Mouse models in leukemia

Human Philadelphia-positive leukemia results from a balanced chromosomal translocation, which fuses the BCR gene on chromosome 22 to the ABL proto-oncogene on chromosome 9. The understanding of Ph-positive leukemogenesis has advanced enormously over the last few decades. Although in vitro assay systems currently used, are not always relevant to human tumor biology, much can and has been learned from studies, employing cell cultures and overexpression of BCR/ABL oncogenes.Another restriction in leukemia research is the availability of primary human tumor material for study. Moreover, such tissues often represent terminally advanced stages of tumorigenesis. Therefore, the importance of in vivo models to study Philadelphia-positive leukemia is manifold. A well defined transgenic mouse model allows for tumorigenesis to be studied from its earliest stages onward and factors and mechanisms that eventually contribute to malignant progression of the leukemic cells can be uncovered. Besides an 'unlimited' provision of tumor material for analysis, more importantly, the availability of a transgenic mouse model provides a means by which cancer treatment regimes can be tested. In addition, identification of cellular components and/or pathways that contribute to the onset or progression of leukemia may eventually lead to the discovery and development of new drugs.In 1990, Heisterkamp and co-workers reported on a transgenic mouse model for Philadelphia-positive acute lymphoblastic leukemia (ALL). Since most of the transgenic animals of an earlier study had succumbed to leukemia, part of the aim of this thesis was to generate BCR/ABL P190 transgenic founder animals de novo and to derive a transgenic animal line(s) which was to be used for future studies. In order to better understand the animal model, leukemogenesis was studied in great detail in transgenic founder animals and their progeny. In the second chapter a cytogenetic study of the mouse model for acute lymphoblastic leukemia is presented. Karyotypic analysis of leukemic bone marrow of a significant number of mice shows, that leukemic cells undergo a clonal development and karyotype evolution toward a more aggressive tumor: a high frequency of aneuploidy is found in advanced leukemia, as occurs in human leukemia, with a preference for gain of chromosomes 10, 12, 14 and 17. These findings are corroborated by experiments that reveal a gain of malignancy of the cancer upon serial transplantation of leukemic bone marrow to irradiated recipient mice and by molecular analysis of lymphomas using immunoglobulin rearrangement as an indicator for tumor clonality. The results suggest that BCR/ABL has a destabilizing effect on the regulation of the proces of mitosis.In the third chapter, a correlation is described between the transcriptional status of the BCR/ABL P190 transgene and the development of leukemia: methylation of particular sequences in BCR exon-1 in the transgene is closely coupled to transgene inactivation, providing additional evidence for a direct role of BCR/ABL in leukemogenesis. A biological dissection of the oncogenic specificity of BCR/ABL is presented in the fourth chapter. Using sensitive molecular biological techniques, it is shown that, although expression of the BCR/ABL transgene is detectable in every tissue, from very early on in mouse development, no other neoplasias than of hematopoietic origin are found. The results strongly suggest that the oncogenicity of BCR/ABL is restricted to nucleated blood cells, which is very likely a reflection of cellular functions of the BCR and or ABL gene in signal transduction specific to hematopoietic lineages. The observations would also explain why the Ph-chromosome, which one would expect to arise by chance in many proliferating tissues, is found only in blood cancers.An analysis of transgenic mouse models for chronic myelogenous leukemia, using BCR/ABL P210 transgenes is presented in the fifth chapter. The clinical disease spectrum includes differentiated and undifferentiated T and B cell leukemias. The myeloid compartment is implicated only sporadically and rather late in the disease process. In some instances, the observed myelo-proliferation is a sequel to deregulation of cytokine production at advanced stages of leukemia. The course of P210 induced leukemia was acute rather than chronic, be it with an on average longer latency period than typical for ALL in BCR/ABL P190 mice. From these studies is was concluded that in the mouse, BCR/ABL P210 evokes a clinically different disease than BCR/ABL P190. Although no evidence for a chronic myeloproliferative disorder in the peripheral blood was found, an imbalance in myelopoiesis in the bone marrow suggests an effect of BCR/ABL P210 on primitive myeloid progenitors.The sixth chapter summarizes an analysis of interferon-α(IFN-α) treatment of the BCR/ABL P190 transgenic mice. (IFN-α) is currently one of the most effective drugs in the treatment of CML. Recently, (IFN-α) was tried in the treatment of ALL. No effect of (IFN-α) on animal survival or disease pattern were noted when administered to the BCR/ABL P190 mice. The conclusion was reached that, at least in a transgenic setting, (IFN-α) does not interfere with BCR/ABL P190 mediated leukemia.In order to study the normal cellular function of the BCR gene and to eventually assess its role in leukemogenesis, studies focussing on the mouse bcr gene function are presented in chapters 7 and 8. The seventh chapter describes the ablation of a functional mouse bcr gene by means of recently developed gene targeting techniques. One of two mouse bcr alleles was inactivated in a mouse embryonic stem cell line through gene interruption by insertional replacement vectors. Ibis genetically altered cell fine was then injected into developing mouse embryos. Through germline transmission of the mutated allele and subsequent breeding both bcr alleles were inactivated. Although bcr -null mutants are phenotypically normal, their neutrophils display impaired regulation of respiratory burst, which becomes apparent when these cells are activated in vivo: an overproduction of superoxide leads to significantly more oxidative tissue damage during experimental endotoxemia. The results connect Bcr in vivo with the regulation of superoxide production by the NADPH- oxidase system of leukocytes and suggest a link between the cell types affected by loss of Bcr function and the those involved in Ph -positive leukemia.Additional information on biological processes that Bcr participates in, are described. in the eighths chapter. Notwithstanding its function in hematopoietic cells, the Bcr protein is normally found in high levels in brain. The expression pattern of bcr in rodent brain was examined by means of in situ hybridization and Northern analysis. Although not directly connected with leukemogenesis, a potentially interesting role for p160Bcr in the brain is discussed as its expression pattern appears to coincide with the functional organization of particularly highly specialized structures in the brain.With the availability of well defined transgenic mouse models for BCR/ABL positive leukemia, an opportunity is created to study the nature of cellular interactions and processes that contribute to the onset and development of Ph -positive leukemia. Ultimately, such investigations are aimed at designing and testing effective therapeutic drugs to fight the disease

Cytosolic enzymes with a mitochondrial ancestry from the anaerobic chytrid Piromyces sp. E2

The anaerobic chytrid Piromyces sp. E2 lacks mitochondria, but contains hydrogen-producing organelles, the hydrogenosomes. We are interested in how the adaptation to anaerobiosis influenced enzyme compartmentalization in this organism. Random sequencing of a cDNA library from Piromyces sp. E2 resulted in the isolation of cDNAs encoding malate dehydrogenase, aconitase and acetohydroxyacid reductoisomerase. Phylogenetic analysis of the deduced amino acid sequences revealed that they are closely related to their mitochondrial homologues from aerobic eukaryotes. However, the deduced sequences lack N-terminal extensions, which function as mitochondrial leader sequences in the corresponding mitochondrial enzymes from aerobic eukaryotes. Subcellular fractionation and enzyme assays confirmed that the corresponding enzymes are located in the cytosol. As anaerobic chytrids evolved from aerobic, mitochondria-bearing ancestors, we suggest that, in the course of the adaptation from an aerobic to an anaerobic lifestyle, mitochondrial enzymes were retargeted to the cytosol with the concomitant loss of their N-terminal leader sequences

A hydrogenosome with pyruvate formate-lyase: Anaerobic chytrid fungi use an alternative route for pyruvate catabolism

The chytrid fungi Piromyces sp. E2 and Neocallimastix sp. L2 are obligatory amitochondriate anaerobes that possess hydrogenosomes. Hydrogenosomes are highly specialized organelles engaged in anaerobic carbon metabolism; they generate molecular hydrogen and ATP. Here, we show for the first time that chytrid hydrogenosomes use pyruvate formate-lyase (PFL) and not pyruvate:ferredoxin oxidoreductase (PFO) for pyruvate catabolism, unlike all other hydrogenosomes studied to date. Chytrid PFLs are encoded by a multigene family and are abundantly expressed in Piromyces sp. E2 and Neocallimastix sp. L2. Western blotting after cellular fractionation, proteinase K protection assays and determinations of enzyme activities reveal that PFL is present in the hydrogenosomes of Piromyces sp. E2. The main route of the hydrogenosomal carbon metabolism involves PFL; the formation of equimolar amounts of formate and acetate by isolated hydrogenosomes ex

A Population-based Study on Lymph Node Retrieval in Patients with Esophageal Cancer: Results from the Dutch Upper Gastrointestinal Cancer Audit

Background: For esophageal cancer, the number of retrieved lymph nodes (LNs) is often used as a quality indicator. The aim of this study is to analyze the number of retrieved LNs in The Netherlands, assess factors associated with LN yield, and explore the association with short-term outcomes. This is a population-based study on lymph node retrieval in patients with esophageal cancer, presenting results from the Dutch Upper Gastrointestinal Cancer Audit. Study Design: For this retrospective national cohort study, patients with esophageal carcinoma who underwent esophagectomy between 2011 and 2016 were included. The primary outcome was the number of retrieved LNs. Univariable and multivariable regression analyses were used to test for association with ≥ 15 LNs. Patients and Results: 3970 patients were included. Between 2011 and 2016, the median number of LNs increased from 15 to 20. Factors independently associated with ≥ 15 LNs were: 0–10 kg preoperative weight loss (versus: unknown weight loss, odds ratio [95% confidence interval]: 0.71 [0.57–0.88]), Charlson score 0 (versus: Charlson score 2: 0.76 [0.63–0.92]), cN2 category (reference: cN0, 1.32 [1.05–1.65]), no neoadjuvant therapy and neoadjuvant chemotherapy (reference: neoadjuvant chemoradiotherapy, 1.73 [1.29–2.32] and 2.15 [1.54–3.01]), minimally invasive transthoracic (reference: open transthoracic, 1.46 [1.15–1.85]), open transthoracic (versus open and minimally invasive transhiatal, 0.29 [0.23–0.36] and 0.43 [0.32–0.59]), hospital volume of 26–50 or > 50 resections/year (reference: 0–25, 1.94 [1.55–2.42] and 3.01 [2.36–3.83]), and year of surgery [reference: 2011, odds ratios (ORs) 1.48, 1.53, 2.28, 2.44, 2.54]. There was no association of ≥ 15 LNs with short-term outcomes. Conclusions: The number of LNs retrieved increased between 2011 and 2016. Weight loss, Charlson score, cN category, neoadjuvant therapy, surgical approach, year of resection, and hospital volume were all associated with increased LN yield. Retrieval of ≥ 15 LNs was not associated with increased postoperative morbidity/mortality

Definition, diagnosis and treatment of oligometastatic oesophagogastric cancer: A Delphi consensus study in Europe.

Local treatment improves the outcomes for oligometastatic disease (OMD, i.e. an intermediate state between locoregional and widespread disseminated disease). However, consensus about the definition, diagnosis and treatment of oligometastatic oesophagogastric cancer is lacking. The aim of this study was to develop a multidisciplinary European consensus statement on the definition, diagnosis and treatment of oligometastatic oesophagogastric cancer. In total, 65 specialists in the multidisciplinary treatment for oesophagogastric cancer from 49 expert centres across 16 European countries were requested to participate in this Delphi study. The consensus finding process consisted of a starting meeting, 2 online Delphi questionnaire rounds and an online consensus meeting. Input for Delphi questionnaires consisted of (1) a systematic review on definitions of oligometastatic oesophagogastric cancer and (2) a discussion of real-life clinical cases by multidisciplinary teams. Experts were asked to score each statement on a 5-point Likert scale. The agreement was scored to be either absent/poor (<50%), fair (50%-75%) or consensus (≥75%). A total of 48 experts participated in the starting meeting, both Delphi rounds, and the consensus meeting (overall response rate: 71%). OMD was considered in patients with metastatic oesophagogastric cancer limited to 1 organ with ≤3 metastases or 1 extra-regional lymph node station (consensus). In addition, OMD was considered in patients without progression at restaging after systemic therapy (consensus). For patients with synchronous or metachronous OMD with a disease-free interval ≤2 years, systemic therapy followed by restaging to consider local treatment was considered as treatment (consensus). For metachronous OMD with a disease-free interval >2 years, either upfront local treatment or systemic treatment followed by restaging was considered as treatment (fair agreement). The OMEC project has resulted in a multidisciplinary European consensus statement for the definition, diagnosis and treatment of oligometastatic oesophagogastric adenocarcinoma and squamous cell cancer. This can be used to standardise inclusion criteria for future clinical trials

Neoadjuvant chemoradiotherapy plus surgery versus active surveillance for oesophageal cancer: A stepped-wedge cluster randomised trial

Background: Neoadjuvant chemoradiotherapy (nCRT) plus surgery is a standard treatment for locally advanced oesophageal cancer. With this treatment, 29% of patients have a pathologically complete response in the resection specimen. This provides the rationale for investigating an active surveillance approach. The aim of this study is to assess the (cost-)effectiveness of active surveillance vs. standard oesophagectomy after nCRT for oesophageal cancer. Methods: This is a phase-III multi-centre, stepped-wedge cluster randomised controlled trial. A total of 300 patients with clinically complete response (cCR, i.e. no local or disseminated disease proven by histology) after nCRT will be randomised to show non-inferiority of active surveillance to standard oesophagectomy (non-inferiority margin 15%, intra-correlation coefficient 0.02, power 80%, 2-sided α 0.05, 12% drop-out). Patients will undergo a first clinical response evaluation (CRE-I) 4-6 weeks after nCRT, consisting of endoscopy with bite-on-bite biopsies of the primary tumour site and other suspected lesions. Clinically complete responders will undergo a second CRE (CRE-II), 6-8 weeks after CRE-I. CRE-II will include 18F-FDG-PET-CT, followed by endoscopy with bite-on-bite biopsies and ultra-endosonography plus fine needle aspiration of suspected lymph nodes and/or PET- positive lesions. Patients with cCR at CRE-II will be assigned to oesophagectomy (first phase) or active surveillance (second phase of the study). The duration of the first phase is determined randomly over the 12 centres, i.e., stepped-wedge cluster design. Patients in the active surveillance arm will undergo diagnostic evaluations similar to CRE-II at 6/9/12/16/20/24/30/36/48 and 60 months after nCRT. In this arm, oesophagectomy will be offered only to patients in whom locoregional regrowth is highly suspected or proven, without distant dissemination. The main study parameter is overall survival; secondary endpoints include percentage of patients who do not undergo surgery, quality of life, clinical irresectability (cT4b) rate, radical resection rate, postoperative complications, progression-free survival, distant dissemination rate, and cost-effectiveness. We hypothesise that active surveillance leads to non-inferior survival, improved quality of life and a reduction in costs, compared to standard oesophagectomy. Discussion: If active surveillance and surgery as needed after nCRT leads to non-inferior survival compared to standard oesophagectomy, this organ-sparing approach can be implemented as a standard of care

Mass transfer phenomena in fluidized beds with horizontally immersed membranes

Mass transfer phenomena in fluidized bed reactors with horizontally immersed membranes have been investigated using a verified and validated Two-Fluid Model. A binary hydrogen-nitrogen mixture was injected into the fluidized bed which was operated in the bubbling fluidization regime, and hydrogen was extracted via horizontally immersed membranes. The hydrogen flux is lowest on top of the membranes and highest at the bottom of the membranes. The main causes for the low flux on top of the membranes are densified zones and insufficient hydrogen replenishment due to the flow pattern of the gas. Gas pockets do not have a negative effect on the mass transfer towards the membranes. In systems with membrane tube banks, the membranes located at the walls perform worst, because solids mostly flow downwards near the walls of a fluidized bed, which causes gas back-mixing, which hinders hydrogen replenishment and thereby decreases the driving force for hydrogen transport. Removing the membranes closest to the wall increases the overall efficiency of the system. Replacing wall membranes with inactive tubes has no significant effect on the system. The membrane tube banks also have a significant effect on the hydrodynamics.ACKNOWLEDGEMENTS. The authors are grateful to TTW and NWO for their financial support through the VIDI project ClingCO2, project number 12365.publishedVersio

A Two‐Fluid model study of hydrogen production via water gas shift in fluidized bed membrane reactors

Fluidized bed membrane reactors have been proposed as a promising reactor concept for the production of ultra-pure hydrogen via Water Gas Shift (WGS). High-flux thin-film dense palladium-based membranes are used to selectively extract hydrogen from the reaction medium, which shifts the thermodynamic equilibrium towards the products’ side, increasing the conversion. A Two-Fluid Model (TFM) has been used to investigate the effect of hydrogen extraction via perm-selective membranes on the WGS reaction rates in the fluidized bed. A thorough TFM verification study was performed, which showed that the model is able to accurately predict the concentration profiles for various types of nth order and equilibrium chemical reactions. Also, the implementation of the WGS reaction rate in the TFM was checked. The results have shown a clear positive effect of the hydrogen permeation on the WGS reaction rates, both for vertically and horizontally immersed membranes. In systems with horizontally immersed membranes, gas pockets that contain a very small amount of catalyst develop underneath the membrane tube, resulting in reduced local reaction rates. Densified zones on top of the membrane tube show increased local reaction rates. Mass transfer limitations from the emulsion phase to the membrane surface is the most pronounced effect that reduces the overall reactor performance. The developed model allows further investigating different configurations and operation modes to further optimize the reactor’s performance

A Two‐Fluid model study of hydrogen production via water gas shift in fluidized bed membrane reactors

Fluidized bed membrane reactors have been proposed as a promising reactor concept for the production of ultra-pure hydrogen via Water Gas Shift (WGS). High-flux thin-film dense palladium-based membranes are used to selectively extract hydrogen from the reaction medium, which shifts the thermodynamic equilibrium towards the products’ side, increasing the conversion. A Two-Fluid Model (TFM) has been used to investigate the effect of hydrogen extraction via perm-selective membranes on the WGS reaction rates in the fluidized bed. A thorough TFM verification study was performed, which showed that the model is able to accurately predict the concentration profiles for various types of nth order and equilibrium chemical reactions. Also, the implementation of the WGS reaction rate in the TFM was checked. The results have shown a clear positive effect of the hydrogen permeation on the WGS reaction rates, both for vertically and horizontally immersed membranes. In systems with horizontally immersed membranes, gas pockets that contain a very small amount of catalyst develop underneath the membrane tube, resulting in reduced local reaction rates. Densified zones on top of the membrane tube show increased local reaction rates. Mass transfer limitations from the emulsion phase to the membrane surface is the most pronounced effect that reduces the overall reactor performance. The developed model allows further investigating different configurations and operation modes to further optimize the reactor’s performance.publishedVersio