published_or_final_version / Medicine / Master / Master of Philosophy

CBL proteins are E3 ubiquitin ligases and adaptor proteins. The mammalian homologs – CBL, CBL-B and CBL-3 show broad tissue expression; accordingly, the CBL proteins play roles in multiple cell types. We have investigated the function of the CBL-B protein in hematopoietic cells and fibroblasts. The causative agent of chronic myeloid leukemia (CML) is BCR-ABL. This oncogenic fusion down-modulates CBL-B protein levels, suggesting that CBL-B regulates either the development or progression of CML. To assess the involvement of CBL-B in CML, bone marrow transduction and transplantation (BMT) studies were performed. Recipients of BCR-ABL-infected CBL-B(-/-) cells succumbed to a CML-like myeloproliferative disease with a longer latency than the wild-type recipients. Peripheral blood white blood cell numbers were reduced, as were splenic weights. Yet despite the reduced leukemic burden, granulocyte numbers were amplified throughout the animals. As well, CBLB(-/-) bone marrow (BM) cells possessed defective BM homing capabilities. From these results we concluded that CBL-B negatively regulates granulopoiesis and that prolonged latency in our CBL-B(-/-) BMT animals was a function of perturbed homing.To develop an in vitro model to study CBL-B function we established mouse embryonic fibroblasts (MEFs) deficient in CBL-B expression. Transduction of the wild-type and CBL-B-deficient MEFs with BCR-ABL did not confer transformation; nevertheless, the role of CBL-B in fibroblasts was evaluated. The CBL-B(-/-) MEFs showed enhanced chemotactic migration toward serum in both Transwell migration and time-lapse video microscopy studies. The biochemical response to serum was extensively evaluated leading to the development of a model. We predict that CBL-B deficiency either: (a) augments GRB2-associated binding protein 2 (GAB2) phosphorylation leading to enhanced extracellular signal-regulated kinase (ERK) and protein kinase B (PKB / Akt) signaling, or (b) alleviates negative control of Vav3 resulting in stimulation of Rho effectors. In either case, our results reveal a negative regulatory role for CBL-B in fibroblast migration. The two studies detailed herein expand our knowledge of CBL-B function. They strongly suggest that CBL-B can modulate granulocyte proliferation and point toward a role for CBL-B in the motility of numerous cell types.

Acute Myeloid Leukemia (AML), the most common form of acute leukemia in adults, is only curable in approximately 30% of all cases. Despite prognostic risk stratification using sub-typing and cytogenetic analysis to direct therapy, the mortality and relapse rate remains high. AML patients with normal karyotypes are defined as intermediate risk and are the most challenging to treat. Somatic mutations may be the key in refining prognostic stratification and providing useful therapeutic targets. The FMS-like tyrosine kinase 3 (FLT3) and Nucleophosmin (NPM1) genes have common mutated forms that are associated with overall survival and response to therapy. We assessed mutations in the FLT3 and NPM1 genes and their levels of expression in twenty eight AML patients in the presence and absence of HIV and their response to induction therapy. Furthermore, we used a novel technique, High Resolution Melting (HRM) Analysis to detect FLT3 Internal Tandem Duplications (ITD) and NPM1 exon 12 mutations. Five of the patients in this study were HIV positive, three of whom did not survive post-induction therapy. Of the AML patients, 17.9% were positive for the NPM1 mutation and 21% had mutated FLT3. Interestingly, the presence of the FLT3 and NPM1 mutations were coupled with an increase in expression levels of FLT3 and NPM1 from presentation to post-induction respectively and the loss of these mutations were coupled with a decrease in levels of expression from presentation to post-induction. However, an increase/decrease from presentation to post-induction did not necessarily denote the presence/absence of a mutation. Therefore, while mutational status of genes may generally confer mRNA levels, our results showed that there existed no definitive trend between mRNA levels of NPM1 and FLT3 expression and mutational status. We found that the HRM method was definitive for the simpler NPM1 mutation however detection of the FLT3-ITD mutation was challenging. There isn’t a clear distinction between mutated and non-mutated FLT3 due to the formation of hetero-duplexes during analysis, making detection highly subjective and error-prone. Sequencing allowed confirmation of mutated FLT3 and non-mutated FLT3 which were not in all instances in concordance with HRM analysis. The prognostic value in terms of overall survival of NPM1 and FLT3 mutations in this study is indefinite. Furthermore, the analysis of the HIV positive AML patients revealed no clear correlation between NPM1 and FLT3 levels of mRNA expression and mutational status. Also, the small number of HIV positive AML patients did not allow for conclusions to be made regarding HIV status and survival when affected with AML. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2012.

Background: Children with Down syndrome (DS) have increased risk for developing AML (DS-AMKL), and they usually experience severe therapy-related toxicities compared to non DS-AMKL. Refractory/ relapsed disease has very poor outcome, and patients would benefit from novel, less toxic, therapeutic strategies that overcome resistance. Relapse/resistance are linked to cancer stem cells with high aldehyde dehydrogenase (ALDH) activity. The purpose of the present work was to study less toxic alternative therapeutic agents for relapsed/refractory DS-AMKL. Methods: Fourteen AML cell lines including the DS-AMKL CMY and CMK from relapsed/refractory AML were used. Cytarabine (Ara-C), bortezomib (BTZ), disulfiram/copper (DSF/Cu2+) were evaluated for cytotoxicity, depletion of ALDH-positive cells, and resistance. BTZ-resistant CMY and CMK variants were generated by continuous BTZ treatment. Cell viability was assessed using CellTiter-Glo((R)), ALDH activity by ALDELUOR(TM), and proteasome inhibition by western blot of ubiquitinated proteins and the Proteasome-Glo(TM) Chymotrypsin-Like (CT-like) assay, apoptosis by Annexin V Fluos/Propidium iodide staining, and mutations were detected using PCR, cloning and sequencing. Results: Ara-C-resistant AML cell lines were sensitive to BTZ and DSF/Cu2+. The Ara-C-resistant DS-AMKL CMY cells had a high percentage of ALDHbright "stem-like" populations that may underlie Ara-C resistance. One percent of these cells were still resistant to BTZ but sensitive to DSF/Cu2+. To understand the mechanism of BTZ resistance, BTZ resistant (CMY-BR) and (CMK-BR) were generated. A novel mutation PSMB5 Q62P underlied BTZ resistance, and was associated with an overexpression of the beta 5 proteasome subunit. BTZ-resistance conferred increased resistance toAra-C due to G1 arrest in the CMY-BR cells, which protected the cells from S-phase damage by Ara-C. CMY-BR and CMK-BR cells were cross-resistant to CFZ and MG-132 but sensitive to DSF/Cu2+. In this setting, DSF/Cu2+ induced apoptosis and proteasome inhibition independent of CT-like activity inhibition. Conclusions: We provide evidence that DSF/Cu2+ overcomes Ara-C and BTZ resistance in cell lines from DS-AMKL patients. A novel mutation underlying BTZ resistance was detected that may identify BTZ-resistant patients, who may not benefit from treatment with CFZ or Ara-C, but may be responsive to DSF/Cu2+. Our findings support the clinical development of DSF/Cu2+ as a less toxic efficacious treatment approach in patients with relapsed/refractory DS-AMKL.

Chromodomain Helicase DNA-Binding Protein 4 (CHD4) is an ATPase that alters the phasing of nucleosomes on DNA and has recently been implicated in DNA double stranded break (DSB) repair. Here, we show that depletion of CHD4 in Acute Myeloid Leukemia (AML) blasts induces a global relaxation of chromatin that renders cells more susceptible to DSB formation, while concurrently impeding their repair. Furthermore, CHD4 depletion renders AML blasts more sensitive both in vitro and in vivo to genotoxic agents used in clinical therapy: daunorubicin (DNR) and cytarabine (ara-C). Sensitization to DNR and ara-C is mediated in part by activation of the ATM pathway, which is preliminarily activated by a Tip60-dependent mechanism in response to chromatin relaxation and further activated by genotoxic-agent induced DSBs. This sensitization preferentially affects AML cells, as CHD4 depletion in normal CD34+ hematopoetic progenitors does not increase their susceptibility to DNR or ara-C. Unexpectedly, we found that CHD4 is necessary for maintaining the tumor formatting behavior of AML cells, as CHD4 depletion severely restricted the ability of AML cells to form xenografts in mice and colonies in soft agar. Taken together, these results provide evidence for CHD4 as a novel therapeutic target whose inhibition has the potential to enhance the effectiveness of genotoxic agents used in AML therapy.

Un des mécanismes d’évasion des cellules tumorales au système immunitaire fait intervenir la famille des molécules de type B7. Ces molécules de costimulation ont aussi un rôle dans les mécanismes de tolérance immunitaire. La molécule B7-H1 (PD-L1 ou CD274) inhibe directement les lymphocytes T cytotoxiques (CTL) et est fortement exprimée à la surface de nombreux types de cellules tumorales. Les mécanismes de régulation de son expression ne sont pas bien connus. Il existe une augmentation d’expression de cette molécule après stimulation par l’IFNγ et récemment les ligands des Toll-Like-Receptor (TLR) 2, 4 et 9 ont été impliqués dans sa régulation au niveau du myélome multiple. L’implication possible des TLR dans l’expression de B7-H1 suggère un rôle possible de pathogènes dans l’échappement des tumeurs au système immunitaireIl n’existe aucune donnée dans la littérature sur l’expression des TLR au niveau des cellules blastiques de patients atteints de leucémies aigues myéloblastique (LAM). Nous avons dans un premier temps étudié l’expression des TLR dans les LAM et l’inductibilité de l’expression de la molécule B7-H1 par les ligands des TLR en cytométrie en flux. Nos résultats montrent que les TLR sont exprimés dans les LAM, avec une grande variabilité suivant les patients. On observe une augmentation significative de l’expression de B7-H1 après stimulation par le LPS (ligand TLR4) alors qu’elle n’est pas significative pour les autres ligands (PGN et ODN). Comme dans les tumeurs solides et le myélome multiple, l’expression de B7-H1 est augmentée par l’IFN γ. Des tests de lyse CTL sont en cours afin de confirmer le rôle fonctionnel de cette expression de B7-H1 via les TLR dans les LAM.Une autre partie de l’étude a été réalisée sur deux lignées murines leucémiques : DA1-3B et WEHI-3B, afin de disposer de modèles expérimentaux d’expression de B7-H1. On retrouve sur ces deux modèles l’augmentation d’expression de B7-H1 après stimulation par l’IFN γ et les ligands des TLR. L’utilisation de différents inhibiteurs chimiques des voies de signalisation suggère le rôle des voies MEK/ERK et de la voie JAK/STAT dans l’expression de cette molécule. La voie PI3kinase/Akt semble au contraire jouer un rôle inhibiteur. Le travail se poursuit avec la transfection de transdominants négatifs des différentes voies et de mutants constitutivement actifs. L’objectif à terme est de tester des stratégies d’immunothérapies des LAM par blocage pharmacologique de l’expression de B7-H1. / B7-H1 (PD-L1) is a B7-related protein that inhibits T-cell responses. B7-H1 participates in the immunoescape of cancer cells and is also involved in the long-term persistence of leukemic cells in a mouse model. B7-H1 can be constitutively expressed by cancer cells but is also induced by various stimuli. We therefore examined the constitutive and inducible expression of B7-H1 and the consequences of expression in human acute myeloid leukemia (AML). We analyzed B7-H1 expression in a cohort of 79 patients with AML. Blast cells were also studied after incubation with interferon-gamma or TLR ligands. Functionality was evaluated by cytotoxic T-cell activity against blast cells. Expression of B7-H1 at diagnosis was high in 18% of patients. Expression of toll-like receptors (TLR) 2, 4, and 9 was detected in one-third of AML samples. Expression of TLR2 and TLR4 ligands or IFN- induced by B7-H1 was found to protect AML cells from CTL-mediated lysis. Spontaneous B7-H1 expression was also found to be enhanced at relapse in some patients. MEK inhibitors including UO126 and AZD6244 reduced B7-H1 expression and restored CTL-mediated lysis of blast cells. In AML, B7-H1 expression by blasts represents a possible immune escape mechanism. The inducibility of B7-H1 expression by IFN- or TLR ligands suggests that various stimuli, either produced during the immune response against leukemia cells or released by infectious microorganisms, could protect leukemic cells from T-cells. The efficacy of MEK inhibitors against B7-H1-mediated inhibition of CTLs suggests a possible cancer immunotherapy strategy using targeted drugs.

La leucémie myéloïde chronique (LMC) est une maladie myéloproliférative, résultant d'une translocation réciproque entre les chromosomes 9 et 22, donnant naissance à une kinase de fusion à activité continue, la BCR-ABL. Les inhibiteurs de la tyrosine kinase (ITK) sont le traitement de choix de la LMC, mais ces inhibiteurs n'offrent pas de cure radicale aux patients, qui, une fois le traitement interrompu, présentent une rechute et une exacerbation de leur condition. En plus, les patients peuvent développer une résistance ou une intolérance aux ITK, d'où la nécessité de nouvelles modalités thérapeutiques. Les imidazoquinoxalines sont de nouveaux composés à visée anticancéreuse, dont la structure est basée sur celle de l'imiquimod, un immunomodulateur connu. EAPB0203, un composé du groupe des imidazo[1,2-α]quinoxalines, a fait preuve d'un pouvoir anticancéreux sur des cellules de mélanome, in vitro et in vivo, ainsi que sur des cellules de leucémie liée au virus HTLV-1, in vitro. EAPB0503 est un composé de la même famille, à pouvoir environ 10 fois supérieur à celui de l'EAPB0203 sur les cellules de mélanome. Dans le cadre de ce projet de thèse, l'activité de l'EAPB0203 a été comparée à celle de l'EAPB0503, et leur effet sur la prolifération de trois lignées cellulaires de LMC a confirmé que l'EAPB0503 est plus efficace que l'EAPB0203. Pour ce, l'EAPB0503 a été adopté pour la suite des expérimentations que nous avons entreprises dans le but de caractériser le mécanisme d'action par lequel ce composé inhibe la croissance cellulaire. Les CI50 de l'EAPB0503 ont été déterminées pour chacune des trois lignées, et adoptées tout au long du travail. On a démontré que l'EAPB0503 provoque un arrêt du cycle cellulaire en PréG0 (accumulation de cellules apoptotiques) et en G2/M. Une nette augmentation du niveau de phosphorylation de l'histone-3 suggère un arrêt des cellules en début de mitose. Cet arrêt du cycle cellulaire s'accompagne d'une diminution du potentiel membranaire mitochondrial, d'une activation modérée du cytochrome c et du clivage de PARP (suggérant l'implication de caspases). On a également démontré que l'EAPB0503 induit l'apoptose comme le montre la fragmentation de l'ADN. Des résultats préliminaires proposent aussi une stabilisation de la protéine pro-apoptotique, bax, et une diminution du niveau de la protéine anti-apoptotique Bcl-2, augmentant ainsi le rapport Bax/Bcl-2 et renversant ainsi l'équilibre du côté apoptotique. En plus, EAPB0503 a potentialisé l'effet de l'imatinib (le premier ITK), et, à deux, ces deux agents ont eu un effet synergique sur la croissance des cellules de LMC. Il a été également très intéressant de montrer que l'EAPB0503 dégrade l'oncoprotéine, BCR-ABL, dans les cellules sensibles et résistantes à l'imatinib. Ensemble, ces résultats suggèrent un effet prometteur de l'EAPB0503 sur la LMC, en monothérapie ou en association avec les ITK. / Chronic myeloid leukemia (CML) is a myeloproliferative disorder that originates in a reciprocal translocation, resulting in the Philadelphia chromosome that harbors the bcr-abl oncogene. This fusion gene codes for a constitutively active tyrosine kinase, BCR-ABL that confers leukemogenesis. Tyrosine kinase inhibitor (TKI), have revolutionized the therapeutic management of CML. Imatinib, the first generation TKI, is highly effective in inducing remissions and prolonging the survival of CML patients. However, failure of this therapy occurs in almost one-third of the patients due to intolerance to treatment, lack of therapeutic response or resistance. Moreover, CML stem cells (LIC) remain insensitive to this therapy, which almost inevitably leads to relapse upon treatment discontinuation. Imidazoquinoxalines are imiquimod derivatives with direct immunomodulatory effect and antitumor activity on melanoma and T-cell lymphoma, attributed to growth inhibition and induction of caspase-dependent apoptosis. We investigated the effects of EAPB0203 and EAPB0503, novel imidazoquinoxaline derivatives, on human CML cell lines. We show that EAPB0203 and EAPB0503 inhibit cell growth of three CML cell lines in a dose- and time-dependent manner. Compared to the previously described EAPB0203, EAPB0503 has a better inhibitory activity on CML cells. We demonstrated that EAPB0503 induced a specific cell cycle arrest in mitosis in CML cells, evidenced by decreased levels of cyclin D1 and increased phosphorylation of histone-3. This was accompanied by the direct activation of apoptosis as demonstrated by increased PreG0 population, DNA breakage, poly(ADP-ribose) polymerase cleavage and breakdown of mitochondrial membrane potential. Preliminary results have shown a stabilization of the pro-apoptotic molecule, bax, versus a decrease in anti-apoptotic bcl-2, shifting the ratio towards the induction of apoptosis. Interestingly, EAPB0503-treated cells had decreased levels of BCR-ABL oncoprotein. The combination of EAPB0503 with imatinib synergizes to inhibit CML cells proliferation and most importantly, EABP0503 inhibited the proliferation of imatinib-resistant CML cells offering a promising therapeutic modality that alone or in combination with TKI would circumvent mutations and resistance to TKI and improve the prognosis of CML.

Les leucémies aiguës myéloïdes (LAM) sont des hémopathies malignes caractérisées par une accumulation dans la moelle et le sang de progéniteurs hématopoïétiques bloqués dans un stade différenciation. La mutation FLT3-ITD, qui entraîne une activation constitutive du récepteur à activité tyrosine kinase FLT3, est retrouvée dans 20-25% des LAM et est associée à un mauvais pronostique. De nombreux inhibiteurs de FLT3 ont été développés et certains sont testés en clinique mais des études mettent en évidence l'apparition de résistance. Une meilleure compréhension des mécanismes oncogéniques de FLT3-ITD est donc nécessaire afin d'améliorer le traitement des LAM. Mes travaux de thèse ont été centrés sur l'analyse du processus autophagique qui correspond à l'un des mécanismes de résistance décrits dans les cellules cancéreuses en réponse aux traitements. Au cours de cette étude, nous avons constaté que l'expression de FLT3-ITD augmente l'autophagie basale des cellules de LAM, et que l'inhibition du récepteur réduit cette autophagie dans des échantillons primaires de LAM et dans des lignées cellulaires. Nous avons pu montrer que l'autophagie est requise pour la prolifération et la survie in vitro et in vivo des cellules de LAM et que sont ciblage permet de surmonté la résistance aux inhibiteurs de FLT3. De plus, nous avons identifié la protéine ATF4 comme un acteur essentiel au processus d'autophagie en aval de FLT3-ITD. Ces résultats suggèrent que le ciblage de l'autophagie ou d'ATF4 chez les patients exprimant les mutations de FLT3 peut représenter une stratégie thérapeutique prometteuse et innovatrice dans les LAM. / Acute myeloid leukemias (AMLs) are a family of hematological malignancies characterized by an accumulation in the marrow and blood of hematopoietic progenitors blocked in their differentiation process. The FLT3-ITD mutation is found in 20-25% of AMLs and is associated with a poor prognosis. Different FLT3 inhibitors have been developed and some of them are clinically tested but resistance to treatment has been observed in many patients. A better understanding of AML biology is necessary in order to improve the treatment of AMLs. My thesis project focused on the analysis of the autophagic process, which is one of the mechanisms described in the resistance of cancer cells. In this study, we found that the FLT3-ITD expression increases basal autophagy in AML cells, and that the receptor inhibition reduced this autophagy in primary patient samples and cell lines. We show that autophagy is required for proliferation and survival in vitro and in vivo of leukemic cells lines and inhibition of autophagy overcomes resistance to FLT3 inhibitors. In addition, we identified the ATF4 protein as a key actor of the autophagy process induced by the FLT3-ITD mutation. These results suggest that targeting autophagy or ATF4 may represent a promising and innovative therapeutic strategy for FLT3 mutated AMLs.

A Leucemia Mieloide Aguda (LMA) é uma neoplasia originada a partir da expansão clonal de blastos da linhagem mieloide em medula óssea, sangue periférico e outros tecidos. Entre as mutações mais frequentemente detectadas nas LMAs, se encontra a mutação do tipo duplicação interna em tandem (FLT3-ITD) que é detectada em aproximadamente 25% dos pacientes adultos. Esta mutação no receptor de tirosina quinase FLT3 é uma inserção de 3 a 400 pares de base na região juxtamembrana do receptor, a qual é responsável pelo controle da atividade enzimática dos domínios tirosina quinase. Quando esta mutação se encontra presente, a região juxtamembrana perde a capacidade de controlar a ativação dos domínios tirosina quinase e o receptor fica constitutivamente ativo conferindo uma vantagem proliferativa ao clone leucêmico. Esta mutação é considerada de mal prognóstico e já foram desenvolvidos inibidores de tirosina quinase específicos para o receptor FLT3 (FLT3 TKI). Porém, os resultados dos primeiros ensaios clínicos não apresentaram a efetividade esperada e continua a busca de novas combinações de drogas que contribuam a aumentar a efetividade destes inibidores. É por isso que este trabalho teve por objetivo testar a combinação de FLT3 TKIs com o ácido trans-retinoico (ATRA) já aplicado no tratamento da Leucemia Promielocítica Aguda (LPA) com PML-RARA. A combinação de FLT3 TKIs com ATRA induziu a morte celular programada de forma precoce tanto na linhagem de LMA MV4-11 como na MOLM-13. Esta morte celular observada foi inibida com pré- tratamento com inibidor de caspases QVD. O tratamento combinado in vivo em camundongos Nod Scid Gamma (NSG) transplantados com células MOLM-13, aumentou a sobrevida dos animais e diminuiu a percentagem de células CD45 humanas em medula óssea, baço e sangue periférico. No seu conjunto, nossos dados sugerem que o ATRA aumenta o efeito citotóxico dos FLT3-TKIs. Este achado poderá ser relevante para o tratamento de pacientes com LMA portadores de mutaçãoes ITD no gene FLT3 / Acute Myeloid Leukemia (LMA) is hematological disease that arises from the clonal expansion of a myeloid blast in bone marrow, peripheral blood and other tissues. Among LMAs mutations most frequently detected, 25% of adult patients carry the FLT3-ITD mutation. This mutation is a pair base insertion of 3 to 400 in the juxtamembrane domain of the receptor and leads to the constitutive activation of the kinase domains. It gives the leukemic clone a proliferative advantage and it is associated with a bag prognosis. FLT3 tyrosine kinase inhibitors (FLT3 TKI) were already developed in order to improved patients\' treatment. However, results from the first clinical trials were not as promising as expected. Therefore, there is still room for testing new drug combinations that could improve FLT3 TKIs efficacy. The main objective of this work was to test FLT3 TKIs in combination with all-trans retinoic acid (ATRA) already used in Acute Promyelocytic Leukemia (APL) with PMLRARA treatment. The combination of FLT3-TKIs and ATRA induced early programmed cell death in both the MV4-11 and MOLM-13 LMA lines. This early cell death was inhibited with QVD caspase inhibitor pre-treatment. In vivo combined treatment in Nod Scid Gamma (NSG) mice transplanted with MOLM-13 cells, increased animals survival and decreased the percentage of human CD45 cells in bone marrow, spleen and peripheral blood. Taken together, our data suggest that ATRA increases the cytotoxic effect of FLT3- TKIs. This finding may be relevant for the treatment of patients with AML with ITD mutations in the FLT3 gene

Myeloid leukemias include malignant diseases characterized by clonal expansion of the myeloid cell lineage. While in case of chronic myeloid leukemia (CML), the main cause of the disease has already been identified - t(9;22) and the aktivity of the fusion product of the translocation BCR-ABL, acute myeloid leukemia (AML) has been associated with plenty of different translocations and mutations. The aim of this work was to contribute to the improvement of monitoring of patients with myeloid leukemias via detailed study of the panleukemic marker Wilms tumor gene 1 (wt1) expression. Prognostic value of wt1 expression has been proved for AML patients, however, it has not yet been confirmed for CML patients. Expression of different wt1 variants (more then 36 protein products) is known very poorly in both, AML and CML as well as in normal hematopoiesis. Most of the study is focused on CML, only limited parts are dedicated to AML. In the first part of the work, we clearly proved prognostic value of total wt1 mRNA expression for CML patients. Statistical evaluations revealed critical wt1 values which enable to specify prognosis of patients responding non-optimally to imatinib. Bcr-abl looses much of its prognostic value in these patients. Further, we have designed and optimized PCRs for selected wt1...