Дисертації з теми "Minimal Residual Disease Detection"
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1
Uzunel, Mehmet. "The methodology and significance of minimal residual disease detection after allogeneic stem cell transplantation /." Stockholm, 2003. http://diss.kib.ki.se/2003/91-7349-619-7.
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2
Steward, Colin Graham. "Technical aspects of minimal residual disease detection in childhood B-lineage acute lymphoblastic leukaemia." Thesis, University of Bristol, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.241085.
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3
Langlands, Kenneth. "Application of molecular analysis to the detection and study of minimal residual disease in haematological neoplasms." Thesis, University of Edinburgh, 1994. http://hdl.handle.net/1842/19913.
Повний текст джерелаАнотація:
The aims of this study are i) to screen tumour from patients with leukaemia and lymphoma and determine the incidence of tumour markers, t(14;18) translocation, T-cell receptor δ (TcRδ) chain and immunoglobulin heavy chain (IgH) gene rearrangements ii) to develop sensitive PCR based techniques using these tumour markers and iii) to analyse serial remission samples and peripheral blood stem cells (PBSC) for residual tumour. Southern bolt analysis showed that 55% of patients with pre-B acute lymphoblastic leukaemia (ALL) had TcR Vδ2-Dδ3 rearrangements and that 85% of patients with B-lineage disease had IgH rearrangements. PCR analysis showed a TcRδ marker in 53% of pre B ALL and a CDRIII marker in 77% of B-lineage disorders therefore these patients were available for further study of minimal disease. Direct sequence analysis of PCR products from TcR Vδ2-Dδ3 and the third complementarity-determining region (CDRIII) of IgH demonstrated sufficient junctional diversity to permit unique clone specific probes of 20 nucleotides to be designed. Junctional diversity was generated by random N- nucleotide insertion, gene segment deletion and addition of other D segments.
4
Papadaki, Christina [Verfasser], and Karsten [Akademischer Betreuer] Spiekermann. "Detection of minimal residual disease in Acute Myeloid Leukemia with t(8;21) translocation / Christina Papadaki ; Betreuer: Karsten Spiekermann." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2017. http://d-nb.info/1138195545/34.
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5
Dang, Raymond K. B. "Molecular detection of minimal residual disease in breast cancer and leukaemias using p53 tumour suppressor gene mutations as markers." Thesis, University of Edinburgh, 2000. http://hdl.handle.net/1842/22132.
Повний текст джерелаАнотація:
The use of peripheral blood progenitor cell (PBPC) transplantation is an important advance in the treatment of breast cancer and acute leukaemias, and these conditions are among the commonest indications for this procedure. Inevitably, there is concern that malignant cells may contaminate progenitor cell harvests and be re-infused during transplantation and cause disease relapse. Various methods are available for the detection of such minimal residual disease (MRD), and the key aim of this project was to evaluate the feasibility of using a tumour-specific marker, namely mutations within the p53 gene, for this purpose. This provided a useful model to assess the feasibility of using subtle genetic changes to detect MRD within PBPC harvests from patients with malignant diseases. The first step involved the use of denaturing gradient gel electrophoresis (DGGE) to screen original tumour tissues for mutations to be used as disease markers, in 5 individually PCR-amplified DNA fragments (A to E) covering exons 5 to 8 of p53. The technique was first optimised using cell lines known to contain p53 mutations in each fragment. Optimisation was performed with respect to electrophoresis temperature, time, voltage and polyacrylamide cross-linker. The sensitivity of DGGE in detecting a mutation in a mixed cell population was determined by diluting tumour cells in wild type (WT) cells. Although the presence of a mutation could be demonstrated when tumour cells occurred as 5% of total, a representation of at least 40% was required for the mutant homoduplex to be isolated for sequencing. Clinical samples studied were from 51 breast cancer patients, 38 of whom had metastatic disease or at high risk of metastasis, and 13 had high risk stage II/III disease randomised in a clinical study investigating PBPC transplantation and adjuvant therapy, and from 29 patients with acute leukaemias. A positive result was obtained in 14 of 51 primary breast cancer patients (1 was positive in 2 different fragments) and 3 of 29 patients with acute leukaemias.
6
Chan, Wai. "Clonal rearrangement of T-cell receptor delta gene in hematological malignancies and applications in detection of minimal residual disease /." Hong Kong : University of Hong Kong, 1995. http://sunzi.lib.hku.hk/hkuto/record.jsp?B1705512X.
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7
Woerner, Sandra Maria [Verfasser], Monika [Akademischer Betreuer] Engelhardt, and Robert [Akademischer Betreuer] Zeiser. "Establishment of a 6-, 8- and 10-color multiparameter flow cytometry assay for the detection of minimal residual disease in multiple myeloma patients: challenging diversity in a straightforward approach." Freiburg : Universität, 2019. http://d-nb.info/1233196715/34.
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8
Thörn, Ingrid. "Minimal Residual Disease Assessment in Childhood Acute Lymphoblastic Leukemia." Doctoral thesis, Uppsala universitet, Institutionen för genetik och patologi, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-101028.
Повний текст джерелаАнотація:
Traditionally, response to treatment in hematological malignancies is evaluated by light microscopy of bone marrow (BM) smears, but due to more effective therapies more sensitive methods are needed. Today, detection of minimal residual disease (MRD) using immunological and molecular techniques can be 100 times more sensitive than morphology. The main aim of this thesis was to compare and evaluate three currently available MRD methods in childhood acute lymphoblastic leukemia (ALL): (i) real-time quantitative PCR (RQ-PCR) of rearranged antigen receptor genes, (ii) multicolor flow cytometry (FCM) of leukemia-associated immunophenotypes and (iii) real-time quantitative PCR of fusion gene transcripts (RT-PCR). In paper I, we assessed the applicability of RQ-PCR in a population-based cohort of childhood ALL diagnosed in Sweden between 2002-2006. Clonal IG/TCR rearrangements were identified in the 96% of the 279 ALL cases. Using RQ-PCR, the quantitative range of 10-3 was reached in 93% of B-cell precursor (BCP) ALL and 86% of T-cell ALL (T-ALL) by at least one target gene. In paper II, we compared MRD detection using both RQ-PCR and FCM in the context of NOPHO ALL-2000 protocol. By applying the stratification threshold of ≥0.1% MRD late during induction therapy (day 29), we could demonstrate that both methods can predict the risk of BM relapse but not extramedullary relapse. However, the threshold of ≥0.2% MRD appears to be more optimal using RQ-PCR in BCP ALL, whilst in T-ALL, the results indicate that RQ-PCR is preferable for MRD assessment. The stability of RNA in vitro is a critical factor when using sensitive molecular techniques such as MRD detection. In paper III, we evaluated the influence on MRD detection when blood is collected in tubes with RNA stabilization reagents (PAX gene Vacutatiner®) compared to collection in EDTA-tubes (non-stabilized). We analyzed 68 matched samples from chronic myeloid leukemia patients and the results indicated that non-stabilized blood processed within 30 hours is preferable for MRD detection. In paper IV, follow-up samples from eight children with Philadelphia positive (Ph+) ALL were evaluated with the three available MRD methods. MRD measured by the fusion gene transcripts (BCR-ABL1) appeared to be the most sensitive method, however, precise quantification can be difficult and the other methods are thus complementary. In conclusion, all three applied MRD methods are useful and correlate to each other, although not necessary exchangeable in individual patients. We also conclude that MRD assessment by RQ-PCR, based on rearranged IG/TCR genes and multicolor FCM are predictive for identification of high risk childhood ALL patients.
9
Thörn, Ingrid. "Minimal Residual Disease Assessment in Childhood Acute Lymphoblastic Leukemia." Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-101028.
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10
Farahat, Nahla Mohamed Gamal. "Minimal residual disease in acute leukaemia by quantitative flow cytometry." Thesis, Institute of Cancer Research (University Of London), 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.244275.
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11
Lin, Feng. "Minimal residual disease after bone marrow transplantation for chronic myeloid leukaemia." Thesis, Imperial College London, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.285196.
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12
Björklund, Elisabet. "Multiparameter flow cytometry and minimal residual disease in patients with acute leukemia /." Stockholm, 2004. http://diss.kib.ki.se/2004/91-7349-624-3.
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13
Major, Attila Louis. "Photodetection and photodynamic therapy of minimal residual disease in the peritoneal cavity." [S.l. : Amsterdam : s.n.] ; Universiteit van Amsterdam [Host], 2001. http://dare.uva.nl/document/60402.
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14
Smith, Brendan Michael. "The determination of minimal residual disease in patients with primary breast cancer." Thesis, Imperial College London, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.408242.
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15
Provan, Andrew. "Antigen receptor rearrangement in the lymphoid malignancies." Thesis, University of Southampton, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.242480.
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16
Eckert, Cornelia [Verfasser]. "Minimal Residual Disease beim Rezidiv der akuten lymphoblastischen Leukämie im Kindes-/Jugendalter / Cornelia Eckert." Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2016. http://d-nb.info/1102197165/34.
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17
Van, Rhee Frits. "Detection and treatment of residual disease in Philadelphia positive leukaemias." Thesis, Imperial College London, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.285168.
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18
Goulden, Nicholas John. "An assessment of the clinical relevance of minimal residual disease in childhood acute lymphoblastic leukaemia." Thesis, University of Bristol, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.245586.
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19
Kasprzyk, Arkadiusz. "Investigation of clonality and minimal residual disease in haematological malignancy using fluorescent in situ hybridization." Thesis, University College London (University of London), 1998. http://discovery.ucl.ac.uk/1317904/.
Повний текст джерелаАнотація:
Cytogenetic analysis of the malignant clone is clinically important in haematological malignancy. Analysis by metaphase cytogenetics is restricted to the small proportion of malignant cells which are actively dividing. This thesis explores the dynamics of malignant clones using the technique of fluorescence in situ hybridization (FISH) to visualize chromosomal abnormalities in interphase (non-dividing) cells. Hyperdiploid (>46 chromosomes) clones have been investigated by interphase FISH in acute lymphoblastic leukaemia (ALL), acute myeloid leukaemia (AML) and myelodysplastic syndrome (MDS) using appropriate chromosome-specific probes. A hyperdiploid clone was detected in interphase cells in 9/65 patients with ALL in whom metaphase cytogenetics had failed or was normal. A single hyperdiploid cell was identified as clonal in one patient with MDS but not in six others with AML, MDS or ALL. The involvement of different cell lineages in the malignant clone was investigated by simultaneous FISH and identification of the cell type by morphology or monoclonal antibodies. In ALL, hyperdiploid clones were restricted to the lymphoid blasts in 9/9 cases, while Philadelphia (Ph) positive clones, (identified by probes to the genes m- BCR or M-BCR and ABL which fuse as a result of the translocation) were found either in lymphoid blasts alone (1/3 cases) or in both lymphoid and myeloid cells (2/3 cases). In AML trisomy 8 (using a chromosome 8-specific probe) and an 11q23 abnormality (which split YAC 13HH4) were both found only in the myeloid blasts, in 3/3 and 2/2 cases respectively. A sensitive method for the detection of hyperdiploid \geq 50 clones in ALL was developed for minimal residual disease detection. Simultaneous probing of three chromosomes enabled detection of one hyperdiploid cell in 10,000. Heterogeneity in the speed with which the clone was eliminated in remission was seen in 16 patients and early relapse was detected in one patient.
20
Rott, Yvonne [Verfasser]. "DNA-Vakzinierung zur Behandlung von minimal residual disease bei Ph+ akuter lymphoblastischer Leukämie im syngenen Mausmodell / Yvonne Rott." Lübeck : Zentrale Hochschulbibliothek Lübeck, 2013. http://d-nb.info/1037455673/34.
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21
Sugg, Timo Franz [Verfasser], and P. [Akademischer Betreuer] Lang. "Durchflusszytometrische Bestimmung der Minimal Residual Disease bei pädiatrischen Patienten mit akuten Leukämien / Timo Franz Sugg ; Betreuer: P. Lang." Tübingen : Universitätsbibliothek Tübingen, 2012. http://d-nb.info/1160517932/34.
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22
Herbster, Andreas [Verfasser], and P. J. [Akademischer Betreuer] Lang. "Minimal Residual Disease und leukämische Progenitorzellen bei der akuten Leukämie des Kindesalters / Andreas Herbster ; Betreuer: P. J. Lang." Tübingen : Universitätsbibliothek Tübingen, 2013. http://d-nb.info/1160754527/34.
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23
Moppett, John Paul. "Prospective analysis of real time minimal residual disease assessment in childhood acute lymphoblastic leukaemia prior to bone marrow transplantation." Thesis, University of Bristol, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.274837.
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24
Woodward, Eleanor. "The use of gene expression profiling to identify novel minimal residual disease markers (MRD) in acute myeloid leukaemia (AML)." Thesis, Cardiff University, 2010. http://orca.cf.ac.uk/55179/.
Повний текст джерелаАнотація:
Acute myeloid leukaemia (AML) is a heterogeneous disorder characterised by the accumulation of immature haematopoietic cells blocked at various stages of differentiation. Despite improved survival rates over the past decade, relapse occurs in approximately 70% patients undergoing chemotherapy. A potential reason for this is that current clinical protocols do not take account of the level of residual disease present at remission. Therefore, one strategy to reduce relapse rates is to monitor minimal residual disease and continue to treat until the patient is minimal residual disease negative. Current minimal residual disease markers are available for patients with characterised fusion genes but approximately 50% of patients have no detectable chromosomal aberration and therefore are without markers. Gene expression profiling is a powerful tool for disease classification, prognosis and therapeutic predictions. This study aimed to investigate the use gene expression profiling to identify novel minimal residual disease markers for specific AML sub-groups. Patient diagnostic samples were profiled to identify genes specific to AML patients with a favourable translocation in order to establish the "proof-of-principle". Several genes identified were followed in patient diagnostic and follow- up samples and compared to the markers currently used. Continuing with normal karyotype AML, genes were identified as specific to this sub-group. Several homeobox (HOX) genes and the Wilms' tumour (WT1) gene were identified and their MRD levels followed in diagnostic and follow-up samples. Only WT1 identified as specific to normal karyotype AML met the necessary criteria to be an MRD marker. Although the majority of genes selected from the GEP in this study proved unsuitable as markers, the identification and validation of a marker already used for MRD monitoring, WT1, demonstrates the ability of gene expression profiling to identify potential minimal residual disease markers in normal karyotype AML.
25
Knechtli, Christopher John Cranstoun. "The application of minimal residual disease analysis in children and adolescents undergoing allogeneic bone marrow transplantation for acute lymphoblastic leukaemia." Thesis, University of Bristol, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.297806.
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26
Radić, Shechter Ksenija [Verfasser], and Martin [Akademischer Betreuer] Jechlinger. "Exploring the metabolic landscape and resulting vulnerabilities of minimal residual disease in breast cancer / Ksenija Radic Shechter ; Betreuer: Martin Jechlinger." Heidelberg : Universitätsbibliothek Heidelberg, 2019. http://d-nb.info/1205371710/34.
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27
Carvalho, Franceli Ramos. "Avaliação de ensaios comerciais de RT-qPCR para monitoramento de doença residual mínima em pacientes com leucemia mielóide crônica." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2017. http://hdl.handle.net/10183/164468.
Повний текст джерелаАнотація:
A utilização de Inibidores da Tirosino Quinase (ITQ) alterou drasticamente a expectativa de vida do paciente com Leucemia Mielóide Crônica (LMC) e o monitoramento da expressão do oncogene BCR-ABL1 tornou-se um fator prognóstico fundamental para avaliação da resposta ao tratamento. Atualmente, a necessidade de desenvolvimento de metodologias moleculares que facilitem a quantificação rápida, barata e sensível, associada à detecção precoce de baixos níveis de BCR-ABL1, tem proporcionado o surgimento de diversos ensaios comerciais para monitoramento molecular. Entretanto, estes kits possuem uma variabilidade na sua composição, execução e parâmetros analíticos, principalmente com relação à sensibilidade, o que torna os resultados, muitas vezes, não comparáveis. Esse trabalho teve como objetivo revisar a literatura buscando identificar as diferentes opções comerciais disponíveis para o monitoramento do BCR-ABL1, além de comparar os resultados de dois destes ensaios com a metodologia de referência. A partir da revisão realizada, identificamos cinco kits comerciais como principais opções disponíveis para monitoramento de BCR-ABL1 na LMC: GeneXpert® BCR-ABL Assay (Cepheid), Ipsogen® BCR-ABL1 Mbcr Fusion Quant Kit (QIAGEN), BCR-ABL1 Quant RUO™ Assay (Asuragen), LightCycler® t(9;22) Quantification Kit (Roche Molecular Biochemicals) e ODK-1201 (Otsuka Pharmaceutical Co. Ltd.). Posteriormente, comparamos os resultados e avaliamos o desempenho dos ensaios GeneXpert® BCR-ABL e do BCR-ABL1 Quant RUO™ com a metodologia de referência a partir de amostras de 60 pacientes com LMC em uso de ITQ. Identificamos uma concordância global ótima, com coeficientes de correlação de 0,97 (GeneXpert® BCR-ABL Assay) e 0,84 (BCR-ABL1 Quant RUO™ Assay). No entanto, na avaliação da concordância relacionada ao alcance ou não de uma Resposta Molecular Maior (RMM), o ensaio BCR-ABL1 Quant RUO™ apresentou melhores resultados, com uma menor discrepância para respostas moleculares profundas. A análise estratificada por subtipos de transcritos de BCR-ABL1 não mostrou diferença de desempenho entre os dois ensaios. A partir das análises comparativas realizadas e respectivas vantagens de cada teste, aliados aos dados obtidos a partir da revisão da literatura, sugere-se que o GeneXpert® BCR-ABL poderia ser utilizado como um teste primário, devido à rapidez do ensaio, enquanto o BCR-ABL1 Quant RUO™, por apresentar resultados associados a uma maior sensibilidade, poderia ser um teste secundário, a fim de confirmar resultados abaixo de uma RMM ou resultados não detectáveis. Fica evidente que a escolha de um ensaio comercial deve atender às necessidades de cada laboratório, mas que, fundamentalmente, esteja alinhada às recomendações internacionais de quantificação.The use of tyrosine kinase inhibitors (TKIs) has drastically changed the life expectancy of patients with chronic myeloid leukemia (CML) and monitoring the expression of the BCR-ABL1 oncogene has become a key prognostic factor for assessing treatment response. The need to development molecular methodologies that facilitate fast, cheap and sensitive quantification associated with the early detection of low levels of BCR-ABL1 has led to the emergence of several commercial assays for molecular monitoring. However, these kits have variability in their composition, performance and analytical parameters, mainly in relation to the sensitivity, which makes the results often not comparable. This work aimed to review the literature in order to identify the different commercial options available for the monitoring of BCR-ABL1, in addition to comparing the results of two of these tests with the reference methodology. From the review, we identified five commercial kits as the main options available for monitoring BCR-ABL1 in the LMC: GeneXpert® BCR-ABL Assay (Cepheid), Ipsogen® BCR-ABL1 Mbcr Fusion Quant Kit (QIAGEN), BCR-ABL1 Quant RUO™ Assay (Asuragen), LightCycler® t (9; 22) Quantification Kit (Roche Molecular Biochemicals) and ODK-1201 (Otsuka Pharmaceutical Co. Ltd.). Subsequently, we compared the results and evaluated the performance of the GeneXpert® BCR-ABL and BCR-ABL1 Quant RUO™ with reference methodology from samples of 60 patients with CML using TKI. We identified an optimal overall agreement for the two trials, with correlation coefficients of 0.97 and 0.84, respectively. However, in the evaluation of the agreement related to the reach of a Major Molecular Response (MMR), the BCR-ABL1 Quant RUO™ assay presented better results, with a smaller discrepancy for deep molecular responses. Analysis stratified by subtypes of BCR-ABL1 transcripts showed no difference in performance between the two assays. From the comparative analyzes performed and the respective advantages of each test, allied to the data obtained from the literature review, it is suggested that GeneXpert® BCR-ABL assay could be used as a primary test, due to the rapidity of the assay, while the BCR-ABL1 Quant RUO™, for presenting results associated with increased sensitivity, could be a secondary test in order to confirm results below an MMR or undetected results. It is clear that the choice of a commercial assay should meet the needs of each laboratory, but that it is fundamentally in line with international quantification recommendations.
28
Tripiraneni, Gopichand. "Minimal residual disease in bone marrow of patients with breast cancer : kinetics, association with occult clinical metastases and the potential role of multi-mutated HSV1 (JS1/34.5-/47-) as a purging agent." Thesis, Imperial College London, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.501431.
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29
Norén, Nyström Ulrika. "Vascular density and bone marrow fibrosis in childhood acute lymphoblastic leukemia." Doctoral thesis, Umeå universitet, Pediatrik, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-1642.
Повний текст джерелаАнотація:
Background: In childhood acute lymphoblastic leukemia (ALL), the cure rate has now reached 80% in the western world. Even so, 15¬–20% will die from the disease or treatment-related causes, among them children who did not present any known unfavorable features at diagnosis. Treatment of childhood ALL is risk-adapted, meaning that certain factors that are related to the child or the leukemic blasts stratifies to more or less intensive treatment. In this thesis, characteristics of the bone marrow (BM) stroma, reflecting the interaction between the leukemic cells and their microenvironment, were evaluated. The aims were to investigate these factors in relation to other known data in order to further understand the biology of leukemia, and to suggest additional risk factors that would further improve decision making for the treatment of individual children diagnosed with ALL. Methods: We retrospectively investigated microvessel density (MVD), blast-congested vessel fraction (BCVF), and degree of fibrosis – reticulin fiber density (RFD) – in sections from diagnostic BM biopsies from children diagnosed in Umeå, Uppsala, and Stockholm. RFD was also studied in BM sections from treatment day 29. Results: RFD had prognostic impact in patients with high-hyperdiploid (HeH) leukemia. Moreover, rapid reduction of RFD during induction treatment was associated with a favorable prognosis compared to slow reduction, in B-cell precursor (BCP) ALL patients. There was also a correlation between RFD at diagnosis and minimal residual disease (MRD) measured by flow cytometry on treatment day 29 in BCP patients. BCP patients with high RFD and high MVD had an unfavorable outcome compared to all other BCP patients. In addition, MVD and RFD were both associated with immunophenotype, and MVD with cytogenetic aberrations. There was a correlation between MVD and WBC count in BCP high-risk patients. There was also a strong correlation between BCVF and WBC count in all BCP patients, but not between BCVF and MVD or RFD. There was a negative correlation between MVD and in vitro cellular resistance to several drugs in BCP patients. A drug-resistance score combining the drugs most strongly correlated to MVD – cytarabine, doxorubicin, and dexametasone (ADD score) – identified the prognostic potential of ADD score in HeH patients with no unfavorable features. Conclusions: Taken together, these studies indicate that stroma factors in leukemia are related to both phenotypic and genotypic features of acute leukemia. Stroma factors also seem to influence the response to induction treatment, in vitro drug resistance, and outcome in certain subgroups of childhood ALL patients. The results emphasize the importance of BM stroma in leukemia and the need for greater use of BM biopsy at diagnosis.
30
Thiede, Christian, Martin Bornhäuser, and Gerhard Ehninger. "Strategies and Clinical Implications of Chimerism Diagnostics after Allogeneic Hematopoietic Stem Cell Transplantation." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2014. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-137505.
Повний текст джерелаАнотація:
Analysis of donor chimerism has become a routine method for the documentation of engraftment after allogeneic hematopoietic stem cell transplantation (HSCT). In recent years several groups have also focused on the application of this technique for the detection of relapsing disease after allogeneic HSCT. This review addresses technical issues (sensitivity, specificity) and discusses the advantages and limitations of methods currently used for chimerism analysis and their usefulness for the detection of MRD. In addition, the potential impact of novel procedures, e.g. subset chimerism or real-time PCR-based procedures, is discussedDieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich
31
Elorza, Álvarez Izaskun. "Enfermedad mínima residual medida mediante citometría de flujo multiparamétrica en niños con leucemia linfoblástica aguda sometidos a trasplante alogénico de progenitores hematopoyéticos." Doctoral thesis, Universitat Autònoma de Barcelona, 2015. http://hdl.handle.net/10803/310609.
Повний текст джерелаАнотація:
El trasplante alogénico de progenitores hematopoyéticos (alo-TPH) logra mejor supervivencia que la quimioterapia en los niños con leucemia linfoblástica aguda (LLA) de alto riesgo. El principal obstáculo para el éxito del trasplante es la recaída y la remisión completa morfológica pre-trasplante es el factor pronóstico principal para la supervivencia libre de recaída (SLR).
La presencia de enfermedad mínima residual (EMR) en médula ósea previa al trasplante, medida mediante técnicas de reacción en cadena de la polimerasa (PCR) ha demostrado ser un factor independiente de menor SLR en niños con LLA.
La citometría de flujo multiparamétrica (CFM) es una técnica ampliamente utilizada para detectar inmunofenotipos anómalos en el estudio de diagnóstico inicial de la LLA así como para su monitorización a lo largo del tratamiento. En varios estudios se comparan las técnicas de PCR y CFM concluyendo que ambas son complementarias.
El objetivo de esta tesis es determinar si existe relación entre la presencia de EMR medida mediante CFM previa a un alo-TPH en niños con LLA y los resultados post-trasplante; se estudiaran también, otros factores pre y postrasplante asociados a recaída y mortalidad.
La EMR fue cuantificada mediante CFM previamente a un alo-trasplante en 80 niños con LLA (rango: 6 meses-19 años). De acuerdo con el nivel de EMR detectado, los pacientes se dividieron en 2 grupos: el grupo EMR-positiva (n= 25) con presencia de blastos igual o mayor de 0,01% respecto a la población total de células y el grupo EMR-negativa (n=55) con menor de 0,01% de blastos.
La SLR en el grupo completo a los 3 años postrasplante fue del 72% siendo la supervivencia global (SG) del 51%. La SLR en el grupo con EMR positiva fue del 50% comparada con el 80 % del grupo EMR negativa (Log Rank 9,5; p=0,002). La SG global en el grupo de EMR positiva fue del 30% comparada con el 59% del grupo EMR positiva (Log Rank 6,5; p=0,01). La presencia de EMR pretrasplante medida mediante CFM identificó a un grupo de pacientes con 5,5 veces mayor riesgo de recaer y 3,4 veces de fallecer, confirmándose la importancia de su presencia previa al trasplante así como la validez de la prueba para su identificación.
El análisis bivariado realizado mostró que el uso de radioterapia y la presencia de EICH aguda postrasplante fueron factores protectores de recaída, y en el caso de la EICH aguda, también de mortalidad. La SLR a los 3 años postrasplante en los que no presentaron EICHa fue del 36%, en los de grado I-II del 79% y III-IV del 81%. La SG a los 3 años postrasplante en los que no presentaron EICHa fue del 23 %, en los de grado I-II del 56 % y III-IV del 57%.
Al estratificar por EMR se observa, que la presencia de EICH agudo favorece más a los pacientes con EMR positiva pre-trasplante. En relación con los estudios de seguimiento postrasplante, se objetivó que los pacientes que presentaron EMR positiva medida mediante CFM recayeron más que en los que se mantuvo negativa, 88% vs 17%. En este trabajo los estudios de quimerismo postrasplante no ofrecieron datos con valor clínico en relación a la recaída.
Se requieren más estudios para definir nuevos protocolos para el subgrupo de pacientes que presentan EMR positiva previa al trasplante. También, se deberá investigar en la valoración de la EMR y quimerismo postrasplante como factores pronóstico de recaída y qué actitud tomar ante sus resultados.Outcomes with allogeneic haematopoietic stem cell transplantation (allo-HST) are better than with chemotherapy in children with high-risk acute lymphoblastic leukaemia (ALL). The main drawback to successful transplant is relapse. The major prognostic factor for long-term relapse-free survival (RFS) is complete morphological remission prior to transplant. Minimal residual disease (MRD) in bone marrow pre-transplant, measured by polymerase chain reaction (PCR) techniques, has proved to be an independent factor of relapse post-transplant and consequently shorter survival in children with ALL. Multiparametric flow cytometry (MFC) is widely used to detect anomalous immunophenotypes in the diagnostic work-up of ALL and its monitoring throughout treatment. Several studies concluded that PCR and MFC are complementary. This thesis aimed to ascertain whether a relationship exists between MRD prior to allo-HST in children with ALL measured by MFC and outcome, assessed as RFS and overall survival (OS). Furthermore, other pre- and post-transplant factors associated with survival were studied. MRD was quantified by MFC prior to allo-HST in 80 children with ALL (age range: 6 months-19 years). According to the MRD level detected, patients were divided into two groups: MRD- positive (n=25) with blast cells ≥ 0.01% compared with total cell population, and MRD-negative (n=55) with blast cell < 0.01%. RFS at 3 years post-transplant was 72%, with OS 51%. RFS in the MRD-positive group was 50% versus 80% in the MRD-negative group (Log Rank 9.5; p=0.002). OS in the MRD-positive group was 30% versus 59% in the MRD-negative group (Log Rank 6.5; p=0.01). The presence of MRD pre-transplant measured by MFC identified a group of patients with a 5.5- fold greater risk of relapse and 3.4-fold of death, which confirmed the importance of its presence prior to transplant and the validity of the test for its identification. Bivariate analysis showed the use of radiotherapy during conditioning and the presence of acute graft-versus-host disease (aGvHD) post-transplant to be protective factors against relapse and, in the case of aGvHD, also mortality. EFS at 3 years post-transplant was 36% in patients without aGvHD, 79% in those with grades I to II and 81% in those with III to IV. OS at 3 years post-transplant was 23% in patients without aGvHD, 56 % in those with grades I to II and 57 % in those with III to IV. When patients were stratified by MRD, aGvHD favored more those with positive MRD pre-transplant. Regarding post-transplant follow-up studies, patients with positive MRD measured by MFC relapsed less than those who remained negative: 87% versus 17%. This work did not show that chimerism studies post-transplant offered relapse-related data of clinical value. Further studies are required to define new protocols for the subgroup of patients with MRD positive prior allo-HST. Furthermore, MRD and chimerism post-transplant should be assessed as prognostic factors of relapse and their interpretation used as a basis for follow-up.
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Thiede, Christian, Martin Bornhäuser, and Gerhard Ehninger. "Strategies and Clinical Implications of Chimerism Diagnostics after Allogeneic Hematopoietic Stem Cell Transplantation." Karger, 2004. https://tud.qucosa.de/id/qucosa%3A27736.
Повний текст джерелаАнотація:
Analysis of donor chimerism has become a routine method for the documentation of engraftment after allogeneic hematopoietic stem cell transplantation (HSCT). In recent years several groups have also focused on the application of this technique for the detection of relapsing disease after allogeneic HSCT. This review addresses technical issues (sensitivity, specificity) and discusses the advantages and limitations of methods currently used for chimerism analysis and their usefulness for the detection of MRD. In addition, the potential impact of novel procedures, e.g. subset chimerism or real-time PCR-based procedures, is discussed.Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.
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Chen, Yu-Hsiang. "Multi-Modality Plasma-Based Detection of Minimal Residual Disease in Triple-Negative Breast Cancer." Diss., 2019. http://hdl.handle.net/1805/20202.
Повний текст джерелаАнотація:
Indiana University-Purdue University Indianapolis (IUPUI)Triple-negative breast cancers (TNBCs) are pathologically defined by the absence of estrogen, progesterone, and HER2 receptors. Compared to other breast cancers, TNBC has a relatively high mortality. In addition, TNBC patients are more likely to relapse in the first few years after treatment, and experiencing a shorter median time from recurrence to death. Detecting the presence of tumor in patients who are technically “disease-free” after neoadjuvant chemotherapy and surgery as early as possible might be able to predict recurrence of patients, and then provide timely intervention for additional therapy. To this end, I applied the analysis of “liquid biopsies” for early detection of minimal residual disease (MRD) on early-stage TNBC patients using next-generation sequencing. For the first part of this study, I focused on detecting circulating tumor DNA (ctDNA) from TNBC patients after neoadjuvant chemotherapy and surgery. First, patient-specific somatic mutations were identified by sequencing primary tumors. From these data, 82% of the patients had at least one TP53 mutation, followed by 16% of the patients having at least one PIK3CA mutation. Next, I sequenced matched plasma samples collected after surgery to identify ctDNA with the same mutations. I observed that by detecting corresponding ctDNA I was able to predict rapid recurrence, but not distant recurrence. To increase the sensitivity of MRD detection, in the second part I developed a strategy to co-detect ctDNA along with circulating tumor RNA (ctRNA). An advantage of ctRNA is its active release into the circulation from living cancer cells. Preliminary data showed that more mutant molecules were identified after incorporating ctRNA with ctDNA detection in a metastatic breast cancer setting. A validation study in early-stage TNBC is in progress. In summary, my study suggests that co-detection of ctDNA and ctRNA could be a potential solution for the early detection of disease recurrence.
2021-08-05
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Al-Mawali, Adhra Hilal Nasser. "Novel multiparameter flow cytometry techniques for the detection of leukaemia associated phenotypes and minimal residual disease monitoring in acute myeloid leukaemia." 2008. http://hdl.handle.net/2440/47916.
Повний текст джерелаАнотація:
Despite high remission rate in acute myeloid leukaemia (AML) after chemotherapy, relapse of the underlying disease remains a major challenge and one of the most frequent causes of treatment failure. In this study, the presence of leukaemiaassociated phenotypes (LAPs) was first studied retrospectively using our standard diagnostic protocol with 3-colour flow cytometry. LAPs were present in 54 (64%) of 84 AML patients analysed between 2002 to 2004. The presence of LAPs was correlated with failure to respond to induction chemotherapy (p <0.05) in univariate analysis. Presence of LAPs was shown to be an independent predictor for failure to respond to induction chemotherapy with a relative risk ratio of 1.6 (p < 0.05, 95% CI, 1.0-2.6) in multivariate analysis. Subsequently, in a prospective study, we used 5-colour multiparametric flow cytometry (MFC) for detection of LAPs to determine if LAPs could be detected in a greater proportion of leukaemic patients and minimal residual disease (MRD) detection could therefore be applied in more patients. In 54 consecutive, newly diagnosed AML patients from 2005 to 2007, LAPs were identified in 51 (94%). Thus, MRD studies were potentially applicable to virtually all patients. The sensitivity and specificity of MFC technique was improved by analysing 10 normal and 5 regenerating bone marrows (BM) for the presence of these LAPs and by determining maximum log difference (LD). CD7, CD19, CD2, CD11b and CD56 were the most sensitive and reliable markers for MRD studies. LAPs were rarely detected in either normal or regenerating BMs. Through dilutional experiments from 50% LAPs to 0.001%, it was determined that 1 leukaemic in 104 and 105 normal cells could be detected using the improved techniques. Of the 54 patients, 31 received chemotherapy, with 27 achieving complete remission (CR). Two were LAP negative and thus 25 were evaluable for MRD post induction and 22-post consolidation chemotherapy. Detection of MRD >0.15% was able to distinguish between two groups of patients according to relapse status. Although, the number of patients was small, detection of MRD post induction > 0.15% was shown to be an independent predictor of adverse prognosis for both relapse free survival (RFS) and overall survival (OS) in a multivariate analysis [p = 0.037 and 0.026, 95% CI (1.1-20.5 and 1.2-22.2), hazard ratio 4.7 and 5.2 respectively]. Post consolidation, there was a trend for patients with higher MRD values to show shorter RFS (p = 0.06). MFC using 5-colour allows us to detect LAPs in virtually all AML patients and our preliminary results suggest the technique is a suitable approach for MRD analysis. However, 5-colour MFC is technically challenging, resource intensive, and may not be feasible in a routine diagnostic laboratory. This led us to assess whether we could identify other potential markers for LAPs. Interleukin-3 alpha receptor- chain IL-3_ (CD123) has been suggested to be a marker of leukaemic stem cells (LSC). These cells are thought to be responsible for initiating and maintaining leukaemic cell growth post chemotherapy and hence to give rise to relapse of the disease. Therefore, we analysed 34 AML patients for expression of CD123 in the blast population and defined a population containing leukaemic stem cells using the immunophenotypic markers CD123+/CD34+/CD38-. Thirty-two (94%) of AML patients expressed CD123. We then used a molecular marker to determine whether CD123 expression was confined to the LSC. Thirtynine patients were screened for the presence of FMS-like tyrosine kinase 3 - internal tandem duplication (FLT3/ITD) as the most common molecular abnormality in AML patients. Of those, 12 (31%) were FLT3/ITD positive. In seven of them, CD34+/CD38-/CD123+ and CD34+/CD38-/CD123- populations were sorted to homogeneity by Fluorescence Activated Cell Sorting (BD FACSAriaTM Cell Sorter) and tested for FLT3/ITD. In six of seven patients with FLT3/ITD positive AML, we could not detect the mutation in the CD34+/CD38-/CD123- fraction, but the mutation was detected in the CD34+/CD38-/CD123+ fraction in all seven patients. This novel finding demonstrates that, the oncogenic event occurs in CD123 positive cells, thus supporting the concept that CD123 is a marker of the LSC in CD123 positive AML. This observation suggests novel treatment approaches employing surface marker CD123-targeting antibodies may be of use in the treatment of AML. In conclusion, we demonstrate that using five-colour MFC improves LAP detection in AML and enables MRD studies using immunophenotyping to be applied to virtually all AML patients. Additionally, it increases the sensitivity of the technique for detecting LAP populations. Moreover, evaluation of MRD post induction chemotherapy is the most sensitive time point for detection of MRD, with MRD levels >0.15% predicting relapse and worse prognosis. As an alternative to using individualised LAPs specific to each patient, CD34+/CD38-/CD123+ cells may in the future serve as a better marker for MRD studies. This marker identifies the putative LSC, which is responsible for regrowth of leukaemia and relapse of the disease. Thus, instead of looking at whole “blast” population which results in huge data analysis and interpretation for the different LAPs which may have different underlying biology, it may be more informative to look at the frequency of LSC after achieving CR using CD34+/CD38-/CD123+ as the single LAP for MRD studies.http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1317088
Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2008
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Yeung, David Tak On. "Prognostic markers associated with tyrosine kinase inhibitor treatment response and maintenance of treatment free remission in chronic myeloid leukaemia." Thesis, 2016. http://hdl.handle.net/2440/119800.
Повний текст джерелаАнотація:
Treatment outcomes in Chronic Phase Chronic Myeloid Leukaemia (CP-CML) have dramatically improved with the introduction of highly active tyrosine kinase inhibitors (TKIs). However, treatment responses are highly heterogeneous. The aim of this thesis is to identify prognostic markers that may help individualise treatment and optimise outcome by stratifying patients into risk groups. Selective treatment intensification is important - more potent treatment may be associated with increased toxicity, and universal adoption of the most potent treatment does not optimally balance risk versus benefit, nor is such a strategy cost effective. This thesis will summarise factors with prognostic significance in CP-CML. Such predictive factors include the metric called the “halving time”, which measures the velocity of BCR-ABL1 decline with initial TKI treatment. This correlates well with future treatment response and risk of disease progression. Conversely, the treatment resistance can be measured by the speed at which the BCR-ABL1 rises, in a similar metric called the “doubling time”. A patient’s Killer Immunoglobulin-like Receptor (KIR) genotype is also correlated with survival as well as molecular outcomes. Whilst the biological basis for this interaction is poorly understand, it is believed to be underpinned by the innate immune system’s role in tumour surveillance and suppression. In the setting of disease resistance mediated by BCR-ABL1 kinase domain mutations, demonstrating an increased number of low level mutants via the use of ultra sensitive mutation detection techniques may help prognosticate patients with a history of the T315I mutation. Early molecular response (EMR, BCR-ABL1 ≤10% at 3 months after starting treatment) is currently acknowledged as one of the strongest prognostic markers, and salvage strategies targeting patients failing to achieve time dependent molecular targets may be an optimal point of intervention. The TIDEL-II study examined such a strategy, firstly by imatinib dose escalation, followed by switching to nilotinib, in patients who fail to achieve time dependent molecular responses. This study also examined the effectiveness of increasing imatinib dose in patients with serum trough levels <1000 ng/mL, a threshold thought to be necessary to achieve cytogenetic response. Although survival and overall molecular response in TIDEL-II is excellent, this strategy was found to be of only marginal benefit in those who failed to achieve EMR. The subsequent study, Pinnacle, aims to study the combination of pegylated interferon and nilotinib in a similar context. Enrolment in this study is ongoing. The same prognostic marker (EMR) may identify patients unlikely to achieve deep molecular responses (DMR), a milestone associated with excellent long term event free survival. It is also commonly stipulated as a pre-requisite to participation in treatment cessation studies. Consistent performance of BCR-ABL1 qRT-PCR assays of a sufficient quality to determine this may be a challenge for some laboratories. To avoid false negatives which may lead to inappropriate reassurance and cessation attempts, we have developed an improved protocol that reliably achieves detection sensitivity required to classify patients in DMR. This assay may also be used as a basis for further enhancing BCR-ABL1 qRT-PCR sensitivity, aimed at identifying patients with an ultra-low level of residual disease. Such patients are hypothesised to have negligible risk of molecular recurrence upon treatment cessation, versus patients who have residual disease just below the current limit of detection. Such ultra-sensitive BCR-ABL1 qRT-PCR may, in this manner, contribute to further elucidation of the prognostic markers of successful treatment cessation. The thesis will conclude with prospects in prognostic markers in CML research and clinical management.Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Medicine, 2016.
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Lin, Pei-Chin, and 林佩瑾. "The incidence of TEL-AML1 and BCR-ABL(p190) fusion transcripts in childhood acute lymphoblastic leukemia of Southern Taiwan and application of the fusion transcripts in the detection of minimal residual disease." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/50080739003767682043.
Повний текст джерелаАнотація:
碩士高雄醫學大學
醫學研究所碩士班
93
Chromosome abnormalities were found in 80% to 90% of childhood acute lymphoblastic leukemia (ALL) cases. These leukemia-specific chromosome aberrations not only have prognostic value but also provide important clues for further investigation of leukogenesis , leukemic cell transformation and proliferation. Since the first fusion gene , BCR-ABL in t(9;22) , was discovered , many other chromosome aberrations with fusion genes have been identified. For instance , t(12;21) with TEL-AML1 fusion gene have been noted as the most frequent rearrangement in child hood ALL, whereas t(4;11) with MLL-AF4 fusion gene has been identified particularly in infant ALL. Advancement in molecular biology leads to accurate detection of these leukemia-specific chromosome aberrations with sensitive techniques, such as fluorescence in situ hybridization (FISH) , Southern blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR). Leukemia-specific fusion genes have been used as molecular markers for minimal residual disease (MRD) monitoring. The remaining leukemic cells below the threshold of cytomorphological techniques (sensitivity around 1%~5%) was referred to as MRD. Several studies showed that existence of MRD during the maintenance phase is a independent poor prognostic factor. Monitoring MRD at consecutive time points can give clinical relevant insight into the effectiveness of treatment. In the present study, we apply RT-PCR technique in the detection of two leukemia-specific chromosome fusion genes, TEL-AML1 fusion gene and BCR-ABL p190 fusion gene. We also monitor the expression level of these fusion genes at sequential time points of the treatment course. Twenty-nine patients were enrolled in the study including 20 newly-diagnosed ALL, 5 relapsed ALL, 2 newly-diagnosed AML , 1 relapsed AML and one JMML. Of total 25 ALL cases, TEL-AML1 fusion gene was detected in 8 patients (6 newly-diagnosed and 2 relapsed cases) and BCR-ABL p190 was detected in only 2 newly-diagnosed cases. The incidence of TEL-AML1 fusion gene and BCR-ABL p190 fusion gene in our cases were 32 % and 10 % respectively. The clinical features of our eight TML-AML1 positive ALL cases were similar to the other studies except two of them were younger than 12 months old. Also, no t(12;21) was detected by the conventional cytogenetic study in our cases as it was identified as a cryptic chromosome translocation. All cases but one (TML-AML1 negative and BCR-ABL p190 negative) achieved cytomorphological remission after induction therapy and one patient among the 8 TML-AML1 positive cases relapsed during the consolidation phase. The other 18 newly-diagnosed ALL patients remained in remission status with follow-up period ranged from 1 month to 24 months. Blotting analysis was used for minimal residual disease detection by TEL-AML1 fusion transcript in the eight TML-AML1 positive ALL patients. Among six patients whose bone marrow or peripheral blood samples were obtained after treatment , reduction of TEL-AML1 expression levels were found in four. For further investigation of the accuracy and clinical implication of our methods , more experience and a large study group will be needed.
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Ross, David Morrall. "Minimal residual disease in chronic myeloid leukaemia after imatinib treatment." 2010. http://hdl.handle.net/2440/60064.
Повний текст джерелаАнотація:
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
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Ross, David Morrall. "Minimal residual disease in chronic myeloid leukaemia after imatinib treatment." Thesis, 2010. http://hdl.handle.net/2440/60064.
Повний текст джерелаАнотація:
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
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Hempel, Katharina. "Vergleichende Analyse von Antigenexpressionsmustern kindlicher cALL-Blasten und gesunden B-Zellvorstufen – Nutzen für die MRD Diagnostik." Doctoral thesis, 2019. https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-179203.
Повний текст джерелаАнотація:
Die MRD Diagnostik ist von erheblicher Bedeutung für die Risikostratifizierung kindlicher Leukämien. Um aber gesunde, sich regenerierende Vorstufen von blastären Zellen unterscheiden zu können ist die genaue Kenntnis des Antigenverlaufs sowohl der Vorstufen der B-Zellreihe als auch der Blasten notwendig. In dieser Arbeit wird eine Vergleichende Analyse von B-Zellvorstufen und Blasten mittels Durchflusszytometrie durchgeführt. Von besonders diskriminativem Wert waren die Vorläufermarker CD10, CD34, sowie die lymphatischen Marker CD19, CD20, CD22, CD45, cyCD79a und cyTdT. Zur Beschreibung des individuellen LAIP eigneten sich vor allem die Marker CD11b, CD38, CD58, CD123 und CD133, sowie die myeloischen Marker CD13 und CD33. Die Bessere Unterscheidung zwischen gesunden und kranken Zellen zusammen mit neuen Entwicklungen in Diagnostik und Therapie muss in Zukunft zur weiteren Verbesserung der Überlebensraten, auch im Rezidiv führenMRD diagnostic is important for the ongoing therapy of cALL in children. For better discrimination between blasts and b-cell progenitors we used flow cytometry to analyse the antigen expression on b cell progenitors with those on cALL blasts
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Schwenke-Pillich, Cornelia [Verfasser]. "Minimal Residual Disease (MRD) : Monitoring bei niedrig-malignen Non-Hodgkin-Lymphomen, Chemotherapie + Rituximab vs. Chemotherapie / vorgelegt von: Cornelia Schwenke-Pillich." 2009. http://d-nb.info/992829739/34.
Повний текст джерела
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Schröder, Katrin [Verfasser]. "Minimal-residual-disease-Status direkt vor und zu verschiedenen Zeitpunkten nach Stammzelltransplantation bei Akuter Lymphatischer Leukämie im Kindesalter / vorgelegt von Katrin Schröder." 2009. http://d-nb.info/997711965/34.
Повний текст джерела
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Carreira, Joana Sofia Camilo. "Detection of leukemia-associated immunophenotypic protein markers in circulating extracellular vesicles from acute myeloid leukemia patients." Master's thesis, 2020. http://hdl.handle.net/10316/94281.
Повний текст джерелаАнотація:
Dissertação de Mestrado em Investigação Biomédica apresentada à Faculdade de MedicinaA leucemia mieloide aguda (LMA) é uma neoplasia hematológica caracterizada pela acumulação de progenitores mieloides leucémicos na medula óssea. A elevada taxa de mortalidade aos 5 anos após o diagnostico está relacionada com a presença de quantidades residuais de células leucémicas que são responsáveis pela recaída e que não são detetadas pelos atuais métodos de diagnostico. Desta forma, a monitorização da doença residual mensurável (DRM), utilizando técnicas moleculares mais sensíveis, permite a identificação dos doentes com risco acrescido de recidiva. Marcadores proteicos imunofenotipicos associados à leucemia (LAIPs) são atualmente úteis para detetar DRM. Infelizmente, esta monitorização não é realizada frequentemente e em tempo-real devido á necessidade de recolha invasiva de um aspirado de medula óssea. Assim, a identificação de biomarcadores de DRM, baseados numa biópsia líquida utilizando sangue periférico dos doentes, permitiria uma monitorização em tempo real e menos invasiva da DRM. Recentemente, as vesículas extracelulares (EVs) foram reconhecidas como uma potencial fonte de biomarcadores do cancro em biópsias líquidas.Neste trabalho procuramos validar um protocolo de isolamento de EVs, usando o sangue periférico de doentes com LMA e ainda verificar a presença de LAIPs nas EVs em circulação ao longo dos diferentes estádios da doença.Para tal, os constituintes do plasma pobre em plaquetas foram separados por tamanho, utilizando um protocolo de cromatografia por exclusão molecular (SEC), sendo as EVs posteriormente concentradas por ultrafiltração (UF). A recuperação da amostra foi analisada nas diferentes fases de isolamento. As EVs eluídas foram caracterizadas de acordo com seu tamanho, concentração e conteúdo proteico. Por fim, a presença de vários LAIPs nas EVs foi analisada e semi-quantificada usando Western Blot. O protocolo implementado permitiu o isolamento de EVs de acordo com o seu tamanho, a partir do sangue periférico de doentes com LMA. As EVs isoladas apresentaram um tamanho que variou entre os 50nm a 300nm, eram abundantes no plasma destes doentes e expressavam marcadores proteicos associados a EVs, tais como a CD63, HSP70, Anexina XI, CD81, CD9 e a Mitofilina. A UF permitiu aumentar a concentração proteica e o número de partículas necessários à etapa seguinte. Por fim, vários marcadores imunofenotipicos clinicamente estabelecidos associados à LMA (LAIPs) foram identificados nas EVs circulantes destes doentes, revelando uma expressão diferencial nos vários estádios da doença. Concluindo, validamos com sucesso um protocolo para o isolamento de EVs a partir do sangue de doentes com LMA. Além disso, os resultados preliminares sugerem que alguns LAIPs poderão ter potencial como biomarcadores de DRM em EVs de plasma sanguíneo, uma vez que seus níveis variam de acordo com o estádio da doença. Trabalho futuro irá confirmar esta hipótese.
Acute myeloid leukaemia (AML) is a hematopoietic stem cell disorder characterized by the accumulation of myeloid progenitors in the bone marrow. The poor 5-year survival rate is related to the undetected residual amounts of leukemic cells by microscopic assessment, which translates into a high frequency of post-treatment relapse. Monitoring this measurable residual disease (MRD) by sensitive molecular techniques allows the identification of patients who are at risk for an early relapse. Many different leukaemia associated immunophenotypic protein markers (LAIPs) are presently useful to detect MRD. Their analysis requires invasive bone marrow aspirates, thus severely hindering real-time monitoring of the disease. Therefore, the identification of innovative liquid biopsy-based biomarkers of MRD in patients’ peripheral blood (PB) would allow minimally invasive and real-time monitoring of MRD. Recently, extracellular vesicles (EVs) have been recognized as a potential source of cancer biomarkers.Here we aimed to implement and validate an EV isolation protocol from the PB of AML patients and perform a comprehensive characterization of LAIPs in the AML patients’ circulating EVs throughout the different stages of the disease.For that, Poor Platelet Plasma constituents were resolved by size, using a size-exclusion chromatography (SEC) method, and were further concentrated by an Ultrafiltration (UF) approach. The recovery yield of the sample was verified in each stage of this two-step protocol. The isolated EVs were characterized accordingly to their size, concentration (by Nanoparticle Tracking Analysis), and protein content (by Western Blot). Finally, the LAIPs presence in EVs was analysed and semi-quantified by WB following protein extraction.This two-step protocol allowed the isolation of intact and size resolved EVs from the PB of AML patients. The isolated EVs had a size ranging from 50nm to 300nm, were highly abundant and expressed EV-associated protein markers such as CD63, HSP70, Annexin XI, CD81, CD9, and Mitofilin. The UF allowed the required protein and particle concentration required for downstream analysis by WB. Importantly, several LAIPs were identified in paired samples of diagnosis, remission and relapse of seven patients’ circulating EVs, revealing a differential expression throughout the disease evolution.Taken together, a two-step protocol was validated for the isolation of EVs from the PB plasma and several EV-associated LAIPs were monitored in the blood of AML patients throughout distinct stages of the disease. The preliminary results indicate that some of these markers may have potential as MRD biomarkers detected in EVs from blood plasma, since their levels vary according to the disease status. Future work will confirm this possibility.
Outro - This work was supported by FEDER – Fundo Europeu de Desenvolvimento Regional through COMPETE 2020 and by FCT - Foundation for Science and Technology, in the framework of project POCI-01-0145-FEDER-030457
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Bielčiková, Zuzana. "Cirkulující nádorové buňky u pacientek s karcinomem prsu." Doctoral thesis, 2017. http://www.nusl.cz/ntk/nusl-370957.
Повний текст джерелаАнотація:
Circulating tumor cells (CTCs) represent a systemic phase of the localised cancer disease. They can be distinguished and enriched from the peripheral blood and so from the surrounding leukocytes by either physical properties (e.g., density and size) or biological properties (e.g., expression of epithelial proteins such as EpCAM or cytokeratins) and are usually further characterized by immunostaining or RT-PCR assays. Selecting patients with the risk of disease relaps at the time of diagnosis is crucial for clinicians in deciding who should, and who should not, receive adjuvant chemotherapy. We know that CTCs are strong prognostic factor in patients with metastatic as well as localized breast cancer (BC). It is also known that the prognostic power of circulating tumor cells in women with BC is independent from the standard prognostic indicators. Testing of CTCs known recently as "liquid biopsy" could be informative not only as predictor of the disease relapse, but also as the predictor of therapy effectiveness. The clinical use of CTCs must be strictly encouraged by clinical trials results. Monitoring of CTCs in time could zoom in the mechanism of therapy resistance and/or may provide the identification of new druggable targets. The purpose of my work was therefore to assess the CTCs positivity rate...
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"Early detection of dementia of the Alzheimer's type: examining the use of cognitive tasks and neuropsychological tests for Chinese with minimal education." Thesis, 2011. http://library.cuhk.edu.hk/record=b6075474.
Повний текст джерелаАнотація:
Chang, Jianfang.Thesis (Ph.D.)--Chinese University of Hong Kong, 2011.
Includes bibliographical references (leaves 183-217).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstract also in Chinese.
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Pokorná, Kateřina. "Preklinický model akutní promyelocytární leukemie: studium anti-leukemického efektu vyvolaného pomocí ATRA a DNA vakcinace." Doctoral thesis, 2012. http://www.nusl.cz/ntk/nusl-309473.
Повний текст джерелаАнотація:
DOCTORAL THESIS 2012 POKORNA Abstract We have used a well characterized transplantable transgenic mouse model which mimics human acute promyelocytic leukemia (APL), both in its biological characteristics and its response to conventional therapeutic drugs. The aim of our study was to better characterize the efficacy of the combined treatment and to determine molecular markers of clinical outcome. We established a minimal residual disease monitoring based on the high sensitivity of detection of PML-RAR transcripts by polymerase chain reaction (PCR) technology in APL mice. We showed that oncogene-specific PCR-based assays allow, like in patients, the diagnosis, follow-up and prediction of disease evolution. Furthermore, PCR assay was used to assess various tissues and organs for the presence of PML-RAR-positive cells in minimal residual disease free long-term survivors. As expected, majority of mice had no measurable tissue level of PML-RAR demonstrating the efficacy of immunotherapy. However, tracking the oncogene-positive cells reveals for the first time that extramedullary PML-RAR-positive cell reservoirs such as the brain may persist and be involved in the leukemia relapse. We aimed at investigating the immune responses involved in the anti-leukemic effect of the combined immutherapy. To evaluate the...
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Monteiro, Marta Filipa Pimenta Rainho. "O papel da Citometria de Fluxo na avaliação da doença residual mínima em cães com Linfoma B difuso de células grandes." Master's thesis, 2019. http://hdl.handle.net/10400.26/29654.
Повний текст джерелаАнотація:
O linfoma representa um grupo heterógeno de neoplasias com origem na alteração maligna
das células linfoides. Dependendo do tipo de células afetadas e do grau de desenvolvimento da doença,
o linfoma pode apresentar diferentes comportamentos biológicos e prognósticos.
Em cães, o linfoma é o tumor hematopoiético mais comum, apresentando várias semelhanças
com o linfoma não-Hodgkin em humanos. Cerca de 70% dos casos de linfoma no cão têm origem em
células B, sendo o linfoma B difuso de células grandes um dos subtipos mais comuns.
Apesar do linfoma B difuso de células grandes constituir uma neoplasia de alto grau, tem-se
verificado melhorias na abordagem terapêutica e consequentemente no prognóstico. Não obstante, a
maioria dos cães que são submetidos a quimioterapia e atingem remissão completa, eventualmente
apresentam uma recidiva tumoral.
A avaliação do número de células neoplásicas que permanecem num paciente durante ou após
ser submetido a tratamento, ou seja, o grau de doença mínima residual, permite correlacionar com a
possibilidade de remissão clínica ou recidiva tumoral. Um aumento da quantificação da doença mínima
residual durante o tratamento quimioterápico é indicativo de uma inadequada eficácia do mesmo. Por
sua vez, caso se verifique o aumento da quantificação da doença mínima residual após tratamento, é
possível inferir acerca de uma possível recidiva.
A deteção da doença mínima residual em animais com linfoma B difuso de células grandes é
feita com recurso a várias técnicas, no entanto, a citometria de fluxo demonstrou ser relevante em cães
com a referida patologia.
Em Medicina Veterinária, a citometria de fluxo tem-se revelado de grande utilidade na análise
e caracterização imunofenotípica do linfoma maligno na espécie canina. Quanto ao linfoma B difuso de
células grandes, esta técnica tem permitido avaliar a infiltração neoplásica em amostras de sangue
periférico, medula óssea e linfonodos.Lymphoma represents a heterogeneous group of neoplasms arising from the transformation of malignant lymphoid cells. Depending upon the type of cells affected and the degree of the development of the disease, lymphoma is associated with different behaviour and prognosis. In dogs, lymphoma is considered the most common hematopoietic malignant neoplasm, presenting several similarities with non-Hodgkin's lymphoma, in humans. About 70% of lymphoma cases may have originated in B cells with diffuse large B-cell lymphoma is one of the most common subtypes. Although diffuse large B-cell lymphoma is a high-grade neoplasia, improvements have been observed nowadays in treatment and therefore in its prognosis. However, most patients that undergo chemotherapy and achieve complete remission then might eventually relapse. The quantification of minimal residual disease which is the neoplastic cells that remain in a patient after or under a chemotherapy treatment, allows to correlate with clinical remission or relapse. If the quantification of minimal residual disease found to be high under therapy that indicates an inadequate treatment response. Otherwise if the quantification of minimal residual disease found to be high after therapy it could represent an eventual relapse. The quantification of minimal residual disease in animals with diffuse large B-cell lymphoma is performed by several different techniques, however, flow cytometry has demonstrated to be relevant in dogs with this specific neoplasm. In veterinary medicine, flow cytometry has shown to be very useful in the immunophenotypic analysis and characterization of lymphoma within dogs. About the diffuse large B cell lymphoma, these technique has allowed the evaluation of neoplasm infiltration in the peripheral blood, bone marrow and lymphnodes.
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Ghahremanlou, Mohsen. "Development and Application of Serum Assay to Monitor Response to Therapy and Predict for Relapse in Acute Myeloid Leukemia." Thesis, 2013. http://hdl.handle.net/1807/42846.
Повний текст джерелаАнотація:
The diagnosis and monitoring of AML relies predominantly on the identification of blast cells in the bone marrow and peripheral blood. While at the time of diagnosis the identification of leukemic cells is relatively easy, during remission the identification of small numbers of blasts is problematic. This is most evident by the fact that patients who achieve complete remission frequently relapse, despite pathologic examination indicating a marked reduction in leukemic cell burden. In this thesis I have explored the potential of using serum proteins secreted by leukemic cells as a means of monitoring disease in patients. To identify proteins that might be useful for monitoring, I took advantage of published gene expression arrays and looked into online bioinformatics databases. Using specific characteristics, I was able to identify approximately 107 candidate proteins secreted by AML cells. RT-PCR analysis and ELISA assays were performed to evaluate the variability of expressions and serum level differences of twelve different proteins in the list.
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Jančušková, Tereza. "Využití nových molekulárních technologií v identifikaci unikátních klonálních markerů pro monitorování minimální reziduální nemoci u akutních leukémií." Doctoral thesis, 2015. http://www.nusl.cz/ntk/nusl-334587.
Повний текст джерелаАнотація:
Acute leukemias (AL) comprise a heterogeneous group of hematologic malignancies, and individual patient responses to treatment can be difficult to predict. Monitoring of minimal residual disease (MRD) is thus very important and holds great potential for improving treatment strategies. Common MRD targets include immunoglobulin heavy chain or T-cell receptor gene rearrangements, recurrent cytogenetic abnormalities and mutations in important hematological genes. Whereas in the majority of adult acute lymphoblastic leukemia patients a suitable MRD target can be identified, in adult acute myeloid leukemia patients well-characterized targets are found in only half of cases. The identification of new specific molecular markers of leukemic blasts for MRD assessment, particularly in AML patients, is therefore highly desirable. Our aim was to develop a flexible strategy for mapping of cytogenetically identified unique clone-specific abnormalities down to the single nucleotide level and, based on the sequence, design a specific real-time PCR assay for MRD assessment in AL patients without any previously described MRD marker. Using a combination of cytogenetic (chromosome banding, chromosome microdissection), molecular cytogenetic (mFISH, mBAND) and molecular biological (next- generation sequencing, long-range...
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Volejníková, Jana. "Prognóza akutní lymfoblastické leukémie u dětí v závislosti na nových klinicko-biologických faktorech." Doctoral thesis, 2013. http://www.nusl.cz/ntk/nusl-328719.
Повний текст джерелаАнотація:
Great progress has been achieved in the diagnostics and therapy of childhood acute lymphoblastic leukemia (ALL) during the last few decades and the permanent cure rate for children and adolescents has risen to nearly 90%. The basic principle of ALL treatment is to split patients into several groups receiving treatment of different intensity according to exactly defined prognostic features. This is aimed at reducing both the risk of relapse and toxic complications of treatment. The development of new diagnostic methods, especially in the field of molecular genetics and flow cytometry, allowed further improvements in the risk stratification - the minimal residual disease (MRD) has become a crucial prognostic factor in modern treatment protocols for pediatric ALL as a sensitive marker of both response to therapy and subclinical leukemic involvement of various tissues of the organism. Nevertheless, there is still an intensive search for new markers that would enable even more precise characterization of the leukemic clone, and treatment strategies reflecting the biology of leukemic cells are being optimized. The first part of our study describes the monitoring and prognostic impact of MRD in peripheral blood of children with ALL with emphasis on very early time points of treatment. MRD was examined by the...
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Kramarzová, Karolina. "Role genu WT1 a jeho izoforem v hematopoeze a leukemogenezi." Doctoral thesis, 2013. http://www.nusl.cz/ntk/nusl-321451.
Повний текст джерелаАнотація:
61 Summary Wilms' tumor gene 1 (WT1) is highly expressed in acute leukemia and other hematological malignancies. It has been therefore suggested as a potential universal marker of minimal residual disease (MRD), particularly in patients with acute myeloid leukemia (AML). Due to controversial results of some of the studies, the role of WT1 in MRD follow-up and WT1 prognostic significance remain unclear. WT1 protein is produced in more than 36 different isoforms. These variants have distinct, partially overlapping functions and their ratio is supposed to influence the final effect of WT1. However, despite the increasing number of studies, the clinical impact of WT1 and its isoforms in acute leukemia have not yet been elucidated. We established a unique qPCR method to assess the expression pattern of the main 4 WT1 isoforms. Using this method, we determined the ratio of WT1 variants in the samples of patients with AML, myelodysplastic syndrome (MDS) and healthy controls. Our data showed that this pattern can distinguish among particular hematological malignancies, but lacks a prognostic significance. Within our international study group we determined the prognostic significance of total WT1 expression in childhood AML. Based on our results of a large cohort of patients we can conclude that WT1 expression at...