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The twenty-first century is beginning with a sharp turn in the field of cancer therapy. Molecular targeted therapies against specific oncogenic events are now possible. The BCR-ABL story represents a notable example of how research from the fields of cytogenetics, retroviral oncology, protein phosphorylation, and small molecule chemical inhibitors can lead to the development of a successful molecular targeted therapy. Imatinib mesylate (Gleevec, STI571, or CP57148B) is a direct inhibitor of ABL (ABL1), ARG (ABL2), KIT, and PDGFR tyrosine kinases. This drug has had a major impact on the treatment of chronic myelogenous leukemia (CML) as well as other blood neoplasias and solid tumors with etiologies based on activation of these tyrosine kinases. Analysis of CML patients resistant to BCR-ABL suppression by Imatinib mesylate coupled with the crystallographic structure of ABL complexed to this inhibitor have shown how structural mutations in ABL can circumvent an otherwise potent anticancer drug. The successes and limitations of Imatinib mesylate hold general lessons for the development of alternative molecular targeted therapies in oncology.

Formalin fixation and paraffin embedding present the standard procedures for conserving clinical tissues for histological analysis. However, molecular analysis is impaired by the cross linking properties of formalin. The PAXgene tissue system (PreAnalytix, Switzerland) is a new formalin-free tissue collection device. Aims In this study we aimed to evaluate this new tissue preservation technique in comparison with formalin fixation and fresh frozen tissue samples. Methods 12 melanoma biopsy samples were divided and fixed simultaneously with formalin, PAXgene or fresh frozen in liquid nitrogen and analysed with regard to morphology, immunohistochemistry,  DNA and RNA content and quality. Markers of melanocytic differentiation and tumour cell proliferation were used. Results Morphology was well preserved in PAXPE samples. However, 5 out of 11 immunohistochemical markers showed significantly lower overall staining and staining intensity with PAXPE tissues in comparison with formalin-fixed, paraffin-embedded (FFPE). Increasing membrane permeability through adding a detergent did proportionally increase staining intensity in PAXPE samples. Amplification of different mRNA amplicons showed a direct relationship with the size of the amplicon with greater template integrity observed in PAXPE samples. Sequencing and mutational analysis of DNA samples were comparable for all the different fixation methods, while the level of DNA fragmentation seemed to be lower in PAXPE compared with FFPE tissues. Conclusions The switch from formalin to PAXgene fixation would require a re-evaluation of immunohistochemical markers and staining procedures originally developed for FFPE tissues. Our data demonstrate that PAXPE fixation offers some advantages concerning molecular analysis. However, these advantages would not justify substituting formalin fixation in any routine pathology laboratory.

Cellular transformation by the BCR/ABL oncogene depends on the ABL-encoded tyrosine kinase activity. To block BCR/ABL function, we created a unique tyrosine phosphatase by fusing the catalytic domain of SHP1 (SHP1c) to the ABL binding domain (ABD) of RIN1, an established binding partner and substrate for c-ABL and BCR/ABL. This fusion construct (ABD/SHP1c) binds to BCR/ABL in cells and functions as an active phosphatase. ABD/SHP1c effectively suppressed BCR/ABL function as judged by reductions in transformation of fibroblast cells, growth factor independence of hematopoietic cell lines, and proliferation of primary bone marrow cells. In addition, the leukemogenic properties of BCR/ABL in a murine model system were blocked by coexpression of ABD/SHP1c. Both the \"escort\" function provided by ABD and the inhibitor function provided by the phosphatase of SHP1c were necessary for effective BCR/ABL interference. Expression of ABD/SHP1c also reversed the transformed phenotype of K562, a human leukemia-derived cell line. These results have direct implications for leukemia therapeutics and suggest an approach to block aberrant signal transduction in other pathologies through the use of appropriately designed escort/inhibitors.

BCR-ABL expression led to a dramatic up-regulation of the IL-3, IL-5, and granulocyte-macrophage colony-stimulating factor receptor β common (IL-3Rβc) and IL-3 receptor β (IL-3Rβ) chains in murine embryonic stem cell-derived hematopoietic cells coincident with an expansion of multipotent progenitors and myeloid elements. This up-regulation required BCR-ABL tyrosine kinase activity and led to IL-3Rβc/β chain tyrosine phosphorylation in the absence of detectable IL-3 production. These results suggested that cytokine-independent IL-3 receptor activation could be a dominant signaling component in BCR-ABL-induced leukemogenesis. To unambiguously define the significance of IL-3 receptor-dependent signaling in BCR-ABL-induced leukemogenesis, BCR-ABL-transduced bone marrow cells deficient in either IL-3Rβc chain or both IL-3Rβc/β chain expression were examined for their ability in generating myeloproliferative disease (MPD). These BCR-ABL-expressing knockout cells were capable of generating MPD similar to control cells, demonstrating that IL-3 receptor activation is not essential for BCR-ABL-induced MPD. However, the IL-3Rβc/β chain could act as a cofactor in BCR-ABL-induced leukemogenesis by activation of its many known oncogenic signaling pathways.

The Ph chromosome has been genetically linked to CML and ALL. Its chimeric fusion gene product, BCR-ABL, can generate leukemia in mice. This review will discuss selected model systems developed to study BCR-ABL induced leukemia and focuses on what we have learned about the human disease from these models. Five main experimental approaches will be discussed including: (i) Reconstitution of mice with bone marrow cells retrovirally transduced with BCR-ABL; (ii) Transgenic mice expressing BCR-ABL; (iii) Knock-in mice with BCR-ABL expression driven from the endogenous bcr locus; (iv) Development of CML-like disease in mice with loss of function mutations in heterologous genes; and (v) ES in vitro hematopoietic differentiation coupled with regulated BCR-ABL expression.

BCR-ABL is a chimeric fusion gene product of the Philadelphia chromosome whose heightened tyrosine kinase activity is essential for the generation of most chronic myelogenous leukemias (CML) and 5–15% of acute B cell lymphoblastic leukemias (B-ALL). Suppression of BCR-ABL's tyrosine kinase activity with the inhibitor imatinib mesylate is effective in inducing hematological remission in most CML patients. However, a significant portion of CML patients relapse from imatinib mesylate treatment due to mutations in BCR-ABL rendering the protein resistant to drug inhibition (Chapter 1). Identification of signaling molecules critical to BCR-ABL oncogenesis could reveal therapeutic targets for imatinib mesylate resistant patients. We have developed an embryonic stem (ES) cell in vitro differentiation system with tetracycline-regulated BCR-ABL induction to study its effects during defined stages of hematopoiesis (Chapter 2). BCR-ABL suppressed apoptosis in ES-derived hematopoietic progenitors coincident with up regulation of the antiapoptotic molecule BCL-XL and inhibition of the proapoptotic molecule p38 MAPK (Chapter 3). Suppression of p38 MAPK with the inhibitor SB203580 was sufficient to expand ES-derived hematopoietic progenitors to levels approaching that seen following BCR-ABL induction (Chapter 3). Interleukin 3 receptor beta common and beta (IL-3Rβc/β) chains have been shown to contribute to hematopoietic progenitor expansion and myelopoiesis, and were activated upon BCR-ABL induction in ES-derived multipotent and myeloid cells (Chapter 4). However, IL-3Rβc/β chains double knockout progenitors were capable of generating BCR-ABL induced murine myeloproliferative disease (MPD) similar to control cells demonstrating that IL-3 receptor activation is not essential in this leukemogenesis model (Chapter 4). Numerous animal models have been developed to study BCR-ABL oncogenesis (Chapter 5). Using a B-ALL model, we demonstrated that RIN1 expression potentiated BCR-ABL leukemogenesis (Chapter 6). A fusion of the BCR-ABL binding region of RIN1 to the tyrosine phosphatase domain of SHP1 generated a chimeric protein (ABD/SHP1c) that selectively inhibited BCR-ABL activity leading to suppression of B-ALL development (Chapter 7). Positron emission tomography of mice transplanted with BCR-ABL transduced G2A deficient and wild type cells revealed that G2A is a negative modifier of B-ALL (Chapter 8). These studies reveal BCR-ABL regulated signaling pathways that could be therapeutic targets for imatinib mesylate resistant patients (Chapter 9).

Aims/hypothesisFew studies examine the association between age at diagnosis and subsequent complications from type 2 diabetes. This paper aims to summarise the risk of mortality, macrovascular complications and microvascular complications associated with age at diagnosis of type 2 diabetes.MethodsData were sourced from MEDLINE and All EBM (Evidence Based Medicine) databases from inception to July 2018. Observational studies, investigating the effect of age at diabetes diagnosis on macrovascular and microvascular diabetes complications in adults with type 2 diabetes were selected according to pre-specified criteria. Two investigators independently extracted data and evaluated all studies. If data were not reported in a comparable format, data were obtained from authors, presented as minimally adjusted ORs (and 95% CIs) per 1 year increase in age at diabetes diagnosis, adjusted for current age for each outcome of interest. The study protocol was recorded with PROSPERO International Prospective Register of Systematic Reviews (CRD42016043593).ResultsData from 26 observational studies comprising 1,325,493 individuals from 30 countries were included. Random-effects meta-analyses with inverse variance weighting were used to obtain the pooled ORs. Age at diabetes diagnosis was inversely associated with risk of all-cause mortality and macrovascular and microvascular disease (all p < 0.001). Each 1 year increase in age at diabetes diagnosis was associated with a 4%, 3% and 5% decreased risk of all-cause mortality, macrovascular disease and microvascular disease, respectively, adjusted for current age. The effects were consistent for the individual components of the composite outcomes (all p < 0.001).Conclusions/interpretationYounger, rather than older, age at diabetes diagnosis was associated with higher risk of mortality and vascular disease. Early and sustained interventions to delay type 2 diabetes onset and improve blood glucose levels and cardiovascular risk profiles of those already diagnosed are essential to reduce morbidity and mortality.

Vaccines and other alternative products can help minimize the need for antibiotics by preventing and controlling infectious diseases in animal populations, and are central to the future success of animal agriculture. To assess scientific advancements related to alternatives to antibiotics and provide actionable strategies to support their development, the United States Department of Agriculture, with support from the World Organisation for Animal Health, organized the second International Symposium on Alternatives to Antibiotics. It focused on six key areas: vaccines; microbial-derived products; non-nutritive phytochemicals; immune-related products; chemicals, enzymes, and innovative drugs; and regulatory pathways to enable the development and licensure of alternatives to antibiotics. This article, part of a two-part series, synthesizes and expands on the expert panel discussions regarding opportunities, challenges and needs for the development of vaccines that may reduce the need for use of antibiotics in animals; new approaches and potential solutions will be discussed in part 2 of this series. Vaccines are widely used to prevent infections in food animals. Various studies have demonstrated that their animal agricultural use can lead to significant reductions in antibiotic consumption, making them promising alternatives to antibiotics. To be widely used in food producing animals, vaccines have to be safe, effective, easy to use, and cost-effective. Many current vaccines fall short in one or more of these respects. Scientific advancements may allow many of these limitations to be overcome, but progress is funding-dependent. Research will have to be prioritized to ensure scarce public resources are dedicated to areas of potentially greatest impact first, and private investments into vaccine development constantly compete with other investment opportunities. Although vaccines have the potential to improve animal health, safeguard agricultural productivity, and reduce antibiotic consumption and resulting resistance risks, targeted research and development investments and concerted efforts by all affected are needed to realize that potential.