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Pharmacogenetic profiling of VEGF-inhibitors forcancer therapy and assessing the vascular effects of VEGF during neurodegeneration.
Mismatch repair (MMR) deficiency represents a well-established cause ofLynch syndrome, which is an autosomal dominantly inherited disorder of cancer susceptibility triggered by loss-of-function mutations in MMR genes (MLH1, MSH2, MSH6 or PMS2).(URL:#_ENREF_1) </>Lynch syndrome is </>responsible for 2-5% of endometrial (EM) or colorectal (CRC) tumors. </>Additionally, epigenetic silencing of MLH1 </>contributes to another 15-28% of these tumors.(URL:#_ENREF_2)</> </>Deficiency of the MMR machinery leads to DNA replicationerrors in the tumor </>tissue, but not in the normal surrounding tissue. In particular, errors often </>accumulate as insertion/deletion (indel) mutations in mono- and di-nucleotide </>repeats - a </>phenomenon referred to as microsatellite instability (MSI).(URL:#_ENREF_3)</>
MMR-deficient </>tumors constitute a specific subtype due to their different prognosis and </>clinical outcome.(URL:#_ENREF_4)</> For instance, untreated </>CRC cancer patients with MMR-deficient tumors have a modestly better prognosis, </>but do not seem to benefit from 5-fluorouracil-based adjuvant chemotherapy, </>which is the first-choice chemotherapy for CRC cancer. In particular, in MMR-deficient </>tumors, mismatches induced by 5-fluorouracil are tolerated, leading to failure </>to induce cell death.(URL:#_ENREF_5) </>MMR-deficient </>tumors are also resistant to cisplatin and carboplatin, which are frequently </>used chemotherapies in EM cancer.(URL:#_ENREF_5) </>Furthermore, </>MMR-deficient tumors can be resistant to targeted therapies, because they </>acquire secondary mutations in genes that activate alternative or downstream </>signaling pathways (e.g. PIK3CA). </>Another possibility is that epigenetic silencing of MLH1 coincides with particular mutations, such as the BRAF V600E mutation(URL:#_ENREF_6)</>, </>which represents an established negative predictor of response to targeted </>anti-EGFR therapies in advanced CRC cancer.(URL:#_ENREF_7)</> </>There have been some efforts to individualize the treatment of MMR-deficient </>cancers. These focused on identifying synthetic lethal interactions with the </>MMR pathway or targeting secondary mutations occurring as a result of MMR-deficiency. </>However, until now, these efforts failed to translate into clinically effective </>treatment options.
The </>objectives of this thesis were to generate a more comprehensive picture of the mutation spectra arising in </>MMR-deficient tumors and to more rationally predict their response to </>therapies. Firstly, whole-exome sequencing was applied to characterize </>the unique somatic mutation patterns underlying MMR-deficient tumors. Each MMR-deficient tumor </>exhibited a clear hypermutator phenotype, containing on average 50 times more </>novel somatic mutations than MMR-proficient tumors. The mutations in </>MMR-deficient tumors were mostly occurring as indels in homopolymers. Indels </>were clearly less frequent in exons, suggesting </>that they are loss-of-function mutations undergoing negative clonal selection </>during tumorigenesis. On the other hand, a large set of recurrent indels </>affecting MMR-deficient tumors was identified implying positive clonal </>selection. Secondly, a novel MSI panel was designed to detect MMR-deficiency in </>clinical tumor samples by high-throughput profiling of the selected recurrent </>indels. This Sequenom-based panel was tested on more than 400 tumor samples, </>fromendometrial, colorectal and ovarian cancer patients, and demonstrated that </>it outperforms the standard diagnostic panel (i.e. the revised Bethesda panel). </>Thirdly, pathway analyses revealed that the recurrent indels in MMR-deficient </>tumors preferentiallyaffect the DNA </>double-strand break repair (DSBR) by homologous recombination (HR) pathway, </>containing on average 3.3 indels per tumor. The biological relevance of these </>secondary mutations was validated by performing functional studies in 11 </>primary tumor cultures. Double immunostaining for γH2AX and RAD51, as a measure </>of DSBs and ongoing HR, respectively, showed that MMR-deficient tumors </>mobilized less RAD51-positive foci, after DSB formation. BrdU and PI cell cycle </>analysis using FACS, as well as Western blots were used to exclude a reduced </>S/G2 phase or a reduced RAD51 expression as confounding factors for areduced DSBR by HR activity. Thereby, it is confirmed that the DSBR by HR pathway is also functionally </>affected in MMR-deficient tumors. Moreover, DSB inducers, such as the PARP </>inhibitor olaparib, triggered a dose-dependent reduction in proliferation in </>primary cultures of MMR-deficient tumors, identifying a synthetic lethal </>interaction. In addition, to more accurately measure the level of HR-deficiency </>in MMR-deficient tumors, the level of knock-down of BRCA1, BRCA2 and ATR needed </>to achieve an olaparib sensitivity similar to that observed in MMR-deficient </>cells was assessed. It is established that the loss of DSBR by HR activity in MMR-deficient tumors corresponds to a loss </>of about 70-80% BRCA1 or -2 expression, indicating that the sensitivity to </>olaparib that is observed in MMR-deficient tumors is intermediateto that of cells heterozygous and homozygous-deficient for BRCA1.
Insummary, this </>thesis not only provides the first comprehensive catalogue of somatic mutations </>accumulating in MMR-deficient tumors, but also demonstrates the relevance of </>these mutations in the detection of MSI within the diagnostic setting, and potentially also the targeted treatment of these tumors.
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MMR-deficient </>tumors constitute a specific subtype due to their different prognosis and </>clinical outcome.(URL:#_ENREF_4)</> For instance, untreated </>CRC cancer patients with MMR-deficient tumors have a modestly better prognosis, </>but do not seem to benefit from 5-fluorouracil-based adjuvant chemotherapy, </>which is the first-choice chemotherapy for CRC cancer. In particular, in MMR-deficient </>tumors, mismatches induced by 5-fluorouracil are tolerated, leading to failure </>to induce cell death.(URL:#_ENREF_5) </>MMR-deficient </>tumors are also resistant to cisplatin and carboplatin, which are frequently </>used chemotherapies in EM cancer.(URL:#_ENREF_5) </>Furthermore, </>MMR-deficient tumors can be resistant to targeted therapies, because they </>acquire secondary mutations in genes that activate alternative or downstream </>signaling pathways (e.g. PIK3CA). </>Another possibility is that epigenetic silencing of MLH1 coincides with particular mutations, such as the BRAF V600E mutation(URL:#_ENREF_6)</>, </>which represents an established negative predictor of response to targeted </>anti-EGFR therapies in advanced CRC cancer.(URL:#_ENREF_7)</> </>There have been some efforts to individualize the treatment of MMR-deficient </>cancers. These focused on identifying synthetic lethal interactions with the </>MMR pathway or targeting secondary mutations occurring as a result of MMR-deficiency. </>However, until now, these efforts failed to translate into clinically effective </>treatment options.
The </>objectives of this thesis were to generate a more comprehensive picture of the mutation spectra arising in </>MMR-deficient tumors and to more rationally predict their response to </>therapies. Firstly, whole-exome sequencing was applied to characterize </>the unique somatic mutation patterns underlying MMR-deficient tumors. Each MMR-deficient tumor </>exhibited a clear hypermutator phenotype, containing on average 50 times more </>novel somatic mutations than MMR-proficient tumors. The mutations in </>MMR-deficient tumors were mostly occurring as indels in homopolymers. Indels </>were clearly less frequent in exons, suggesting </>that they are loss-of-function mutations undergoing negative clonal selection </>during tumorigenesis. On the other hand, a large set of recurrent indels </>affecting MMR-deficient tumors was identified implying positive clonal </>selection. Secondly, a novel MSI panel was designed to detect MMR-deficiency in </>clinical tumor samples by high-throughput profiling of the selected recurrent </>indels. This Sequenom-based panel was tested on more than 400 tumor samples, </>fromendometrial, colorectal and ovarian cancer patients, and demonstrated that </>it outperforms the standard diagnostic panel (i.e. the revised Bethesda panel). </>Thirdly, pathway analyses revealed that the recurrent indels in MMR-deficient </>tumors preferentiallyaffect the DNA </>double-strand break repair (DSBR) by homologous recombination (HR) pathway, </>containing on average 3.3 indels per tumor. The biological relevance of these </>secondary mutations was validated by performing functional studies in 11 </>primary tumor cultures. Double immunostaining for γH2AX and RAD51, as a measure </>of DSBs and ongoing HR, respectively, showed that MMR-deficient tumors </>mobilized less RAD51-positive foci, after DSB formation. BrdU and PI cell cycle </>analysis using FACS, as well as Western blots were used to exclude a reduced </>S/G2 phase or a reduced RAD51 expression as confounding factors for areduced DSBR by HR activity. Thereby, it is confirmed that the DSBR by HR pathway is also functionally </>affected in MMR-deficient tumors. Moreover, DSB inducers, such as the PARP </>inhibitor olaparib, triggered a dose-dependent reduction in proliferation in </>primary cultures of MMR-deficient tumors, identifying a synthetic lethal </>interaction. In addition, to more accurately measure the level of HR-deficiency </>in MMR-deficient tumors, the level of knock-down of BRCA1, BRCA2 and ATR needed </>to achieve an olaparib sensitivity similar to that observed in MMR-deficient </>cells was assessed. It is established that the loss of DSBR by HR activity in MMR-deficient tumors corresponds to a loss </>of about 70-80% BRCA1 or -2 expression, indicating that the sensitivity to </>olaparib that is observed in MMR-deficient tumors is intermediateto that of cells heterozygous and homozygous-deficient for BRCA1.
Insummary, this </>thesis not only provides the first comprehensive catalogue of somatic mutations </>accumulating in MMR-deficient tumors, but also demonstrates the relevance of </>these mutations in the detection of MSI within the diagnostic setting, and potentially also the targeted treatment of these tumors.
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Date:1 Oct 2008 → 2 Jan 2014
Keywords:VEGF inhibitors
Disciplines:Neurosciences, Biological and physiological psychology, Cognitive science and intelligent systems, Developmental psychology and ageing, Genetics, Systems biology, Molecular and cell biology, Morphological sciences, Oncology
Project type:PhD project