Exploring the role of homologous recombination deficiency and BRCA1/2 mutations in endometrial cancer

BRCA1 en BRCA2 zijn tumorsuppressor genen die betrokken zijn bij homologe recombinatie reparatie. Een kiembaanmutatie in BRCA1 of BRCA2 leidt tot het erfelijke borst- en eierstokkanker syndroom (HBOC syndroom). Of vrouwen met een BRCA1/2 mutatie ook een verhoogd risico hebben op het ontwikkelen van endometriumcarcinoom was tot nu toe onzeker.In dit proefschrift hebben middels functionele analyse mechanistisch bewijs geleverd dat een zeldzame subgroep van het endometriumcarcinoom, namelijk de sereuze of p53-abnormale/SCNA-hoge endometriumcarcinomen, vaak homoloog recombinatie deficiënt is. Daarnaast hebben we in een groot landelijk cohort: Hereditair Borst- en Eierstokkanker Onderzoek Nederland (HEBON), laten zien dat vrouwen met een BRCA1/2 mutatie (1) vaker endometriumcarcinomen van de p53-abnormale/SCNA-hoge subgroep ontwikkelen en dat deze tumoren ook verlies van het wildtype allel tonen, wat ondersteunt dat deze tumoren gBRCA1/2 geassocieerd zijn, en (2) een verhoogd risico hebben op het ontwikkelen van endometriumcarcinomen, waarbij het hoogste risico werd gevonden voor sereuze endometriumcarcinomen en endometriumcarcinomen van de p53-abnormale/SCNA-hoge subgroep bij BRCA1 mutatie draagsters. Tot slot, door aan te tonen dat BRCA1/2 mutaties betrouwbaar kunnen worden gedetecteerd in diagnostisch tumorweefsel, hebben we een basis gelegd voor een efficiëntere genetische work-up van eierstokkanker patiënten, die ook kan worden uitgebreid naar andere tumortypen.The HEBON study (Chapter 4, Chapter 5) is supported by the Dutch Cancer Society grants NKI1998-1854, NKI2004-3088, NKI2007-3756, the Netherlands Organisation of Scientific Research grant NWO 91109024, the Pink Ribbon grants 110005 and 2014-187.WO76, the BBMRI grant NWO 184.021.007/CP46 and the Transcan grant JTC 2012 Cancer 12-054. The author received an International Travel Grant from the “Leids Universiteits Fonds/ Fonds Van Trigt” and the “René Vogels Foundation” for the study described in Chapter 4. AstraZeneca provided financial support for next-generation sequencing performend in Chapter 6. Financial support for the production and printing of this thesis was provided by the department of Pathology of the Leiden University Medical Center, MRC-Holland and ChipSoft

Degradation of free amino acids in liquidfeeding conditions

The aim of the current study was to determine the degradation of free amino acids in freshly produced liquid feeds. It can be concluded that the level of degradation of free amino acids in liquid feeds might be limited, provided the conditions applied in the current study.---Het doel van deze studie was na te gaan in welke mate vrije aminozuren gevoelig zijn voor afbraak onder brijvoercondities. Onder de toegepaste condities in dit onderzoek trad er slechts in beperkte mate degradatie van vrije aminozuren in de brijvoeders op

The effect of leucovorin rescue therapy on methotrexate-induced oral mucositis in the treatment of paediatric ALL: A systematic review

Introduction: This study aimed to determine the efficacy of different Leucovorin regimens to reduce oral mucositis in children with acute lymphoblastic leukemia after high-dose Methotrexate (HD-MTX). Methods: Twelve articles were included in a systematic literature review. Articles were categorized into low/medium/high risk of bias. Results: As no randomized controlled trial assessing the effect of Leucovorin has been performed, the efficacy of Leucovorin to reduce oral mucositis remains unknown. Leucovorin was initiated at 24, 36 or 42 h after HD-MTX at a dose of 15 or 30 mg/m2. No meta-analysis could be performed as treatment regimens differed. When comparing studies with similar HD-MTX doses, we observed lower oral mucositis rates in regimens with higher cumulative doses of Leucovorin and early initiation of Leucovorin after MTX. Conclusion: Even though future studies are necessary, higher cumulative Leucovorin doses and early initiation of Leucovorin after start of MTX seem to reduce oral mucositis

Tomo-seq identifies SOX9 as a key regulator of cardiac fibrosis during ischemic injury

Background: Cardiac ischemic injury induces a pathological remodeling response, which can ultimately lead to heart failure. Detailed mechanistic insights into molecular signaling pathways relevant for different aspects of cardiac remodeling will support the identification of novel therapeutic targets. Methods: While genome-wide transcriptome analysis on diseased tissues has greatly advanced our understanding of the regulatory networks that drive pathological changes in the heart, this approach has been disadvantaged by the fact that the signals are derived from tissue homogenates. Here we used tomo-seq to obtain a genome-wide gene expression signature with high spatial resolution spanning from the infarcted area to the remote to identify new regulators of cardiac remodeling. Cardiac tissue samples from patients suffering from ischemic heart disease were used to validate our findings. Results: Tracing transcriptional differences with a high spatial resolution across the infarcted heart enabled us to identify gene clusters that share a comparable expression profile. The spatial distribution patterns indicated a separation of expressional changes for genes involved in specific aspects of cardiac remodeling, like fibrosis, cardiomyocyte hypertrophy, and calcium-handling (Col1a2, Nppa, and Serca2). Subsequent correlation analysis allowed for the identification of novel factors that share a comparable transcriptional regulation pattern across the infarcted tissue. The strong correlation between the expression levels of these known marker genes and the expression of the co-regulated genes could be confirmed in human ischemic cardiac tissue samples. Follow-up analysis identified SOX9 as common transcriptional regulator of a large portion of the fibrosis-related genes that become activated under conditions of ischemic injury. Lineage-tracing experiments indicated the majority of COL1-positive fibroblasts to stem from a pool of SOX9-expressing cells and in vivo loss of Sox9 blunted the cardiac fibrotic response upon ischemic injury. The co-localization between SOX9 and COL1 could also be confirmed in patients suffering from ischemic heart disease. Conclusions: Based on the exact local expression cues, tomo-seq can serve to reveal novel genes and key transcription factors involved in specific aspects of cardiac remodeling. Using tomo-seq we were able to unveil the unknown relevance of SOX9 as key regulator of cardiac fibrosis, pointing to SOX9 as potential therapeutic target for cardiac fibrosis

Global methylation in relation to methotrexate-induced oral mucositis in children with acute lymphoblastic leukemia

Background Children with acute lymphoblastic leukemia (ALL) often suffer from toxicity of chemotherapeutic drugs such as Methotrexate (MTX). Previously, we reported that 20% of patients receiving high-dose MTX developed oral mucositis. MTX inhibits folate metabolism, which is essential for DNA methylation. We hypothesize that MTX inhibits DNA methylation, which results into adverse effects. We studied DNA methylation markers during high-dose methotrexate treatment in pediatric acute lymphoblastic leukemia (ALL) in relation to developing oral mucositis. Materials & methods S-Adenosyl-Methionine (SAM) and S-Adenosyl-Homocysteine (SAH) levels and LINE1 DNA methylation were measured prospectively before and after high-dose methotrexate (HD-MTX 4 x 5g/m2) therapy in 82 children with ALL. Methotrexate-induced oral mucositis was registered prospectively. Oral mucositis (grade 3 National Cancer Institute Criteria) was used as clinical endpoint. Results SAM levels decreased significantly during methotrexate therapy (-16.1 nmol/L (-144.0 – +46.0), p<0.001), while SAH levels and the SAM:SAH ratio did not change significantly. LINE1 DNA methylation (+1.4% (-1.1 –+6.5), p<0.001) increased during therapy. SAM and SAH levels were not correlated to LINE1 DNA methylation status. No association was found between DNA methylation markers and developing oral mucositis. Conclusions This was the first study that assessed DNA methylation in relation to MTX-induced oral mucositis in children with ALL. Although global methylation markers did change during methotrexate therapy, methylation status was not associated with developing oral mucositis

Workers' intrinsic work motivation when job demands are high:The role of need for autonomy and perceived opportunity for blended working

Work overload or work pressure may undermine workers' intrinsic motivation. In the present research, we tested the conditions under which this may (not) occur, including the perceived opportunity to blend on-site and off-site working through the effective use of computers and modern information and communication technology. Our sample consisted of 657 workers (51% female) representing a variety of industries. As hypothesized, it is not high job demands per se, but high demands in combination with a high need for autonomy and a lack of perceived opportunities for blended working that undermines intrinsic work motivation. When workers high in need for autonomy perceived opportunities for blended working, their intrinsic work motivation was not negatively affected by increasing job demands. This main finding suggests that, particularly for workers high in need for autonomy, the perceived opportunity for blended working is an effective, contemporary resource to cope with the increasing job demands typically observed in today's workplace. Theoretically, these findings contribute to the refinement and extension of influential demands-resource models and Person-Job Fit theory. Practically, our findings may show managers how to effectively keep workers intrinsically motivated and productive in their jobs when job demands are high

A U-HPLC-ESI-MS/MS-based stable isotope dilution method for the detection and quantitation of methotrexate in plasma

INTRODUCTION: High-dose methotrexate (MTX) is used in the treatment of proliferative diseases such as acute lymphoblastic leukemia. Therapeutic drug monitoring of plasma MTX is important to monitor efficacy and adverse events. The authors aimed to develop a liquid chromatography, electrospray ionization, tandem mass spectrometry (LC-ESI-MS/MS)-based method to determine MTX in plasma for therapeutic drug monitoring and pharmacokinetic studies. METHODS: Samples were analyzed using a Waters Acquity UPLC and Quattro Premier XE. A Waters Acquity UPLC BEH C18 column (2.1 mm x 100 mm, 1.7 μm) was used running an isocratic mobile phase of 21% methanol and 10 mM ammonium bicarbonate. The electrospray was operated in the positive ionization mode monitoring the following mass transitions: m/z 455.2 > 308.2 for MTX and m/z 458.2 > 311.2 for MTXd3. The analysis combined straightforward sample preparation, consisting of dilution and protein precipitation, with a 3-minute run time. RESULTS: The method was linear up to 50 μM (r > 0.99), and the coefficient of variation was 1:10, was 5 nM. Method comparison with the Abbott TDx fluorescent polarization immunoassay (FPIA) showed excellent agreement, and a small but significant negative constant bias was detected (LC-MS/MS = 0.98 x FPIA - 7.3). CONLUSIONS: The authors developed a specific and sensitive stable isotope dilution LC-ESI-MS/MS method to monitor MTX concentrations in plasma within the clinically relevant range. The method can be easily applied in clinical laboratories because it combines straightforward sample pretreatment with LC-MS/MS. Copyrigh

Patient-derived oral mucosa organoids as an in vitro model for methotrexate induced toxicity in pediatric acute lymphoblastic leukemia

We have recently established a protocol to grow wildtype human oral mucosa organoids. These three-dimensional structures can be maintained in culture long-term, do not require immortalization, and recapitulate the multilayered composition of the epithelial lining of the oral mucosa. Here, we validate the use of this model to study the effect of Leucovorin (LV) on Methotrexate (MTX)-induced toxicity. MTX is a chemotherapeutic agent used in the treatment of pediatric acute lymphoblastic leukemia. Although effective, the use of MTX often results in s