Publications by authors named "Mike A Scott"

29 Publications

  • Page 1 of 1

Contribution of immunoglobulin lambda light chain gene rearrangement analysis in the diagnosis of B-cell neoplasms.

Br J Haematol 2019 04 25;185(2):261-265. Epub 2019 Jan 25.

Haematopathology and Oncology Diagnostics Service, Addenbrooke's Hospital, Cambridge University NHS Foundation Trust, Cambridge, UK.

Identification of clonal IGH, IGK and IGL gene rearrangements offers diagnostic adjunct in suspected B-cell neoplasms. However, many centres omit IGL analysis as its value is uncertain. A review of 567 cases with IGH, IGK and IGL rearrangement assessed using BIOMED-2 assays showed clonal immunoglobulin gene rearrangement in 54% of cases, of which 24% had a clonal IGL rearrangement. In two cases, the clonal rearrangement was detected exclusively by IGL analysis. This finding demonstrates the added value of IGL analysis for clonality assessment, especially in suspected B-cell neoplasms in which a clonal IGH and/or IGK rearrangement is not detected or is equivocal.
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http://dx.doi.org/10.1111/bjh.15762DOI Listing
April 2019

Targeting MEK in vemurafenib-resistant hairy cell leukemia.

Leukemia 2019 02 19;33(2):541-545. Epub 2018 Oct 19.

Department of Haematology, University of Cambridge, Cambridge, UK.

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http://dx.doi.org/10.1038/s41375-018-0270-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6365378PMC
February 2019

Leukemia-associated somatic mutations drive distinct patterns of age-related clonal hemopoiesis.

Cell Rep 2015 Mar 26;10(8):1239-45. Epub 2015 Feb 26.

Haematological Cancer Genetics, Wellcome Trust Sanger Institute, Cambridge CB10 1SA, UK; Department of Haematology, Cambridge Biomedical Campus, University of Cambridge, Cambridge CB2 0XY, UK; Department of Haematology, Cambridge University Hospitals NHS Trust, Cambridge CB2 0QQ, UK. Electronic address:

Clonal hemopoiesis driven by leukemia-associated gene mutations can occur without evidence of a blood disorder. To investigate this phenomenon, we interrogated 15 mutation hot spots in blood DNA from 4,219 individuals using ultra-deep sequencing. Using only the hot spots studied, we identified clonal hemopoiesis in 0.8% of individuals under 60, rising to 19.5% of those ≥90 years, thus predicting that clonal hemopoiesis is much more prevalent than previously realized. DNMT3A-R882 mutations were most common and, although their prevalence increased with age, were found in individuals as young as 25 years. By contrast, mutations affecting spliceosome genes SF3B1 and SRSF2, closely associated with the myelodysplastic syndromes, were identified only in those aged >70 years, with several individuals harboring more than one such mutation. This indicates that spliceosome gene mutations drive clonal expansion under selection pressures particular to the aging hemopoietic system and explains the high incidence of clonal disorders associated with these mutations in advanced old age.
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http://dx.doi.org/10.1016/j.celrep.2015.02.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4542313PMC
March 2015

Hairy cell leukemia - immunotargets and therapies.

Immunotargets Ther 2014 24;3:107-20. Epub 2014 Jun 24.

Department of Haematology, Addenbrookes Hospital, University of Cambridge, Cambridge, UK.

Hairy cell leukemia (HCL) is an indolent low-grade B-cell lymphoproliferative disorder that is reasonably sensitive to standard first-line purine analog therapy. However, in many cases, repeat relapses occur, requiring multiple courses of purine analog therapy, promoting eventual drug resistance. This, coupled with the concerning side effects of repeated purine analog exposure, has prompted the search for alternative targets and therapies that may provide deeper remissions. Novel strategies employing immune-mediated targeting via monoclonal antibody therapies and recombinant immunotoxins appear promising in HCL and are currently under investigation. More recently, the concept of targeted kinase inhibition using small-molecule inhibitors in HCL has emerged as another potentially viable option. As a deeper understanding of the aberrant molecular pathways contributing to the pathogenesis of HCL develops, the landscape of management for HCL, particularly in the relapse setting, may change significantly in the future as a result of these promising immunotargets and therapies.
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http://dx.doi.org/10.2147/ITT.S31425DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4918239PMC
July 2016

Pitfalls in the diagnosis of anaplastic large cell lymphoma with a small cell pattern.

Case Rep Hematol 2013 10;2013:840253. Epub 2013 Oct 10.

Department of Haematology, Cambridge University Hospitals NHS Foundation Trust, Hills Road, Cambridge CB20QQ, UK.

Anaplastic large cell lymphoma with a small cell pattern is a rare T-cell lymphoma. This condition is more frequently seen in younger patients and should be considered when patients present with leucocytosis and constitutional symptoms. In this report, we describe our diagnostic work-up for one such case using blood, lymph node, and bone marrow aspirate samples, highlighting the variability of antigen expression seen in different sample types and methodologies. This case shows the importance of having a high index of suspicion and assessing CD30 and anaplastic lymphoma kinase expression in all suspected T-cell neoplasms even though this rare condition is not necessarily expected.
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http://dx.doi.org/10.1155/2013/840253DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3810341PMC
November 2013

Leukaemia update. Part 2: managing patients with leukaemia in the community.

BMJ 2013 Apr 9;346:f1932. Epub 2013 Apr 9.

Department of Pathology, Division of Molecular Histopathology, University of Cambridge, Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK.

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http://dx.doi.org/10.1136/bmj.f1932DOI Listing
April 2013

Leukaemia update. Part 1: diagnosis and management.

BMJ 2013 Mar 28;346:f1660. Epub 2013 Mar 28.

Department of Pathology, Division of Molecular Histopathology, University of Cambridge, Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK.

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http://dx.doi.org/10.1136/bmj.f1660DOI Listing
March 2013

Detection of cytoplasmic nucleophosmin expression by imaging flow cytometry.

Cytometry A 2012 Oct 11;81(10):896-900. Epub 2012 Sep 11.

Haemato-Oncology Diagnostics Service, Department of Haematology, Addenbrooke's Hospital, Cambridge, United Kingdom.

Mutations within the nucleophosmin NPM1 gene occur in approximately one-third of cases of acute myeloid leukemia (AML). These mutations result in cytoplasmic accumulation of the mutant NPM protein. NPM1 mutations are currently detected by molecular methods. Using samples from 37 AML patients, we investigated whether imaging flow cytometry could be a viable alternative to this current technique. Bone marrow/peripheral blood cells were stained with anti-NPM antibody and DRAQ5 nuclear stain, and data were acquired on an ImageStream imaging flow cytometer (Amnis Corp., Seattle, USA). Using the similarity feature for data analysis, we demonstrated that this technique could successfully identify cases of AML with a NPM1 mutation based on cytoplasmic NPM protein staining (at similarity threshold of 1.1 sensitivity 88% and specificity 90%). Combining data of mean fluorescence intensity and % dissimilar staining in a 0-2 scoring system further improved the sensitivity (100%). Imaging flow cytometry has the potential to be included as part of a standard flow cytometry antibody panel to identify potential NPM1 mutations as part of diagnosis and minimal residual disease monitoring. Imaging flow cytometry is an exciting technology that has many possible applications in the diagnosis of hematological malignancies, including the potential to integrate modalities.
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http://dx.doi.org/10.1002/cyto.a.22116DOI Listing
October 2012

EDTA and temperature dependent neutrophil aggregation.

Eur J Haematol 2012 Nov 17;89(5):435. Epub 2012 Aug 17.

Department of Haematology, Addenbrooke's Hospital, Cambridge, UK.

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http://dx.doi.org/10.1111/j.1600-0609.2012.01820.xDOI Listing
November 2012

High resolution melting analysis for detection of BRAF exon 15 mutations in hairy cell leukaemia and other lymphoid malignancies.

Br J Haematol 2011 Dec 13;155(5):609-12. Epub 2011 Sep 13.

Department of Haematology, Haemato-Oncology Diagnostic Service, Addenbrooke's Hospital Department of Histopathology, Addenbrooke's Hospital, Cambridge, UK.

The BRAF V600E mutation has recently been described in all cases of hairy cell leukaemia (HCL). We have developed and validated a rapid and sensitive high-resolution melting analysis (HRMA) assay that detects BRAF exon 15 mutations when hairy cells are as low as 5-10% in a sample. All 48 HCL patients were positive for the BRAF V600E mutation, while 114 non-HCL cases were all V600E negative. Interestingly, we detected a novel BRAF D594N mutation in one patient with multiple myeloma. The HRMA assay offers a useful tool to aid the laboratory diagnosis of HCL.
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http://dx.doi.org/10.1111/j.1365-2141.2011.08868.xDOI Listing
December 2011

PML protein analysis using imaging flow cytometry.

J Clin Pathol 2011 May 22;64(5):447-50. Epub 2010 Dec 22.

Haemato-Oncology Diagnostics Service, Department of Haematology, Addenbrooke's Hospital, Cambridge, UK.

Acute promyelocytic leukaemia (APML) can be promptly diagnosed by detecting abnormal diffuse staining patterns of PML bodies in abnormal promyelocytes using immunofluorescence microscopy. However, this technique is subjective, with low sensitivity. Using samples from 18 patients with acute myeloid leukaemia (AML) (including four with APML), the authors investigated whether imaging flow cytometry could be a viable alternative to this current technique and improve sensitivity levels. Bone marrow/peripheral blood cells were stained with an antibody to PML, and data were acquired on an ImageStream (Amnis Corporation, Seattle, Washington, USA). Using the modulation feature for data analysis, the authors demonstrated that this technique could successfully identify cases of APML. Imaging flow cytometry, by analysing greater numbers of cells and with the potential to include disease-specific antigens, increases the sensitivity of the current immunofluorescence technique. Imaging flow cytometry is an exciting technology that has many possible applications in the diagnosis of haematological malignancies, including the potential to integrate modalities.
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http://dx.doi.org/10.1136/jcp.2010.085662DOI Listing
May 2011

AT9283, a potent inhibitor of the Aurora kinases and Jak2, has therapeutic potential in myeloproliferative disorders.

Br J Haematol 2010 Jul 7;150(1):46-57. Epub 2010 May 7.

Department of Haematology, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK.

Constitutive activation of Janus kinase (Jak) 2 is the most prevalent pathogenic event observed in the myeloproliferative disorders (MPD), suggesting that inhibitors of Jak2 may prove valuable in their management. Inhibition of the Aurora kinases has also proven to be an effective therapeutic strategy in a number of haematological malignancies. AT9283 is a multi-targeted kinase inhibitor with potent activity against Jak2 and Aurora kinases A and B, and is currently being evaluated in clinical trials. To investigate the therapeutic potential of AT9283 in the MPD we studied its activity in a number of Jak2-dependent systems. AT9283 potently inhibited proliferation and Jak2-related signalling in Jak2-dependent cell lines as well as inhibiting the formation of erythroid colonies from haematopoietic progenitors isolated from MPD patients with Jak2 mutations. The compound also demonstrated significant therapeutic potential in vivo in an ETV6-JAK2 (TEL-JAK2) murine leukaemia model. Inhibition of both Jak2 and Aurora B was observed in the model systems used, indicating a dual mechanism of action. Our results suggest that AT9283 may be a valuable therapy in patients with MPD and that the dual inhibition of Jak2 and the Aurora kinases may potentially offer combinatorial efficacy in the treatment of these diseases.
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http://dx.doi.org/10.1111/j.1365-2141.2010.08175.xDOI Listing
July 2010

Clinical utility of routine MPL exon 10 analysis in the diagnosis of essential thrombocythaemia and primary myelofibrosis.

Br J Haematol 2010 Apr 11;149(2):250-7. Epub 2010 Feb 11.

Department of Haematology, Haemato-Oncology Diagnostic Service, Addenbrooke's Hospital, Cambridge, UK.

Approximately 50% of essential thrombocythaemia and primary myelo-fibrosis patients do not have a JAK2 V617F mutation. Up to 5% of these are reported to have a MPL exon 10 mutation but testing for MPL is not routine as there are multiple mutation types. The ability to routinely assess both JAK2 and MPL mutations would be beneficial in the differential diagnosis of unexplained thrombocytosis or myelofibrosis. We developed and applied a high resolution melt (HRM) assay, capable of detecting all known MPL mutations in a single analysis, for the detection of MPL exon 10 mutations. We assessed 175 ET and PMF patients, including 67 that were JAK2 V617F-negative by real time polymerase chain reaction (PCR). Overall, 19/175 (11%) patients had a MPL exon 10 mutation, of whom 16 were JAK2 V617F-negative (16/67; 24%). MPL mutation types were W515L (11), W515K (4), W515R (2) and W515A (1). One patient had both W515L and S505N MPL mutations and these were present in the same haemopoietic colonies. Real time PCR for JAK2 V617F analysis and HRM for MPL exon 10 status identified one or more clonal marker in 71% of patients. This combined genetic approach increases the sensitivity of meeting the World Health Organization diagnostic criteria for these myeloproliferative neoplasms.
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http://dx.doi.org/10.1111/j.1365-2141.2010.08083.xDOI Listing
April 2010

Phospho-STAT5 and phospho-Akt expression in chronic myeloproliferative neoplasms.

Br J Haematol 2009 Nov 31;147(4):495-506. Epub 2009 Aug 31.

Department of Haematology, Addenbrooke's Hospital, Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK.

The majority of Myeloproliferative Neoplasms (MPNs) are characterised by mutations in genes encoding molecules or receptors involved in cell signalling, the most common being the JAK2 V617F mutation. This mutation leads to ligand-independent activation of downstream signalling pathways by constitutive phosphorylation. The signalling pathways affected include the Janus kinase-signal transducers and activators of transcription (JAK-STAT) and phosphotidylinositide-3 kinase (PI3K) pathways, which regulate cell survival and apoptosis respectively. Monoclonal antibodies to phospho-STAT5 and phospho-Akt were generated and assessed by immunocytochemistry on bone marrow biopsies of MPN patients with JAK2 V617F, JAK2 exon 12, MPL exon 10 and KIT D816V mutations. JAK2 V617F mutation was associated with significantly increased levels of phosphorylated STAT5 and Akt in haemopoietic cells, most marked in megakaryocytes. In contrast, JAK2 exon 12 and MPL exon 10 mutations did not affect the level of phosphorylation. In systemic mastocytosis with KIT D618V mutation there was significantly increased expression of phosphorylated STAT5 and Akt in neoplastic mast cells although there was no change in the expression in other haemopoietic cells. JAK2 V617F is associated with upregulated phosphorylation of STAT5 and Akt in megakaryocytes, and to a lesser extent in other haemopoietic cells. Immunocytochemistry of bone marrow trephines for these phospho-proteins can be used as a supplementary diagnostic test with a high negative predictive value.
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http://dx.doi.org/10.1111/j.1365-2141.2009.07870.xDOI Listing
November 2009

A practical strategy for the routine use of BIOMED-2 PCR assays for detection of B- and T-cell clonality in diagnostic haematopathology.

Br J Haematol 2007 Jul;138(1):31-43

Department of Histopathology, Addenbrooke's Hospital, Cambridge, UK.

BIOMED-2 polymerase chain reaction (PCR) assays for clonality analysis of immunoglobulin (IG) and T-cell receptor (TCR) gene rearrangements were evaluated in routine haematopathological practice where paraffin-embedded tissues constitute the majority of specimens. One hundred and twenty-five fresh/frozen and 316 paraffin specimens were analysed for DNA quality and clonality. Seventy-nine per cent of paraffin specimens yielded PCR products of over 300 bp. These specimens and all fresh/frozen specimens were analysed with the complete set of BIOMED-2 reactions for IG (8 reactions) and/or TCR (6 reactions) gene rearrangements. The rate of detection of clonality was 96% in mature B-cell neoplasms and 98% in mature T-cell neoplasms and there were no significant differences in these rates between paraffin and fresh/frozen specimens. As the value of sole use of any individual BIOMED-2 reaction in clonality detection was limited, we assessed combinations of reactions that gave the greatest sensitivity with fewest reactions and were applicable for both fresh/frozen and paraffin specimens. For IG gene rearrangements, three reactions combining one targeting the IG heavy chain framework-2 region and two targeting the IG kappa locus achieved a 91% detection rate. For TCR gene rearrangements, the two TCR gamma reactions gave a 94% detection rate. We therefore recommend this strategy as the first-line assays for routine B- and T-cell clonality analysis in diagnostic haematopathology.
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http://dx.doi.org/10.1111/j.1365-2141.2007.06618.xDOI Listing
July 2007

The incidence, indications and outcome for the non-operative management of breast cancer.

J Surg Oncol 2007 Aug;96(2):137-43

Breast Unit, Bedford Hospital, Bedford, United Kingdom.

Background And Objectives: This study aimed to identify the proportion of patients with breast cancer who do not undergo primary operative treatment, to identify the reasons surgery is not performed, and to determine the outcome for this group of patients.

Methods: Data was obtained from the Bedford Breast Cancer Registry for all non-metastatic patients presenting between January 1990 and December 2004 who were initially treated non-operatively. Robust diagnostic, therapeutic, and follow-up data on all patients was collected prospectively during this period.

Results: One hundred and eighty-five out of 2110 episodes of breast cancer were treated non-operatively during this period. Sixty-eight percent of patients were unfit for surgery, 15% had inoperable tumours, and 17% refused surgical intervention. Median survival and 5-year survival rate for all non-operative patients were 3.7 years and 41.2%. Median survival for inoperable patients was 3.7 years, compared with 3.5 years for those unfit for surgery and 4.2 years for those who refused surgery. The 5-year survival rate for patients refusing surgery was 43%, compared with 61% for a matched group of patients undergoing standard surgical therapy.

Conclusions: This study provides useful data on the reasons for, and outcome of, the non-operative management of breast cancer.
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http://dx.doi.org/10.1002/jso.20789DOI Listing
August 2007

JAK2 exon 12 mutations in polycythemia vera and idiopathic erythrocytosis.

N Engl J Med 2007 Feb;356(5):459-68

University of Cambridge, Cambridge, United Kingdom.

Background: The V617F mutation, which causes the substitution of phenylalanine for valine at position 617 of the Janus kinase (JAK) 2 gene (JAK2), is often present in patients with polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis. However, the molecular basis of these myeloproliferative disorders in patients without the V617F mutation is unclear.

Methods: We searched for new mutations in members of the JAK and signal transducer and activator of transcription (STAT) gene families in patients with V617F-negative polycythemia vera or idiopathic erythrocytosis. The mutations were characterized biochemically and in a murine model of bone marrow transplantation.

Results: We identified four somatic gain-of-function mutations affecting JAK2 exon 12 in 10 V617F-negative patients. Those with a JAK2 exon 12 mutation presented with an isolated erythrocytosis and distinctive bone marrow morphology, and several also had reduced serum erythropoietin levels. Erythroid colonies could be grown from their blood samples in the absence of exogenous erythropoietin. All such erythroid colonies were heterozygous for the mutation, whereas colonies homozygous for the mutation occur in most patients with V617F-positive polycythemia vera. BaF3 cells expressing the murine erythropoietin receptor and also carrying exon 12 mutations could proliferate without added interleukin-3. They also exhibited increased phosphorylation of JAK2 and extracellular regulated kinase 1 and 2, as compared with cells transduced by wild-type JAK2 or V617F JAK2. Three of the exon 12 mutations included a substitution of leucine for lysine at position 539 of JAK2. This mutation resulted in a myeloproliferative phenotype, including erythrocytosis, in a murine model of retroviral bone marrow transplantation.

Conclusions: JAK2 exon 12 mutations define a distinctive myeloproliferative syndrome that affects patients who currently receive a diagnosis of polycythemia vera or idiopathic erythrocytosis.
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http://dx.doi.org/10.1056/NEJMoa065202DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2873834PMC
February 2007

Identifying gene regulatory elements by genomic microarray mapping of DNaseI hypersensitive sites.

Genome Res 2006 Oct 8;16(10):1310-9. Epub 2006 Sep 8.

Department of Haematology, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, CB2 2XY, United Kingdom.

The identification of cis-regulatory elements is central to understanding gene transcription. Hypersensitivity of cis-regulatory elements to digestion with DNaseI remains the gold-standard approach to locating such elements. Traditional methods used to identify DNaseI hypersensitive sites are cumbersome and can only be applied to short stretches of DNA at defined locations. Here we report the development of a novel genomic array-based approach to DNaseI hypersensitive site mapping (ADHM) that permits precise, large-scale identification of such sites from as few as 5 million cells. Using ADHM we identified all previously recognized hematopoietic regulatory elements across 200 kb of the mouse T-cell acute lymphocytic leukemia-1 (Tal1) locus, and, in addition, identified two novel elements within the locus, which show transcriptional regulatory activity. We further validated the ADHM protocol by mapping the DNaseI hypersensitive sites across 250 kb of the human TAL1 locus in CD34+ primary stem/progenitor cells and K562 cells and by mapping the previously known DNaseI hypersensitive sites across 240 kb of the human alpha-globin locus in K562 cells. ADHM provides a powerful approach to identifying DNaseI hypersensitive sites across large genomic regions.
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http://dx.doi.org/10.1101/gr.5373606DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1581440PMC
October 2006

The application of CD antigen proteomics to pharmacogenomics.

Pharmacogenomics 2006 Jul;7(5):759-71

University of Cambridge School of Clinical Medicine, Addenbrooke's Hospital, Hills Road, Box 11, Cambridge CB2 2SP, UK.

The advent of multiplexing technologies has raised the possibility that disease states can be defined using discrete genomic and proteomic patterns or signatures. However, this emerging area has been limited by the 'content problem', arising from the uncertainty of which molecules to focus on. The human cluster of differentiation (CD) antigens are expressed on cells of the human immune system (leukocytes) and on other cell types. These heterogeneous molecules perform a host of roles essential to immune function and to the physiology of other lineages. The 339 defined CD antigens and their, as yet, undefined counterparts constitute key components of the expressed human cell surface proteome. We propose that CD antigen expression patterns will form the basis of a rational, discrete and generalized diagnostic and prognostic system. Furthermore, disease-specific CD antigen proteomic signatures are likely to be more robust than corresponding genomic signatures and will also help to identify molecular targets for therapeutic intervention.
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http://dx.doi.org/10.2217/14622416.7.5.759DOI Listing
July 2006

Progenitors homozygous for the V617F mutation occur in most patients with polycythemia vera, but not essential thrombocythemia.

Blood 2006 Oct 13;108(7):2435-7. Epub 2006 Jun 13.

Department of Haematology, Cambridge Institute for Medical Research, Hills Road, Cambridge CB2 2XY, United Kingdom.

An acquired V617F JAK2 mutation occurs in patients with polycythemia vera (PV) or essential thrombocythemia (ET). In a proportion of V617F-positive patients, mitotic recombination produces mutation-homozygous cells that come to predominate with time. However, the prevalence of homozygosity is unclear, as previous reports studied mixed populations of wild-type, V617F-heterozygous, and V617F-homozygous mutant cells. We therefore analyzed 1766 individual hematopoietic colonies from 34 patients with PV or ET in whom granulocyte sequencing demonstrated that the mutant peak did not predominate. V617F-positive erythroid burst-forming units (BFU-Es) were more frequent in patients with PV compared with patients with ET (P = .022) and, strikingly, V617F-homozygous BFU-Es were detected in all 17 patients with PV, but in none of the patients with ET (P < .001). Moreover, mutation-homozygous cells were present in 2 patients with ET after polycythemic transformation. These results demonstrate that V617F-homozygous erythroid progenitors are present in most patients with PV but occur rarely in those with ET.
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http://dx.doi.org/10.1182/blood-2006-04-018259DOI Listing
October 2006

Osteoclastogenesis during infective exacerbations in patients with cystic fibrosis.

Am J Respir Crit Care Med 2006 Aug 4;174(3):306-11. Epub 2006 May 4.

Department of Haematology, NHS Foundation Trust, Addenbrooke's Hospital, Cambridge, United Kingdom.

Rationale: Adults with cystic fibrosis (CF) are at increased risk of developing osteoporosis. During infective exacerbations, increased production of proinflammatory cytokines and markers of bone resorption have been reported.

Objective: The aim of this study is to investigate the growth and proliferation of potential osteoclast precursor cells before, during, and after intravenous antibiotic treatment of infective exacerbations in patients with CF.

Methods: Hematopoietic precursor cell growth was examined using colony formation assays using Methocult culture medium. Circulating potential osteoclast precursors were identified using four-color flow cytometry by CD14, CD33, CD34, and CD45 expression.

Results: At the start of an infective exacerbation increases in hematopoietic precursor colony formation (15.42 colonies/10(5) cells plated, p = 0.025), proliferation (28.5%, p < 0.001), and the numbers of circulating potential osteoclast precursors (6.5%, p < 0.001) were seen in comparison with baseline levels. These increases declined after treatment with intravenous antibiotics to a level close to baseline.

Conclusions: The results demonstrate an increase in the production of potential osteoclast precursors in the peripheral blood during CF infective exacerbations. This may result in increased bone resorption and contribute to bone loss in patients with CF.
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http://dx.doi.org/10.1164/rccm.200512-1943OCDOI Listing
August 2006

Definition of subtypes of essential thrombocythaemia and relation to polycythaemia vera based on JAK2 V617F mutation status: a prospective study.

Lancet 2005 Dec;366(9501):1945-53

Department of Haematology, University of Cambridge, UK.

Background: An acquired V617F mutation in JAK2 occurs in most patients with polycythaemia vera, but is seen in only half those with essential thrombocythaemia and idiopathic myelofibrosis. We aimed to assess whether patients with the mutation are biologically distinct from those without, and why the same mutation is associated with different disease phenotypes.

Methods: Two sensitive PCR-based methods were used to assess the JAK2 mutation status of 806 patients with essential thrombocythaemia, including 776 from the Medical Research Council's Primary Thrombocythaemia trial (MRC PT-1) and two other prospective studies. Laboratory and clinical features, response to treatment, and clinical events were compared for V617F-positive and V617F-negative patients with essential thrombocythaemia.

Findings: Mutation-positive patients had multiple features resembling polycythaemia vera, with significantly increased haemoglobin (mean increase 9.6 g/L, 95% CI 7.6-11.6 g/L; p<0.0001), neutrophil counts (1.1x10(9)/L, 0.7-1.5x10(9)/L; p<0.0001), bone marrow erythropoiesis and granulopoiesis, more venous thromboses, and a higher rate of polycythaemic transformation than those without the mutation. Mutation-positive patients had lower serum erythropoietin (mean decrease 13.8 U/L; 95% CI, 10.8-16.9 U/L; p<0.0001) and ferritin (n=182; median 58 vs 91 mug/L; p=0.01) concentrations than did mutation-negative patients. Mutation-negative patients did, nonetheless, show many clinical and laboratory features that were characteristic of a myeloproliferative disorder. V617F-positive individuals were more sensitive to therapy with hydroxyurea, but not anagrelide, than those without the JAK2 mutation.

Interpretation: Our results suggest that JAK2 V617F-positive essential thrombocythaemia and polycythaemia vera form a biological continuum, with the degree of erythrocytosis determined by physiological or genetic modifiers.
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http://dx.doi.org/10.1016/S0140-6736(05)67785-9DOI Listing
December 2005

The rectus sling to prevent loop colostomy retraction: a case series.

Int Semin Surg Oncol 2005 Oct 20;2:22. Epub 2005 Oct 20.

Department of Gastrointestinal Surgery, Barnet General Hospital, Wellhouse Lane, Barnet, Herts, EN4 3DJ, UK.

Diverting stomas are being used increasingly in the management of rectal cancer, particularly with low anterior resection following neoadjuvant therapy. We describe a simple anchorage method for loop colostomy using a rectus fascial sling. This has been used successfully in fifteen patients with no complications or evidence of significant spill over of faecal contents into the efferent loop.
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http://dx.doi.org/10.1186/1477-7800-2-22DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1277842PMC
October 2005

Conservation of unique cell-surface CD antigen mosaics in HIV-1-infected individuals.

Blood 2005 Aug 12;106(3):1003-7. Epub 2005 Apr 12.

University of Cambridge School of Clinical Medicine, Addenbrooke's Hospital, Hills Road, Box 111, Cambridge CB2 2SP, United Kingdom.

Cluster of differentiation (CD) antigens are expressed on cells of myeloid and lymphoid lineages. As most disease processes involve immune system activation or suppression, these antigens offer unique opportunities for monitoring host responses. Immunophenotyping using limited numbers of CD antigens enables differentiation states of immune system cells to be determined. Extended phenotyping involving parallel measurement of multiple CD antigens may help identify expression pattern signatures associated with specific disease states. To explore this possibility we have made a CD monoclonal antibody array and scanner, enabling the parallel immunophenotyping of leukocyte cell suspensions in a single and rapid analysis. To demonstrate this approach, we used the specific example of patients infected with human immunodeficiency virus type-1 (HIV-1). An invariant HIV-induced CD antigen signature has been defined that is both robust and independent of clinical outcome, composed of a unique profile of CD antigen expression levels that are both increased and decreased relative to internal controls. The results indicate that HIV-induced changes in CD antigen expression are disease specific and independent of outcome. Their invariant nature indicates an irreversible component to retroviral infection and suggests the utility of CD antigen expression patterns in other disease settings.
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http://dx.doi.org/10.1182/blood-2004-12-4642DOI Listing
August 2005

Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders.

Lancet 2005 Mar 19-25;365(9464):1054-61

Department of Haematology, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK.

Background: Human myeloproliferative disorders form a range of clonal haematological malignant diseases, the main members of which are polycythaemia vera, essential thrombocythaemia, and idiopathic myelofibrosis. The molecular pathogenesis of these disorders is unknown, but tyrosine kinases have been implicated in several related disorders. We investigated the role of the cytoplasmic tyrosine kinase JAK2 in patients with a myeloproliferative disorder.

Methods: We obtained DNA samples from patients with polycythaemia vera, essential thrombocythaemia, or idiopathic myelofibrosis. The coding exons of JAK2 were bidirectionally sequenced from peripheral-blood granulocytes, T cells, or both. Allele-specific PCR, molecular cytogenetic studies, microsatellite PCR, Affymetrix single nucleotide polymorphism array analyses, and colony assays were undertaken on subgroups of patients.

Findings: A single point mutation (Val617Phe) was identified in JAK2 in 71 (97%) of 73 patients with polycythaemia vera, 29 (57%) of 51 with essential thrombocythaemia, and eight (50%) of 16 with idiopathic myelofibrosis. The mutation is acquired, is present in a variable proportion of granulocytes, alters a highly conserved valine present in the negative regulatory JH2 domain, and is predicted to dysregulate kinase activity. It was heterozygous in most patients, homozygous in a subset as a result of mitotic recombination, and arose in a multipotent progenitor capable of giving rise to erythroid and myeloid cells. The mutation was present in all erythropoietin-independent erythroid colonies.

Interpretation: A single acquired mutation of JAK2 was noted in more than half of patients with a myeloproliferative disorder. Its presence in all erythropoietin-independent erythroid colonies demonstrates a link with growth factor hypersensitivity, a key biological feature of these disorders.

Relevance To Practice: Identification of the Val617Phe JAK2 mutation lays the foundation for new approaches to the diagnosis, classification, and treatment of myeloproliferative disorders.
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http://dx.doi.org/10.1016/S0140-6736(05)71142-9DOI Listing
April 2005

Estrogen stimulates differentiation of megakaryocytes and modulates their expression of estrogen receptors alpha and beta.

J Cell Biochem 2004 May;92(2):249-57

University of Cambridge School of Clinical Medicine, Addenbrooke's Hospital, Cambridge, United Kingdom.

Estrogen has multifunctional effects influencing growth, differentiation, and function in many tissues. High-dose estrogen has been shown to produce anabolic skeletal effects in the skeleton of postmenopausal women with increased megakaryocyte (MK) population in the bone marrow, suggesting a possible role for these cells in bone remodelling. To investigate if estrogen stimulates megakaryocytopoiesis and affects on estrogen receptor (ER) expression, CD34(+) cells were cultured for 6, 9, and 14 days plus or minus low-dose or high-dose 17 beta estradiol (E). Cells were immunolocalised for CD61, CD41, ER alpha and beta. ER mRNA expression was assessed by RT-PCR. Cells formed more CD61 positive MK colonies with low- and high-dose E treatment (P < 0.001) at 6 and 9 days. CD41 expression was increased dose-dependently in MK (3- and 5-fold P < 0.001) at 9 days. E-stimulated ER alpha expression at 6 days (P < 0.001) whilst ER beta was dose-dependently increased only at 9 days (P < 0.01). ER alpha mRNA was increased at 6 days but not at 14 days whilst ER beta mRNA expression was only increased at 14 days with E treatment. These results demonstrate that E stimulates the colony forming potential of CD34(+) cells to a more megakaryocytic phenotype in vitro. This finding together with the stimulation of ER protein and mRNA expression adds to the increasing evidence for a role for MKs in estrogen-induced bone formation.
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http://dx.doi.org/10.1002/jcb.20035DOI Listing
May 2004

Derivative chromosome 9 deletions in chronic myeloid leukemia: poor prognosis is not associated with loss of ABL-BCR expression, elevated BCR-ABL levels, or karyotypic instability.

Blood 2002 Jun;99(12):4547-53

Department of Hematology, University of Cambridge, United Kingdom.

Deletions of the derivative chromosome 9 have recently been reported in chronic myeloid leukemia. These deletions are large, occur at the time of the Philadelphia (Ph) translocation, span the translocation breakpoint, and represent a powerful prognostic indicator. However, the molecular mechanisms responsible for the poor prognosis associated with deletions are obscure, and several possible models are investigated here. First, we demonstrate that all derivative chromosome 9 deletions detected by fluorescence in situ hybridization were associated with an absence of ABL-BCR expression. However, loss of ABL-BCR expression also occurred without an overt deletion, suggesting the existence of other mechanisms by which ABL-BCR transcription can be abolished. Furthermore, analysis of survival in 160 patients demonstrated that loss of ABL-BCR expression, in contrast to deletion status, was not an indicator of poor prognosis. Second, we addressed the possibility that concomitant small deletions of the Ph chromosome modulate BCR-ABL transcription. Real-time reverse-transcription polymerase chain reaction was used to demonstrate that derivative chromosome 9 deletions were not accompanied by altered levels of BCR-ABL transcripts. Third, deletions may represent a consequence of genetic instability within the target cell at the time of the Ph translocation, with the poor prognosis reflecting a predisposition to subsequent additional genetic alterations. However, patients with deletions do not exhibit an increased frequency of secondary cytogenetic changes following disease progression. Taken together, these data support a model in which deletions of the derivative chromosome 9 result in rapid disease progression as a result of the loss of one or more genes within the deleted region.
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http://dx.doi.org/10.1182/blood.v99.12.4547DOI Listing
June 2002