Chromothripsis orchestrates leukemic transformation in blast phase MPN through targetable amplification of DYRK1A.

Chromothripsis, the process of catastrophic shattering and haphazard repair of chromosomes, is a common event in cancer. Whether chromothripsis might constitute an actionable molecular event amenable to therapeutic targeting remains an open question. We describe recurrent chromothripsis of chromosome 21 in a subset of patients in blast phase of a myeloproliferative neoplasm (BP-MPN), which alongside other structural variants leads to amplification of a region of chromosome 21 in ∼25% of patients ('chr21amp'). We report that chr21amp BP-MPN has a particularly aggressive and treatment-resistant phenotype. The chr21amp event is highly clonal and present throughout the hematopoietic hierarchy. DYRK1A , a serine threonine kinase and transcription factor, is the only gene in the 2.7Mb minimally amplified region which showed both increased expression and chromatin accessibility compared to non-chr21amp BP-MPN controls. We demonstrate that DYRK1A is a central node at the nexus of multiple cellular functions critical for BP-MPN development, including DNA repair, STAT signalling and BCL2 overexpression. DYRK1A is essential for BP-MPN cell proliferation in vitro and in vivo , and DYRK1A inhibition synergises with BCL2 targeting to induce BP-MPN cell apoptosis. Collectively, these findings define the chr21amp event as a prognostic biomarker in BP-MPN and link chromothripsis to a druggable target.

The genomic landscape of familial glioma.

Glioma is a rare brain tumor with a poor prognosis. Familial glioma is a subset of glioma with a strong genetic predisposition that accounts for approximately 5% of glioma cases. We performed whole-genome sequencing on an exploratory cohort of 203 individuals from 189 families with a history of familial glioma and an additional validation cohort of 122 individuals from 115 families. We found significant enrichment of rare deleterious variants of seven genes in both cohorts, and the most significantly enriched gene was HERC2 (P = 0.0006). Furthermore, we identified rare noncoding variants in both cohorts that were predicted to affect transcription factor binding sites or cause cryptic splicing. Last, we selected a subset of discovered genes for validation by CRISPR knockdown screening and found that DMBT1, HP1BP3, and ZCH7B3 have profound impacts on proliferation. This study performs comprehensive surveillance of the genomic landscape of familial glioma.

Genome sequencing reveals underdiagnosis of primary ciliary dyskinesia in bronchiectasis.

BACKGROUND: Bronchiectasis can result from infectious, genetic, immunological and allergic causes. 60-80% of cases are idiopathic, but a well-recognised genetic cause is the motile ciliopathy, primary ciliary dyskinesia (PCD). Diagnosis of PCD has management implications including addressing comorbidities, implementing genetic and fertility counselling and future access to PCD-specific treatments. Diagnostic testing can be complex; however, PCD genetic testing is moving rapidly from research into clinical diagnostics and would confirm the cause of bronchiectasis. METHODS: This observational study used genetic data from severe bronchiectasis patients recruited to the UK 100,000 Genomes Project and patients referred for gene panel testing within a tertiary respiratory hospital. Patients referred for genetic testing due to clinical suspicion of PCD were excluded from both analyses. Data were accessed from the British Thoracic Society audit, to investigate whether motile ciliopathies are underdiagnosed in people with bronchiectasis in the UK. RESULTS: Pathogenic or likely pathogenic variants were identified in motile ciliopathy genes in 17 (12%) out of 142 individuals by whole-genome sequencing. Similarly, in a single centre with access to pathological diagnostic facilities, 5-10% of patients received a PCD diagnosis by gene panel, often linked to normal/inconclusive nasal nitric oxide and cilia functional test results. In 4898 audited patients with bronchiectasis, <2% were tested for PCD and <1% received genetic testing. CONCLUSIONS: PCD is underdiagnosed as a cause of bronchiectasis. Increased uptake of genetic testing may help to identify bronchiectasis due to motile ciliopathies and ensure appropriate management.

Routine vaccination practice after adult and paediatric allogeneic haematopoietic stem cell transplant: a survey of UK NHS programmes.

This corrects the article DOI: 10.1038/bmt.2016.362.

Provision of long-term monitoring and late effects services following adult allogeneic haematopoietic stem cell transplant: a survey of UK NHS-based programmes.

Despite international guidelines, optimal delivery models of late effects (LE) services for HSCT patients are unclear from the clinical, organizational and economic viewpoints. To scope current LE service delivery models within the UK NHS (National Health Service), in 2014, we surveyed the 27 adult allogeneic HSCT centres using a 30-question online tool, achieving a 100% response rate. Most LE services were led and delivered by senior physicians (>80% centres). Follow-up was usually provided in a dedicated allograft or LE clinic for the first year (>90% centres), but thereafter attrition meant that only ~50% of patients were followed after 5 years. Most centres (69%) had a standard operating procedure for long-term monitoring but access to a LE Multi-Disciplinary Team was rare (19% centres). Access to medical specialities necessary for LE management was good, but specialist interest in long-term HSCT complications was uncommon. Some screening (endocrinopathy, cardiovascular) was near universal, but other areas were more limited (mammography, cervical smears). Funding of extra staff and investigations were the most commonly perceived barriers to implementation of LE services. This survey shows variation in the long-term follow-up of allogeneic HSCT survivors within the UK NHS and further work is warranted to optimize effective, sustainable and affordable models of LE service delivery among this group.

The immunodeficiency of chronic lymphocytic leukaemia.

INTRODUCTION: Patients with chronic lymphocytic leukaemia (CLL) have progressive immunodeficiency and infection is the commonest cause of death. This review seeks to identify the extent of the abnormality, its cause, clinical significance and any possible remedy. SOURCES OF DATA: TJH has studied CLL for the past 40 years and has scanned or read every paper he could find published on the topic since 1970 and most of those of historical importance published before that date. He has read around the subject, covering relevant articles on immunology, cell biology, oncology and genetics. Furthermore, he has attended most major meetings dealing with CLL in this time and has written many reviews to update the state of knowledge about the topic. He receives weekly updates of papers published on CLL from PubMed and Science Direct with the keywords 'chronic lymphocytic leukaemia'. AREAS OF AGREEMENT: The immunodeficiency chiefly manifests as hypogammaglobulinaemia but involves all elements of the immune system. It is caused by the interpolation of tumour cells among immunological cells and mediated by bi-directional cell contact and secretion of cytokines, which both sustain and invigorate the tumour and suppress immunity. CLL treatment generally makes the immunodeficiency worse. Intravenous immunoglobulin is clinically effective but not cost-effective, while prophylactic antibiotics are useful in appropriate circumstances. Vaccination against infectious disease is usually ineffective. AREAS OF CONTROVERSY: Exactly how the presence of tumour cells in the immune organs renders the patient immunodeficient is controversial as is the clinical significance of minor degrees of immunodeficiency in early or indolent cases. The immunosuppressive effect of most forms of treatment is agreed, but how much this should figure in the choice of treatment is a matter of dispute. GROWING POINTS: The study of tumour-stromal interactions is an area of intense research. AREAS TIMELY FOR DEVELOPING RESEARCH: There has been little done to develop better vaccination strategies in patients with CLL, and although effective antimicrobials have been developed to protect against opportunistic infections, many are both expensive and inconvenient. More work is necessary to define precisely which patients should be offered them and when.

Biochemical and functional assessment of equine lymphocyte phosphodiesterases and protein kinase C.

Lymphocytes play an important role in allergic inflammation and have been implicated in the pathogenesis of equine allergic skin and respiratory disease. Targeting intracellular signalling pathways in human lymphocytes has demonstrated a role for both phosphodiesterase and protein kinase C in cell activation. The aim of this study was to measure total cyclic nucleotide hydrolysing phosphodiesterase activity and to identify the phosphodiesterase and protein kinase C isoenzymes present in equine lymphocytes. The functional significance of these isoenzymes was then investigated by examining their role in peripheral blood mononuclear cell proliferation using isoenzyme selective inhibitors. Total cyclic adenosine monophosphate hydrolysing phosphodiesterase activity was double that of cyclic guanosine monophosphate (30+/-2 pmol/min mg versus 16+/-3 pmol/min mg for cyclic adenosine and cyclic guanosine monophosphate phosphodiesterase activity, respectively). Evidence for the presence of PDE1, 3, 4 and 5 was obtained and PKCalpha, beta, delta, eta, iota, theta and zeta were identified. Selective inhibitors of PDE4, PKCdelta and conventional PKCs alpha and beta caused significant inhibition of mitogen-induced peripheral blood mononuclear cell proliferation. This study demonstrates a functional role for specific signalling isoenzymes and suggests that, in the context of allergic inflammation, targeting inflammatory cells involved in disease pathogenesis with relevant isoenzyme inhibitors may have therapeutic potential.

Role of the chemokine eotaxin in the pathogenesis of equine sweet itch.

The chemokine eotaxin is involved in the recruitment of eosinophils and T helper 2 lymphocytes in human allergic diseases, and drugs that block its activity, including eotaxin receptor (CCR3) antagonists, are being developed. The authors have recently cloned the horse ortholog of eotaxin and shown that it can induce equine eosinophil migration and activation in vitro. Moreover, eotaxin mRNA expression was upregulated in cultured horse dermal fibroblasts exposed to equine interleukin-4, suggesting a possible source of this eosinophil chemoattractant in equine skin. The results of this study show that eotaxin and monocyte chemoattractant protein (MCP) 1, but not MCP-2 or MCP-4, mRNA expression is upregulated in skin biopsies of sweet itch lesions when eosinophils are present, when compared with clinically normal skin from the same ponies.

Antigen presentation of Type II collagen in rats.

Collagen-induced arthritis (CIA) is a T-cell dependent disease of rats which follows immunization with bovine type II collagen (bCII). Susceptibility to CIA is linked to the genes encoding the major histocompatibility complex (MHC), suggesting that antigen presentation is important in disease pathogenesis. Antigen-presenting cells (APC) (macrophages, dendritic cells (DC) and B cells) were prepared from WA/KIR/KCL rats and presentation of antigen, in the form of native protein (bCII) or synthetic peptide (bCII:184-198), was assessed in T-cell proliferation assays. Whilst macrophages inhibited proliferative responses to bCII, splenic or thymic low density cells, enriched for DC, presented both bCII and bCII(184-198) peptide. However, bone marrow-derived DC, which stimulated T-cell responses to OVA, failed to present bCII, suggesting differences in processing of these two antigens. B-cell depletion from lymph node cells abrogated the proliferative response to bCII and reconstitution of a T-cell population with B cells restored the proliferative response, indicating that B cells are important for stimulating T-cell responses to bCII. B cells play a critical role in CIA by producing pathogenic anti-bCII antibodies, and we propose that B cells are also important APC which present bCII to CD4+ T cells.

Equine macrophage identification with an antibody (Ki-M6) to human CD68 and a new monoclonal antibody (JB10).

Monoclonal antibodies (mAbs) recognizing equine macrophages are scarce. The present study compared the immunocytochemical staining of various equine tissues (lymphoid tissue, lung, liver, small intestine, skin and blood leucocytes) by an antibody, Ki-M6, which detects CD68 in human macrophages and dendritic cells, and by a new anti-equine mAb, JB10, with staining produced by two previously described anti-equine macrophage mAbs, CZ2.2 and CZ3.3. Ki-M6 was shown to identify equine macrophages, which had a distribution different from those identified by CZ2.2 and CZ3.3. JB10 identified equine macrophages with a distribution similar to those identified by Ki-M6, but additionally bound to polymorphonuclear leucocytes. Flow cytometry of peripheral blood leucocyte subpopulations and tissue immunocytochemistry were used to compare staining by JB10 with that of CZ2.2 and CVS19; the latter identifies the myeloid antigen, EqCD13, found on polymorphonuclear leucocytes. The staining by JB10 differed from that of both CZ2.2 and CVS19, suggesting that JB10 detects a different molecule. These additional mAbs should prove useful for the future study of new, defined, populations of macrophages in equine immune responses and pathology, and, in the case of Ki-M6 antibody, may make possible an analysis of the structure, distribution and function of the CD68 molecule in the horse.

Characterisation of lymphocyte subpopulations in the skin and circulation of horses with sweet itch (Culicoides hypersensitivity).

Circulating lymphocyte numbers are elevated in horses with the allergic skin disease sweet itch and skin lesions are typified by an infiltrate of eosinophils and mononuclear cells, the latter of which have not been fully characterised. The aim of the present study was to characterise the lymphocyte subpopulations in the circulation and skin of ponies with sweet itch by flow cytometry and a newly developed modified alkaline phosphatase immunohistochemical technique. Sweet itch ponies were found to have significantly greater numbers of circulating CD5+ and CD4+ T-lymphocytes than normal animals. Increased numbers of CD3+ T-lymphocytes, most of which were CD4+, and eosinophils were present in the skin of these animals following intradermal injection of a Culicoides antigen extract (97 +/- 21 vs. 449 +/- 49 CD3+ T-lymphocytes/mm2 in deep dermis of vehicle vs. antigen injected sites; 83 +/- 8% CD4+ T-lymphocytes at antigen injected site). T-lymphocytes, which are thought to be important in the pathogenesis of human allergic skin disease, may therefore contribute to the development of sweet itch lesions via the release of cytokines which can cause eosinophil accumulation and activation. An understanding of the pathology of this disease may lead to a more rational approach to therapy.

Mammalian granulocyte-macrophage colony-stimulating factor and some CpG motifs have an effect on the immunogenicity of DNA and subunit vaccines in fish.

A eukaryotic plasmid DNA carrying the AACGTT CpG motif in its ampR gene is a 'danger' signal for mice and caused an increase in the specific antibody titres of fish and mice after immunization with beta-galactosidase (beta-gal). A second pUC-based plasmid, which is inactive in mice and contains the GACGTC CpG motif in its cytomegalovirus (CMV) promoter, had no effect on antibody responses to beta-gal in either fish or mice. A synthetic oligonucleotide, which contains the GACGTT motif, potentiated antibody responses to co-administered beta-gal protein in mice, but not in fish. This is early evidence that lower and higher vertebrates recognize different unmethylated CpG motifs as 'danger' signals. In addition, plasmid DNA expressing mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) had a marked effect on cytotoxic T-cell-like activity in fish by reducing the average number of myofibres that expressed beta-gal, 28 days after co-injection with plasmid DNA expressing beta-gal. Although the mechanism by which the mouse GM-CSF exerted its biological effects in fish is unknown, this finding might have important implications for fish vaccination, particularly when cytotoxic T cells may play a critical role.

Epithelial lymphocyte and macrophage distribution in the adult and fetal equine lung.

Leucocytes in the lung epithelium play an important role in the ability of an animal to respond appropriately to inhaled pathogens. The distribution of lymphoid and myeloid cells associated with the lung epithelium was examined immunohistochemically throughout the respiratory tract of four horses, comprising two adults from an abattoir, one pregnant mare, and her fetus (in the final stage of gestation). Cross and tangential cryosections were labelled with monoclonal antibodies against T-cell, B-cell, macrophage/dendritic myeloid cell, and major histocompatibility Class (MHC) II surface antigens. Cell numbers were determined by microscopy. In the three adult horses, epithelial CD3+ T-cell numbers decreased progressively from the upper to the lower respiratory tract, but in the fetus there were low numbers of T cells (at most, 10% of those seen in the adult airways) and little variation in different parts of the respiratory tract. MHC Class II was expressed on the airway epithelium of the two abattoir horses, but not that of the mare and her fetus. In these two animals occasional large, mostly irregularly-shaped, Class II-positive cells were seen. Very few epithelium-associated cells in any animal were labelled by anti-CD21 antibody, which identifies B cells, or anti-myeloid cell antibodies; an anti-rat macrophage antibody (ED2) was shown, for the first time, to identify mature equine alveolar macrophages. Despite the small number of animals, the results suggest that in normal adult horses the greatest numbers of epithelial T cells are found where there is greatest contact with airborne antigens, and that there is constitutive epithelial MHC Class II expression. The low level of MHC Class II expression in the fetus, together with the reduced numbers of T cells, was consistent with the suggestion that the fetal immune system requires exposure to airborne stimuli for full development. The low level of MHC Class II expression in the mare may have reflected the immunosuppression that accompanies pregnancy.

Altered expression of CD11/CD18 on the peripheral blood phagocytes of patients with tuberculosis.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is characterized by granulomatous lesions made up of epithelioid cells, giant cells and mononuclear leucocytes. Cell-cell adhesion is important in granuloma formation and in the leucocyte migration which accompanies it. We have recently shown increased expression of the adhesion molecules CD11/CD18 (LeuCAMs, beta 2 integrins) on peripheral blood leucocytes from patients with sarcoidosis (Shakoor & Hamblin, 1992). Here we have studied the expression of CD11/CD18 and CD29 (VLA beta 1 integrin) on the peripheral blood leucocytes of 10 TB patients by flow cytometry. The density (expressed as mean fluorescence intensity) of CD11b on monocytes and polymorphs was increased (P < 0.005), as was CD11c (P < 0.005) and CD18 (P < 0.05) on polymorphs. CD11a expression was significantly reduced on polymorphs (P < 0.05). No differences were found in the expression of CD29, the percentages of cells expressing any molecule and, in contrast to sarcoidosis, the density of any molecule on lymphocytes. Although the cytokine tumour necrosis factor (TNF) has been implicated in the process of up-regulation, an ELISA for TNF failed to detect significant levels in plasma. The results suggest increased peripheral phagocyte CD11/CD18 expression is a feature of TB, which may contribute to the pathological processes involved.

Circulating adhesion molecules in sarcoidosis.

Sarcoidosis is a disease of unknown etiology characterized by non-caseating granulomata together with a number of systemic abnormalities. We have recently shown these include increased expression of the integrins CD11/CD18 on peripheral blood leucocytes. Here we have measured serum levels of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1), E-selectin and vascular cell adhesion molecule-1 (VCAM-1) in 23 patients and 14 normal controls using antigen capture sandwich ELISAs. Median circulating E-selectin levels in the patients were nearly three times those of the controls (P < 0.0001, Mann-Whitney U-test), whilst ICAM-1 but not VCAM-1 levels were only slightly elevated. These results show that endothelial cell activation and shedding of E-selectin into the circulation are additional features of the pathology of sarcoidosis.

Increased CD11/CD18 expression on the peripheral blood leucocytes of patients with HIV disease: relationship to disease severity.

In HIV disease increased adhesion between leucocytes themselves and between leucocytes and endothelium may contribute to cell loss and viral spread. Using a novel method for the preparation of blood leucocytes for flow cytometry, we report increased expression of leucocyte adhesion molecules (LeuCAMs) (CD11/CD18) on peripheral blood leucocytes of patients with HIV disease compared with normal controls. Patients were divided into two groups on the basis of CD4 T lymphocyte numbers (those with > 0.5 x 10(9)/l and those with < 0.2 x 10(9)/l), and assessed for p24 antigen expression, viral load and serum tumour necrosis factor (TNF) levels as well as LeuCAM expression. Patients with < 0.2 x 10(9)/lCD4 cells had more p24 antigen and more HIV infectious virus and more serum TNF than those with > 0.5 x 10(9)/l. Whilst the percentages of only monocytes and polymorphs expressing CD11b were significantly increased in patients with the least CD4 cells, the density of LeuCAMs, expressed as mean fluorescence intensity (MFI), was significantly increased on all leucocytes, with the most significant increases being seen on patients with the fewest CD4 T cells. Our findings are consistent with leucocyte activation by a soluble factor, although we could find no correlation between levels of TNF and LeuCAM expression. The increased expression of adhesion molecules on peripheral blood leucocytes could play a role in the cellular extravasation and aggregation seen in HIV disease.