zytotoxischen T-Zellen generiert werden. Diese zytotoxischen CD8+ T-Zellen zeigten in Chromfreisetzungsuntersuchungen lytische Aktivität gegen eine Vielzahl von Zelllinien, die PRAME endogen prozessieren sowie gegen das spezifische PRAME-Peptid. Des Weiteren wurden die hier generierten T-Zellen auf ihre zytotoxische Aktivität gegen akute myeloische Leukämie Blasten hin untersucht, und diese Untersuchungen zeigten AML-Reaktivität der PRAME-spezifischen sowie der als Vergleich genutzten p53- und HLA-A2-spezifischen T-Zellen. Das Potenzial der PRAME-spezifischen ZTL die GVL-Immunität in vivo zu erhöhen ohne das Vorkommen einer GVHD wurde in einem Tumor-Protektions-Model unter der Nutzung von NOD/SCIDgcnull Mäusen untersucht. Die PRA100- bzw. p53-ZTL wurden adoptiv in NOD/SCIDgcnull Rezipienten transferiert und gleichzeitig wurden die Tiere mit PRAME-, oder p53-exprimierende Tumorzelllinien inokuliert. Die Reduktion des Tumorwachstums bestätigte die Spezifität der T-Zellen auch in vivo. In weiteren in vivo Experimenten wurden NOD/SCIDgcnull Mäuse mit AML-Blasten rekonstituiert. Durch die Applikation von nur CD34 positiven Zellen aus einer AML-Probe, oder einer CD56 depletierten Probe, konnten Rekonstitutionen in 95 % aller Versuche erfolgreich beendet werden. Wurde eine Rekonstitution mittels PCR- und FACS-Analysen diagnostiziert, so folgten mehrere Applikationen der PRAME- oder p53-spezifischen ZTL. In diesen Untersuchungen konnten wir in einem therapeutischen AML-in vivo-Modell zeigen, dass die in diesen Untersuchungen generierten/verwandten ZTL in der Lage sind AML-Blasten in vivo zu bekämpfen und so die leukämische Last der Tiere im Blut sowie in der Milz auf unter 1 % zu regulieren. Der prozentuale Anteil humaner AML Zellen im Knochenmark konnte deutlich gesenkt werden (< 10 %). Zusammenfassend sind die von uns generierten PRAME-spezifischen T-Zellen in der Lage, in vitro und auch in vivo, endogen prozessiertes Protein auf Zelllinien und AML-Blasten zu erkennen und zu lysieren. Auch die p53-ZTL, welche als eine weitere Antigen-spezifische ZTL-Population in vivo getestet wurden, zeigten GVL-Effekte. Die Kenntnis von Tumor- bzw. Leukämie assoziierten Antigenen und die daraus erwachsene Möglichkeit der Generierung krankheitsspezifischer ZTL bietet die Grundlage für eine spezifische Immuntherapie maligner Erkrankungen.

SMO inhibitor specifically targets the Hedgehog Pathway and reverts the drug-resistance of Leukemic Stem Cells

Abnormal Hedgehog signaling is associated with human malignancies. Smo, a key player of that signaling, is the most suitable target to inhibit this pathway. To this aim several molecules, antagonists of Smo, have been synthesized, and some of them have started the phase I in clinical trials. Our hospital participated to one of these studies which investigated the oral administration of a new selective inhibitor of Smo (SMOi). To evaluate ex vivo SMOi efficacy and to identify new potential clinical biomarkers of responsiveness, we separated bone marrow CD34+ cells from 5 acute myeloid leukemia (AML), 1 myelofibrosis (MF), 2 blastic phases chronic myeloid leukemia (CML) patients treated with SMOi by immunomagnetic separation, and we analysed their gene expression profile using Affimetrix HG-U133 Plus 2.0 platform. This analysis, showed differential expression after 28 days start of therapy (p-value ≤ 0.05) of 1,197 genes in CML patients and 589 genes in AML patients. This differential expression is related to Hedgehog pathway with a p-value = 0.003 in CML patients and with a p-value = 0.0002 in AML patients, suggesting that SMOi targets specifically this pathway. Among the genes differentially expressed we observed strong up-regulation of Gas1 and Kif27 genes, which may work as biomarkers of responsiveness of SMOi treatment in CML CD34+ cells whereas Hedgehog target genes (such as Smo, Gli1, Gli2, Gli3), Bcl2 and Abca2 were down-regulated, in both AML and CML CD34+ cells. It has been reported that Bcl-2 expression could be correlated with cancer therapy resistance and that Hedgehog signaling modulate ATP-binding (ABC) cassette transporters, whose expression has been correlated with chemoresistance. Moreover we confirmed that in vitro SMOi treatment targets Hedgehog pathway, down-regulate ABC transporters, Abcg2 and Abcb1 genes, and in combination with tyrosine kinase inhibitors (TKIs) could revert the chemoresistance mechanism in K562 TKIs-resistant cell line.

In vivo consequences of AML1-ETO fusion protein expression for hematopoiesis

The t(8;21) (q22;q22) translocation fusing the ETO (also known as MTG8) gene on human chromosome 8 with the AML1 (also called Runx1 or CBFα) gene on chromosome 21 is one of the most common genetic aberrations found in acute myeloid leukemia (AML). This chromosomal translocation occurs in 12 % of de novo AML cases and in up to 40 % of the AML-M2 subtype of the French-American-British classification. To date, the in vivo function of aberrant AML1-ETO fusion protein expression has been investigated by several groups. However, in these studies, controversial results were reported and some key issues remain unknown. Importantly, the consequences of aberrant AML1-ETO expression for self-renewing hematopoietic stem cells (HSCs), multipotent hematopoietic progenitors (MPPs) and lineage-restricted precursors are not known. rn The aim of this thesis was to develop a novel experimental AML1-ETO in vivo model that (i) overcomes the current lack of insight into the pre-leukemic condition of t(8;21)-associated AML, (ii) clarifies the in vivo consequences of AML1-ETO for HSCs, MPPs, progenitors and more mature blood cells and (iii) generates an improved mouse model suitable for mirroring the human condition. For this purpose, a conditional tet on/off mouse model expressing the AML1-ETO fusion protein from the ROSA26 (R26) locus was generated. rn Aberrant AML1-ETO activation in compound ROSA26/tetOAML1-ETO (R26/AE) mice caused high rates of mortality, an overall disruption of hematopoietic organs and a profound alteration of hematopoiesis. However, since the generalized activity of the R26 locus did not recapitulate the leukemic condition found in human patients, it was important to restrict AML1-ETO expression to blood cell lineages. Therefore, bone marrow cells from non-induced R26/AE mice were adoptively transplanted into sublethal irradiated RAG2-/- recipient mice. First signs of phenotypical differences between AML1-ETO-expressing and control mice were observed after eight to nine months of transgene induction. AML1-ETO-expressing mice showed profound changes in hematopoietic organs accompanied by manifest extramedullary hematopoiesis. In addition, a block in early erythropoiesis, B- and T-cell maturation was observed and granulopoiesis was significantly enhanced. Most interestingly, conditional activation of AML1-ETO in chimeric mice did not increase HSCs, MPPs, common lymphoid precursors (CLPs), common myeloid progenitors (CMPs) and megakaryocyte-erythrocyte progenitors (MEPs) but promoted the selective amplification of granulocyte-macrophage progenitors (GMPs). rn The results of this thesis provide clear experimental evidence how aberrant AML1-ETO modulates the developmental properties of normal hematopoiesis and establishes for the first time that AML1-ETO does not increase HSCs, MPPs and common lineage-restricted progenitor pools but specifically amplifies GMPs. The here presented mouse model not only clarifies the role of aberrant AML1-ETO for shaping hematopoietic development but in addition has strong implications for future therapeutic strategies and will be an excellent pre-clinical tool for developing and testing new approaches to treat and eventually cure AML.rn

Leucemia Acuta Mieloide e signaling purinergico: possibili sviluppi terapeutici

I nucleotidi trifosfato sono, dal punto di vista evoluzionistico, tra le molecole più antiche e conservate tra le specie. Oltre al ruolo che ricoprono nella sintesi degli acidi nucleici e nel metabolismo energetico della cellula, negli ultimi anni è emerso sempre di più il loro coinvolgimento nella regolazione di numerose funzioni cellulari. Questi importanti mediatori cellulari sono presenti nel microambiente e cambiamenti nella loro concentrazione extracellulare possono modulare la funzionalità cellulare. I nucleotidi trifosfato ATP e UTP, presenti nel microambiente midollare, sono dei potenti stimolatori dei progenitori emopoietici. Essi stimolano la proliferazione e l’attecchimento delle cellule staminali emopoietiche, così come la loro capacità migratoria, attraverso l’attivazione di specifici recettori di membrana, i recettori purinergici (P2R). In questo studio abbiamo dimostrato che ATP e UTP esercitano un effetto opposto sul compartimento staminale leucemico di leucemia acuta mieloide (LAM). Abbiamo dimostrato che le cellule leucemiche esprimono i recettori P2 funzionalmente attivi. Studi di microarray hanno evidenziato che, a differenza di ciò che avviene nelle CD34+, la stimolazione di cellule leucemiche con ATP induce la down-regolazione dei geni coinvolti nella proliferazione e nella migrazione, mentre up-regola geni inibitori del ciclo cellulare. Abbiamo poi confermato a livello funzionale, mediante test in vitro, gli effetti osservati a livello molecolare. Studi di inibizione farmacologica, ci hanno permesso di capire che l’attività inibitoria dell’ATP sulla proliferazione si esplica attraverso l’attivazione del recettore P2X7, mentre i sottotipi recettoriali P2 prevalentemente coinvolti nella regolazione della migrazione sono i recettori P2Y2 e P2Y4. Esperimenti di xenotrapianto, hanno evidenziato che l’esposizione ad ATP e UTP sia dei blasti leucemici sia delle cellule staminali leucemiche CD38-CD34+ diminuisce la loro capacità di homing e di engraftment in vivo. Inoltre, il trattamento farmacologico con ATP, di topi ai quali è stata indotta una leucemia umana, ha diminuito lo sviluppo della leucemia in vivo.

Untersuchungen zu Migration, Homing und Effektor-Funktionen humaner T-Lymphozyten in NOD.Cg-Prkdc scid Il2r gtm1Wjl, SzJ-Mäusen im Rahmen der allogenen hämatopoetischen Stammzelltransplantation

Die akute myeloische Leukämie (AML) zählt zu den aggressivsten neoplastischen Erkrankungenrnder Hämatopoese. Die Mehrheit der Patienten mit AML erreicht nach Induktions-rnChemotherapie den Zustand der kompletten Remission, jedoch erleiden mehr als die Hälfterndieser Patienten anschließend einen Rückfall und versterben an den Folgen der Erkrankungrn[1]. Die allogene hämatopoetische Stammzelltransplantation (engl.: hematopoietic stem cellrntransplantation, HSCT) stellt die einzig putativ kurative Behandlungsform für rezidierendernPatienten und solche mit schlechter Prognose dar. Jedoch birgt diese Form der Therapiernauch eine Vielzahl an Risiken. Insbesondere das Auftreten einer akuten Transplantat-gegen-rnWirt-Erkrankung (engl.: graft-versus-host disease, GvHD) stellt die Hauptursache für transplantationsassoziierternMortalität und Morbidität dar [2]. Die Depletion von alloreaktiven zytotoxischenrnT Lymphozyten (CTL) aus dem Transplantat ermöglicht zwar die Prävention derrnEntstehung einer GvH-Erkrankung, jedoch häufig unter gleichzeitigem Verlust des förderlichen,rnanti-leukämischen Transplantat-gegen-Leukämie-Effekts (engl.: graft-versus-leukemia,rnGvL) [3]. Um den GvL-Effekt unter Vermeidung einer GvH-Erkrankung zu erhalten, bietetrnsich der gezielte adoptive Transfer von Leukämie-spezifischen, nicht alloreaktiven CTL alsrnattraktive Strategie der Immuntherapie für AML-Patienten nach allogener HSCT an. In derrnvorliegenden Arbeit konnte erfolgreich ein prä-klinisches murines AML-Modell unter Einsatzrndes stark immundefizienten NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ- (NSG-) Mausstamms und primärenrnAML-Blasten durch die Optimierung bereits publizierter Protokolle etabliert werden.rnBei zehn von 17 transplantierten primären AML-Proben konnte ein erfolgreiches Engraftmentrnder humanen Zellen und eine Rekonstitution der humanen Neoplasie in den NSG-Mäusenrnerzielt werden. Die Engraftment-Rate betrug somit 58,82% und lag etwas unter dem aus derrnLiteratur bekannten Wert von 65-70% [4, 5]. Es ließen sich gut, intermediär und schlecht anwachsendernAML-Proben anhand der Engraftment-Stärke und -Reproduzierbarkeit voneinanderrnunterscheiden. Anhand der Analyse von für das Engraftment kritischer Parameter konnternein Zusammenhang zwischen Engraftment-Rate in der Maus und Flt3-Mutationsstatus sowiernFAB-Klassifikation des Patienten hergestellt und somit Angaben aus der Literatur bestätigtrnwerden. Für zwei Patienten-spezifische AML-Modelle, MZ580 und MZ308, konnten in vitrornerfolgreich AML-reaktive, über einzelne bzw. duale HLA-Diskrepanzen restringierte CTLPopulationenrngeneriert und über einen Zeitraum von bis zu 70 Tagen expandiert werden.rnDeren adoptiver Transfer in zuvor mit humanen AML-Blasten inokulierte NSG-Mäuse führternzu einer nahezu vollständigen Eradikation der AML-Blasten und Remission der Versuchstiere.rnAnhand unterschiedlich langer in vitro Kultur-Zeiträume konnte ein für die in vivo ausgeübtenrnEffektor-Funktionen optimaler Reifungszustand der CTL-Populationen von maximalrn28 Tagen bestimmt werden. Die kinetische Analyse der lytischen Aktivität in vivo deutete auf eine relativ schnelle Ausübung der Effektor-Funktionen durch die CTL-Populationen innerhalbrnvon zwei bis 24 Stunden nach adoptivem Transfer hin. Durch die Verwendung von inrnvitro generierten EBV-reaktiven CTL aus einem irrelevanten Spender konnte zudem die Spezifitätrnder in vivo ausgeübten Effektor-Funktionen nachgewiesen werden. Die ex vivo Re-rnIsolation adoptiv transferierter CTL und deren in vitro Analyse in einem IFNγ ELISpot wiesrneine konstante Reaktivität der Zellen ohne Induktion einer Xeno-Reaktivität nach. Die zurrnVerbesserung der Persistenz humaner CTL-Populationen eingesetzten autologen CD4+ TrnZellen zeigten nur im AML MZ308-System eine positive Wirkung. Generell konnte die Persistenzrnin vivo jedoch trotz initialer Substitution mit den Zytokinen IL-2 und IL-7 nicht über einenrnZeitraum von sieben Tagen hinaus aufrechterhalten werden.rnZur Untersuchung des Extravasations-Mechanismus humaner T Zellen über murines Endothelrnwurden sowohl Flusskammer- als auch Transwell-Studien durchgeführt, um die molekularenrnGrundlagen des Adhäsions- und Transmigrationsprozesses aufzuklären. Durch denrnparallelen Einsatz humaner und muriner T Zellen auf murinen Endothelzellen unter Zusatzrnfunktionsblockierender monoklonaler Antikörper konnte gezeigt werden, dass derrnExtravasations-Mechanismus beider Spezies auf Interaktionen homologer Adhäsionsmolekül-rnPaare, nämlich VLA-4–VCAM-1 und LFA-1–ICAM-1, beruht. Für einzelne Moleküle konntenrnin Abhängigkeit der eingesetzten Endothelzellen Unterschiede in der Funktionalität zwischenrnden Spezies identifiziert werden. Der Adhäsionsprozess war durch die Blockade derrnVLA-4–VCAM-1-Interaktion stärker inhibierbar als durch die Blockade von LFA-1–ICAM-1.rnDie Transmigration hingegen war durch die Blockade beider Adhäsionsmolekül-Paare vergleichbarrnstark inhibierbar.

Next-Generation Sequencing-Based Mutations Scanning Strategy of the BCR-ABL Kinase Domain in Patients with PhiladelPhia-Chromosome Positive Leukemias Treated with Tyrosine Kinase Inhibitors

In chronic myeloid leukemia and Philadelphia-positive acute lymphoblastic leukemia patients resistant to tyrosine kinase inhibitors (TKIs), BCR-ABL kinase domain mutation status is an essential component of the therapeutic decision algorithm. The recent development of Ultra-Deep Sequencing approach (UDS) has opened the way to a more accurate characterization of the mutant clones surviving TKIs conjugating assay sensitivity and throughput. We decided to set-up and validated an UDS-based for BCR-ABL KD mutation screening in order to i) resolve qualitatively and quantitatively the complexity and the clonal structure of mutated populations surviving TKIs, ii) study the dynamic of expansion of mutated clones in relation to TKIs therapy, iii) assess whether UDS may allow more sensitive detection of emerging clones, harboring critical 2GTKIs-resistant mutations predicting for an impending relapse, earlier than SS. UDS was performed on a Roche GS Junior instrument, according to an amplicon sequencing design and protocol set up and validated in the framework of the IRON-II (Interlaboratory Robustness of Next-Generation Sequencing) International consortium.Samples from CML and Ph+ ALL patients who had developed resistance to one or multiple TKIs and collected at regular time-points during treatment were selected for this study. Our results indicate the technical feasibility, accuracy and robustness of our UDS-based BCR-ABL KD mutation screening approach. UDS was found to provide a more accurate picture of BCR-ABL KD mutation status, both in terms of presence/absence of mutations and in terms of clonal complexity and showed that BCR-ABL KD mutations detected by SS are only the “tip of iceberg”. In addition UDS may reliably pick 2GTKIs-resistant mutations earlier than SS in a significantly greater proportion of patients.The enhanced sensitivity as well as the possibility to identify low level mutations point the UDS-based approach as an ideal alternative to conventional sequencing for BCR-ABL KD mutation screening in TKIs-resistant Ph+ leukemia patients