A introdução do Mesilato de Imatinibe (MI) como primeiro inibidor específico de BCR-ABL1 na prática clínica revolucionou o tratamento da Leucemia Mieloide Crônica (LMC), tornando-se a terapia padrão para o tratamento desta doença. Porém, cerca de 30% dos pacientes com LMC não respondem à terapia com MI e um número substancial destes casos de resistência não tem causa conhecida. O MI interage com transportadores de membrana ABCG2 e SLCO1A2. Este estudo teve como objetivo investigar a relação da expressão gênica de ABCG2 e de SLCO1A2 com marcadores de resposta ao tratamento com MI, em indivíduos com LMC e avaliar a influência dos polimorfismos ABCG2 c.421C>A e ABCG2 c.-19-99G>A na resposta ao MI. Foram incluídos 118 pacientes com LMC os quais foram classificados em dois grupos: Grupo Respondedor, constituído por 70 pacientes com resposta citogenética completa com a dose padrão de MI (400 mg/dia) por até 18 meses e, Grupo não Respondedor constituído por 48 pacientes sem resposta citogenética completa à dose inicial de 400 mg/dia de MI ou que perderam esta resposta ao longo do tratamento e foram reescalonados para doses de 600 ou 800 mg/dia. A resposta ao tratamento foi avaliada segundo os critérios da European LeukemiaNet. Foram excluídos pacientes com alterações citogenéticas diferentes do cromossomo Ph e mutações no gene BCR-ABL1. Amostras de sangue periférico foram utilizadas para: extração do RNA total para quantificação dos transcritos BCR-ABL1 e expressão gênica de ABCG2 e SLCO1A2; extração de DNA e análise citogenética de banda G. A expressão do gene ABCG2 e SLCO1A2 e as análises dos polimorfismos foram feitas por PCR em tempo real. A expressão de ABCG2 foi maior no grupo de não respondedores ao MI (P=0,028). Este resultado foi influenciado pelos pacientes com resistência primária (N= 34 P=0,029), mas não pelos que apresentaram resistência secundária (N=14 P=0,249) quando comparado com respondedores (N=70). A elevada expressão do gene ABCG2 foi também associada àqueles pacientes que não tiveram resposta molecular maior (número de transcritos BCR-ABL1 ≤ 0,1%) (P=0,027) quando todos os pacientes foram analisados. O gene estudado não foi associado com a resposta molecular completa (número de transcritos BCR-ABL1 ≤ 0,032%). Com relação ao gene SLCO1A2 não foi possível determinar sua expressão devido à baixa concentração do RNA obtido. Os polimorfismos c.421C>A e c.-19-99G>A não foram associados com a expressão do gene ABCG2 e a resposta ao MI. A RMC (no grupo de respondedores) foi associada com o genótipo 421CC e houve tendência a maior frequencia de portadores do genótipo -19-99GG neste mesmo grupo. Portadores do genótipo -19-99AA apresentaram tendência ao risco de ter LMC. Os resultados deste estudo nos permitem concluir que a maior expressão de ABCG2 está associada com a resistência primária ao MI podendo então ser um mediador da resistência ao MI. Os polimorfismos do gene ABCG2 não influenciaram na expressão gênica de ABCG2, mas impactaram na RMC no grupo respondedor ao MI. / The introduction of imatinib mesylate (IM) as the first specific inhibitor of BCR-ABL1 in clinical practice has revolutionized the treatment of chronic myeloid leukemia (CML), becoming the standard therapy for this disease. However, about 20% of CML patients do not respond to therapy with IM and a substantial number of these cases of resistance have no known cause. The MI interacts with membrane transporters ABCG2 and SLCO1A2. The aim of this study was to investigate the relationship of ABCG2 and SLCO1A2 gene expression with markers of response to MI in individuals with CML and evaluate the influence of polymorphisms ABCG2 c.421C> A and c. ABCG2-19-99G> A in response to the MI. One hundred and eighteen patients in chronic phase of CML were studied and classified in two groups: Responder Group comprised 70 patients who had a complete cytogenetic response within 18 months of treatment. The non-responder group comprised 48 patients who did not have a complete cytogenetic response with the initial dose (400 mg/day) of IM or who relapsed during treatment and were submitted to higher doses of 600 or 800 mg/day. Criteria of failed response to treatment were established by European LeukemiaNet. Patients with cytogenetic patterns other than the Philadelphia chromosome and patients with mutations in the BCR-ABL1 gene were excluded from this study. Blood samples were obtained for: total RNA extraction for quantification of BCR-ABL1 and gene expression of ABCG2 and SLCO1A2; genomic DNA extraction and band G cytogenetic analysis. The gene expression and the analysis of the polymorphisms were performed by real time PCR. Expression of ABCG2 in non-responder group was higher than in responder group (P=0.028). This result was influenced by patients with primary resistance (n= 34 P=0.029) but not secondary resistance (n=14 P=0.249) when compared with responders (n=70). The higher expression of ABCG2 gene was also associated with those patients who had major molecular response (number of BCR-ABL1 . 0.1%) (P=0.027) when all patients were analyzed. The studied gene was not associated with the complete molecular response (number of BCR-ABL1 .0.0032). Regarding to the gene SLCO1A2 was not possible to determine its expression due to low concentration of RNA obtained. The c.421C>A e c.-19-99G>A were not associated neither with the ABCG2 gene expression and MI response. CMR in responders group was associated with the 421CC genotype ant there was a trend for higher frequency of carriers of genotype -19-99GG in the same group. Carriers of 19-99AA genotype tended to the risk of having CML. The results of this study allow us to conclude that the higher expression of ABCG2 is associated with primary resistance to IM and may be a mediator of resistance to IM. The ABCG2 polymorphisms did not influence the gene expression of ABCG2 but impacted in CMR of the responders to IM.

A leucemia mieloide crônica (LMC) é uma expansão clonal da célula tronco hematopoética, traduzindo-se por hiperplasia mieloide, leucocitose, neutrofilia, basofilia e esplenomegalia. O cromossomo Filadélfia é característico da doença, sendo produto da translocação t(9:22)(q34;q11), resultando na fusão dos genes ABL e BCR. Esta fusão gera um gene híbrido que codifica uma proteína com elevada atividade tirosinoquinase e tem um papel central na patogenia da LMC. O mesilato de imatinibe (MI) é um derivado da fenilaminopirimidina que inibe a proteína tirosinoquinase BCR-ABL1 in vitro e in vivo. O MI interage com transportadores de membrana de influxo, como o organic carion solute carrier 22 ,member 1 (SLC22A1,hOCT1); e de efluxo, como ATP binding cassette B1 (ABCB1, MDR1, P-gp). Os polimorfismos ABCB1 c.1236C>T, C.3435C>T e c.2677G>T/A têm sido associados com a alteração da função da P-gp. Este estudo teve por objetivo investigar a relação da expressão do RNAm de ABCB1 e SLC22A1 com marcadores de resposta ao tratamento com MI e avaliar a atividade funcional da P-gp em células mononucleares de pacientes com diferentes haplótipos para os polimorfismos ABCB1 c.1236C>T, c.3435C>T e c.2677G>T/A. Foram incluídos 118 pacientes com LMC para o estudo da expressão do RNAm de SLC22A1 e ABCB1 e para o estudo da atividade da P-gp foram selecionados 28 pacientes de acordo com os haplótipos dos polimorfismos ABCB1 c.1236C>T, c.3435C>T e c.2677G>T/A. Para o estudo da expressão do RNAm de SLC22A1 e ABCB1 foram constituídos dois grupos: Grupo 1 com 70 pacientes com resposta citogenética completa com a dose padrão de MI (400 mg/dia de MI) em até 18 meses e, Grupo 2 com 48 pacientes sem resposta citogenética completa com a dose inicial de 400 mg/dia de MI ou que perderam esta resposta ao longo do tratamento. Para o estudo da atividade funcional da P-gp, dos 118 pacientes incluídos, foram selecionados 10 pacientes que apresentaram o haplótipo 1236CC/3435CC/2677GG, 10 pacientes que apresentaram o haplótipo 1236CT/3435CT/2677GT e 8 pacientes que apresentaram o haplótipo 1236TT/3435TT/2677TT. A resposta ao tratamento foi avaliada segundo os critérios da European LeukemiaNet. Amostras de sangue foram obtidas para: quantificação de BCR-ABL1, extração do RNAm total, análise citogenética de banda G, dosagem da concentração plasmática de MI e análise da atividade e expressão da P-gp. A análise da expressão dos genes ABCB1 e SLC22A1 foi feita por PCR em tempo real, a análise da atividade e expressão da P-gp foram feitas por citometria de fluxo e a dosagem da concentração plasmática de MI foi realizada por eletroforese capilar. Resultados: A expressão de ABCB1 e SLC22A1 foi analisada nos 118 pacientes incluídos e foi similar entre os grupos de resposta. A elevada expressão do gene SLC22A1 foi associada àqueles pacientes que alcançaram a resposta molecular maior (RMM) no grupo respondedor (P=0,009). Não houve associação entre a expressão de ABCB1 e a resposta ao MI. Nenhum dos genes foi associado à resposta molecular completa (RMC). No estudo da atividade da P-gp foi observada uma maior atividade nos pacientes que apresentavam o haplótipo 1236CC/3435CC/2677GG quando comparado àqueles que possuíam o haplótipo com alelo mutado. Não houve diferença na expressão do RNAm dos genes SLC22A1 e ABCB1, expressão da P-gp e concentração plasmástica de MI entre os grupos de haplótipos. Os pacientes que não alcançaram a RMM apresentaram uma maior taxa de efluxo mediado pela P-gp quando comparado aos indivíduos que alcançaram esta resposta (64,7% vs. 45,7%; P=0,001). Os indivíduos que alcançaram a RMM e RMC apresentaram maior mediana de expressão do gene SLC22A1. Os pacientes sem RMM apresentaram menor concentração plasmática de MI quando comparados aos que alcançaram esta resposta (0,51 µg/mL vs. 1,42 µg/mL; P=0,001). Não foi observada associação entre a concentração plasmática de MI e a RMC. Em conclusão os pacientes respondedores a dose padrão de 400mg/dia de MI e que alcançaram a RMM apresentam maior expressão de RNAm de SLC22A1 e os portadores dos haplótipos 1236CT/3435CT/2677GT e 1236TT/3435TT/2677TT exibem menor efluxo mediado pela P-gp apresentando maior frequência de RMM. / Chronic myeloid leukemia (CML) is a clonal expansion of hematopoietic stem cell, translating into myeloid hyperplasia, leukocytosis, neutrophilia, basophilia and splenomegaly. The Philadelphia chromosome is characteristic of the disease, being the product of the translocation t(9:22)( q34,q11), resulting in the fusion of the BCR and ABL genes. This fusion generates a hybrid gene that encodes a protein with elevated tyrosine kinase activity and plays a central role in the pathogenesis of CML. Imatinib mesylate (IM) is a derivative of fenilaminopirimidine that inhibits BCR-ABL1 fusion protein tyrosine kinase in vitro and in vivo. IM interacts with uptake membrane transporters, such as cation organic solute carrier 22, member 1 (SLC22A1, hOCT1) and efflux as ATP binding cassette B1 (ABCB1, MDR1,P-gp). ABCB1 polymorphisms c.1236C>T,c.3435C>T and c.2677G>T/A have been associated with altered function of P-gp. This study aimed to investigate the relationship between mRNA expression of ABCB1 and SLC22A1 with markers of response to treatment with IM and evaluate the functional activity of P-gp in mononuclear cells of patients with different haplotypes for ABCB1 c.1236C>T, c.3435C>T and c.2677G>T/A polymorphisms. This study included 118 patients with CML to study the mRNA expression of SLC22A1 and ABCB1 and to study the P-gp activity, 28 patients were selected according to the haplotypes of ABCB1 c.1236C>T, c.3435C>T and c.2677G>T/A polymorphisms. To study the mRNA expression of SLC22A1 and ABCB1, two groups were constituted: Group 1 with 70 patients with a complete cytogenetic response with standard-dose IM (400 mg/day) in 18 months, and group 2 with 48 patients without complete cytogenetic response with the initial dose of IM (400 mg/day) or have lost this response during treatment. To study the P-gp functional activity, 10 patients with haplotype 1236CC/3435CC/2677GG, 10 patients with haplotype 1236CT/3435CT/2677GT and 8 patients with haplotype 1236TT/3435TT/2677TT were enrolled. Treatment response was assessed according to European LeukemiaNet criteria. Blood samples were obtained for: quantification of BCR-ABL1, mRNA extraction, G band cytogenetic analysis, measurement of IM plasma levels and P-gp activity and expression. The ABCB1 and SLC22A1 gene expression analysis was made by real-time PCR, analysis of P-gp activity and protein expression were performed by flow cytometry and determination of plasma Levels of IM was performed by capillary electrophoresis. Results: Expression of ABCB1 and SLC22A1 were analyzed in 118 patients included and was similar between the response groups. Higher expression of the SLC22A1 gene was associated with those patients who achieved a major molecular response (MMR) in the responder group (P=0.009). There was no association between ABCB1 expression and IM response. None of the studied genes was associated with complete molecular response (CMR). In the study of P-gp activity we observed greater activity mediated by P-gp in patients with 1236CC/3435CC/2677GG haplotype when compared to those with the mutated allele. There was no difference in mRNA expression of SLC22A1 and ABCB1 genes, P-gp expression and IM plasma levels between haplotypes groups. Patients who did not achieve MMR showed a higher rate of efflux mediated by P-gp compared to individuals who did achieve this response (64.7% vs. 45.7%, P=0.001). Individuals who achieved MMR and CMR had higher median of SLC22A1 expression. Patients without MMR had lower IM plasma levels compared with those who achieved this response (0.51 µg/mL vs. 1.42 µg/mL, P=0.001). No association was observed between IM plasma levels and CMR. In conclusion patients responders to standard dose of IM (400 mg/day) and who achieved MMR have higher SLC22A1 mRNA expression and the carriers of 1236CT/3435CT/2677GT 1236TT/3435TT/2677TT haplotypes exhibit lower efflux mediated by P-gp with higher frequency of MMR.

Étude phytochimique et activité cytotoxique des métabolites secondaires de Ferula elaeochytris Korovin et Ferula lycia Boiss. (Apiacées) Les plantes du genre Ferula (Apiacées) sont des herbacées vivaces répandues dans l'Asie centrale, la région méditerranéenne et l'Afrique du Nord. Des études récentes ont montré l'intérêt de certains composés isolés des espèces de ce genre comme agents chimiopréventifs ainsi que pour surmonter la résistance aux anticancéreux. Dans ce cadre deux plantes du genre Ferula récoltées dans deux régions différentes de la Turquie ont été choisies pour ce travail : Ferula elaeochytris Korovin et Ferula lycia Boiss. Vingt esters sesquiterpéniques dont sept structures nouvelles, deux acides phénoliques et un saponoside ont été isolés. Toutes ces stuctures ont été établies par méthodes spectrales (SM et RMN). Sur le plan pharmacologique, toutes les molécules isolées ont été testées pour leurs activités cytotoxiques vis-à-vis des lignées cellulaires leucémiques résistantes aux inhibiteurs de tyrosine kinase : K562R (imatinib-résistantes) et DA1-3b/M2 (imatinib et dasatinib-résistantes). L'élaeochytrine A (6-anthraniloyljaeschkeanadiol), s'est révélée être le composé le plus actif et le plus sélectif vis-à-vis des cellules tumorales avec des CI50 de l'ordre de 10 :M.

<p>The objective of this master thesis was to develop an analytical method for the quantification of the cancer drug Imatinib and its main metabolite CGP74588 in plasma. Imatinib is used in the treatment of chronic myeloid leukemia and gastrointestinal stroma tumors. A quantitative analytical method was developed where reversed-phase columns with different stationary phases were studied and the sensitivity was tested with both UV detectors and a mass spectrometric detection. Since the substances were measured in plasma a solid-phase extraction was developed to purify the samples before analysis. The column chosen for the separation was the Max-RP C12 column (100 x 3 mm, 4 μm particle size) manufactured by Phenomenex with a gradient mobile phase with 1% formic acid in methanol and water. The gradient was as follows; 0 min 15:85, 7 min 60:40, 9 min 60:40 with a total runtime of 13.5 min. The internal standard chosen was Opipramol. Mass spectrometric detection using a sonic spray ionization interface in positive mode proved to be about as sensitive as UV detection at 261 nm. The generated (M+H+)+ ions were isolated and fragmented with the use of three mass spectrometric methods; one for Imatinib (transition 494 —› 394), one for CGP74588 (transition 480 —› 394) and one for Opipramol (transition 364 —› 171). For the purification of the plasma samples an Oasis HLB solid-phase extraction cartridge was selected and the recoveries were close to 100%.</p><p>The developed method was partially validated and showed coefficients of variation (CV) for intra-and inter-day precision between 0.4 and 5.4% with UV detection. The validation results for the mass spectrometer were inconclusive.</p>

The objective of this master thesis was to develop an analytical method for the quantification of the cancer drug Imatinib and its main metabolite CGP74588 in plasma. Imatinib is used in the treatment of chronic myeloid leukemia and gastrointestinal stroma tumors. A quantitative analytical method was developed where reversed-phase columns with different stationary phases were studied and the sensitivity was tested with both UV detectors and a mass spectrometric detection. Since the substances were measured in plasma a solid-phase extraction was developed to purify the samples before analysis. The column chosen for the separation was the Max-RP C12 column (100 x 3 mm, 4 μm particle size) manufactured by Phenomenex with a gradient mobile phase with 1% formic acid in methanol and water. The gradient was as follows; 0 min 15:85, 7 min 60:40, 9 min 60:40 with a total runtime of 13.5 min. The internal standard chosen was Opipramol. Mass spectrometric detection using a sonic spray ionization interface in positive mode proved to be about as sensitive as UV detection at 261 nm. The generated (M+H+)+ ions were isolated and fragmented with the use of three mass spectrometric methods; one for Imatinib (transition 494 —› 394), one for CGP74588 (transition 480 —› 394) and one for Opipramol (transition 364 —› 171). For the purification of the plasma samples an Oasis HLB solid-phase extraction cartridge was selected and the recoveries were close to 100%. The developed method was partially validated and showed coefficients of variation (CV) for intra-and inter-day precision between 0.4 and 5.4% with UV detection. The validation results for the mass spectrometer were inconclusive.

Background: GISTs are gastrointestinal mesenchymal tumours that express the type III receptor tyrosine kinase KIT. The KIT proto-oncogene encodes the receptor KIT. Most GISTs have gain-of-function mutations in the KIT or PDGFRA gene. The tyrosine kinase is therefore continuously activated leading to ligand-independent dimerization. Imatinib mesylate (Glivec®) is considered to be the first-line palliative treatment. The activated form of the KIT receptor tyrosine kinase is inhibited by imatinib. The aim of the study was to compare the survival of patients treated with either adjuvant or down-staging imatinib with historic controls treated with radical surgery (R0) only. Methods: A historic control group was chosen from a population-based series from western Sweden (population 1.6 million) that matched the adjuvant (n=23) and down-staging (n=7) groups respectively. Mutation analysis was performed in all cases with bidirectional direct sequencing. The recurrence-free survival was calculated. Results: There was only one recurrence (4 %) in the adjuvant group, and no recurrences in the down-staging study group, compared to 32/48 patients (67 %) in the control group. Tumour size decreased in diameter from 20 cm to 11 cm with down-staging treatment. Conclusion: Adjuvant imatinib improves recurrence-free survival in R0 resected patients. Down-staging treatment with imatinib is recommended for patients with large tumours or metastases. The importance of mutation analysis was established.

In this study, mechanisms of resistance to Imatinib-induced apoptosis in human K562 and Meg-1 chronic myeloid leukemia (CML) cells were examined. Continuous exposure of cells to step-wise increasing concentrations of Imatinib resulted in the selection of 0.2 and 1 &amp / #956 / M imatinib resistant cells. Measurement of endogenous ceramide levels showed that treatment with Imatinib increased the generation of C18-ceramide significantly, which is mainly synthesized by the human longevity assurance gene 1 (hLASS1), in sensitive, but not in resistant cells. Mechanistically, analysis of mRNA and enzyme activity levels of hLASS1 in the absence or presence of Imatinib did not show any significant differences in the resistant cells when compared to its sensitive counterparts, suggesting that accumulation and/or metabolism, but not the synthesis of ceramide, might be altered in resistant cells. iv Indeed, further studies demonstrated that expression levels, and enzyme activity of sphingosine kinase-1 (SK-1), increased significantly in resistant K562 or Meg-1 cells. The expression levels of glucosyl ceramide synthase (GCS) also increased in resistant cells, comparing to the sensitive counterparts, which indicates conversion of pro-apoptotic ceramide to glucosyl ceramide. Expression analyses of BCR-ABL gene demonstrated that expression levels of BCR-ABL gene increased gradually as the cells acquired the resistance. However, Nucleotide sequence analyses of ABL kinase gene revealed that there was no mutation in Imatinib binding region of the gene in resistant cells. There was also an increase in expression levels of MDR1 gene in resistant cells, which transport the toxic substances outside of cells. In conclusion, these data show, for the first time, a role for endogenous ceramide synthesis via hLASS1 in Imatinib-induced apoptosis, and those alterations of the balance between the levels of ceramide and S1P. Mainly the overexpression of SK-1 seems to result in resistance to Imatinib in K562 cells. The cellular resistance may also result from conversion of ceramide to glucosyl ceramide, from overexpression of BCR-ABL and MDR1 genes but not due to mutations in Imatinib binding site of ABL kinase.

<p>Background: GISTs are gastrointestinal mesenchymal tumours that express the type III receptor tyrosine kinase KIT. The KIT proto-oncogene encodes the receptor KIT. Most GISTs have gain-of-function mutations in the KIT or PDGFRA gene. The tyrosine kinase is therefore continuously activated leading to ligand-independent dimerization. Imatinib mesylate (Glivec®) is considered to be the first-line palliative treatment. The activated form of the KIT receptor tyrosine kinase is inhibited by imatinib. The aim of the study was to compare the survival of patients treated with either adjuvant or down-staging imatinib with historic controls treated with radical surgery (R0) only.</p><p>Methods: A historic control group was chosen from a population-based series from western Sweden (population 1.6 million) that matched the adjuvant (n=23) and down-staging (n=7) groups respectively. Mutation analysis was performed in all cases with bidirectional direct sequencing. The recurrence-free survival was calculated.</p><p>Results: There was only one recurrence (4 %) in the adjuvant group, and no recurrences in the down-staging study group, compared to 32/48 patients (67 %) in the control group. Tumour size decreased in diameter from 20 cm to 11 cm with down-staging treatment.</p><p>Conclusion: Adjuvant imatinib improves recurrence-free survival in R0 resected patients. Down-staging treatment with imatinib is recommended for patients with large tumours or metastases. The importance of mutation analysis was established.</p>

Fibroblast growth factors (FGFs) lack signal sequences, and are exported through endoplasmic reticulum (ER)-Golgi-independent non-classical routes. FGFs work as modulators of various cellular activities like mitosis, differentiation, survival etc. Among the FGF family, which comprises of 23 different heparin proteins, human FGF-1 (hFGF-1), a potent angiogenic factors are one of the targets in cancer inhibition, as they are involved in blood vessel formation in tissues. There has been intensive research directed at the development of drugs that could effectively inhibit angiogenesis. In this context, the purpose of this study is to fully understand the molecular principles essential to determine probability of inhibition of hFGF-1 signaling transduction by imatinib. Imatinib, a 2-phenyl amino pyrimidine derivative is a tyrosine kinase inhibitor with antineoplastic activity. Imatinib binds to the intracellular pocket located within tyrosine kinases and inhibit the downstream cell proliferation events, but the exact molecular mechanism is still elusive. In this study, expression of hFGF-1 in recombinant E. coli was carried out, and the expressed protein was purified using heparin affinity column chromatography. The structural interactions governing imatinib-hFGF-1 interaction was studied by monitoring its stability, conformation and binding affinity by equilibrium unfolding using steady state fluorescence and proteolytic digestion assay. These data show that imatinib binds to hFGF-1 and enhances its thermal stability and solvent accessibility. In addition, biacore analysis was carried out to determine the binding affinity of imatinib to hFGF-1.

Around 50% of chronic myeloid leukaemia (CML) patients who remain on imatinib treatment for more than 5 years will achieve a complete molecular response (CMR), defined by undetectable BCR-ABL mRNA in a sensitive reverse transcriptase real-time quantitative PCR (RQ-PCR) assay. Given the increasing importance of CMR on imatinib therapy the primary aim of this study was to improve the accuracy and sensitivity of MRD detection to allow a more accurate estimation of relapse risk when therapy is withdrawn. Firstly, we investigated ways of improving the sensitivity of RT-PCR methods for the detection of BCR-ABL mRNA. Secondly, we investigated the use of the patient-specific BCR-ABL gene for the detection of MRD. Thirdly, we conducted a multi-centre clinical trial of imatinib withdrawal in selected CML patients in a stable CMR. This clinical trial provided patient samples that could be used to test our optimized MRD assays, and provided clinical data on the risk and patterns of relapse after withdrawal of imatinib therapy. The trial is ongoing, but an interim analysis of the study data was performed. In 22 patients the estimated probability of molecular relapse after imatinib withdrawal was 54%, and 60% of relapses occurred within the first 4 months. The average detection limit of BCR-ABL mRNA by RQ-PCR is estimated at around 4.5 log below the level of BCR-ABL prior to commencing treatment. The number of leukaemic cells at diagnosis is around 10¹ ², so the number of residual leukaemic cells in CMR might vary from zero to over a million. We hypothesized that the amount of residual leukaemia in CMR is variable between patients, and that this heterogeneity is a determinant of the risk of relapse when treatment is withdrawn. We developed more sensitive methods for the detection of BCR-ABL and tested these methods in samples from our study patients. We showed that random pentadecamer (15-mer) primers improved the efficiency of reverse transcriptase PCR (RT-PCR), and resulted in a lower detection limit of BCR-ABL mRNA. We also developed a novel nested RT-PCR method using real-time PCR for the second round of the reaction, and this resulted in a lower detection limit of BCR-ABL in patient samples. The utility of this nested RT-PCR method was limited by a false positive rate of 2-3% in the HeLa cell line that we used as our negative control. Consequently, we examined the detection of the patient-specific genomic BCR-ABL sequence as an alternative to RT-PCR. Breakpoints in BCR and ABL1 in CML patients are widely dispersed over 3 kb and 150 kb, respectively. Therefore, the BCR-ABL genomic sequence is essentially unique to each patient. We sequenced the genomic breakpoints of 43 CML patients. We showed that the distribution of breakpoints in BCR and ABL1 was non-random, but we were unable to identify any genomic feature that determined the specific location of individual breakpoints. We developed a novel BCR-ABL DNA Q-PCR method for 12 of the study patients, and in 11 of the patients BCR-ABL DNA was detected when the patient was in a CMR, confirming that this method was more sensitive than RQ-PCR. Contrary to our hypothesis, the detection of BCR-ABL DNA was not predictive of relapse. In most patients who relapsed there was a significant increase in BCR-ABL DNA prior to mRNA relapse. Two patients had stable levels of BCR-ABL DNA measurable on multiple occasions, but remained in remission after 6 months and 15 months, respectively. We have shown that a stable CMR after the withdrawal of imatinib therapy does not necessarily indicate the eradication of leukaemia. / Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2010