Mike A. Scott

Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders

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.

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

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 Councils 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.

Leukemia-Associated Somatic Mutations Drive Distinct Patterns of Age-Related Clonal Hemopoiesis

Summary 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.

Relative Importance of Heat-Labile Enterotoxin in the Causation of Severe Diarrheal Disease in the Gnotobiotic Piglet Model by a Strain of Enterotoxigenic Escherichia coli That Produces Multiple Enterotoxins

ABSTRACT Enterotoxigenic Escherichia coli (ETEC) strains that produce multiple enterotoxins are important causes of severe dehydrating diarrhea in human beings and animals, but the relative importance of these enterotoxins in the pathogenesis is poorly understood. Gnotobiotic piglets were used to study the importance of heat-labile enterotoxin (LT) in infection with an ETEC strain that produces multiple enterotoxins. LT− (ΔeltAB) and complemented mutants of an F4+ LT+ STb+ EAST1+ ETEC strain were constructed, and the virulence of these strains was compared in gnotobiotic piglets expressing receptors for F4+ fimbria. Sixty percent of the piglets inoculated with the LT− mutant developed severe dehydrating diarrhea and septicemia compared to 100% of those inoculated with the nalidixic acid-resistant (Nalr) parent and 100% of those inoculated with the complemented mutant strain. Compared to piglets inoculated with the Nalr parent, the mean rate of weight loss (percent per hour) of those inoculated with the LT− mutant was 67% lower (P < 0.05) and that of those inoculated with the complemented strain was 36% higher (P < 0.001). Similarly, piglets inoculated with the LT− mutant had significant reductions in the mean bacterial colony count (CFU per gram) in the ileum; bacterial colonization scores (square millimeters) in the jejunum and ileum; and clinical pathology parameters of dehydration, electrolyte imbalance, and metabolic acidosis (P < 0.05). These results indicate the significance of LT to the development of severe dehydrating diarrhea and postdiarrheal septicemia in ETEC infections of swine and demonstrate that EAST1, LT, and STb may be concurrently expressed by porcine ETEC strains.

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

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.

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

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.

Cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in human bone

Mutations within the CFTR gene are central to the pathophysiology of cystic fibrosis. CFTR encodes a chloride channel that is located primarily on epithelial cell membranes and is responsible for the regulation of transmembrane chloride and other ion transport. Recent studies indicate a potential association between mutation of the CFTR gene and osteoporosis in patients with CF. Dif et al 1 reported an abnormal skeletal phenotype in CFTR-null mice with striking osteopenia, reduced cortical width and thinning of the trabeculae, while in a study of adults with CF, the ΔF508 mutation was shown to be an independent risk factor for low bone mineral density.2 An association between CFTR mutations and bone disease might be mediated either indirectly by effects of the mutations on other systems (for example, the endocrine system), or it could be due to abnormally …

Analysis of human leukaemias and lymphomas using extensive immunophenotypes from an antibody microarray

A novel antibody microarray has been developed that provides an extensive immunophenotype of leukaemia cells. The assay is a solid phase cell‐capture technique in which 82 antigens are studied simultaneously. This paper presents the analysis of 733 patients with a variety of leukaemias and lymphomas from peripheral blood and bone marrow. Discriminant Function Analysis of the expression profiles from these 733 patients and 63 normal subjects were clustered and showed high levels of consistency with diagnoses obtained using conventional clinical and laboratory criteria. The overall levels of consensus for classification using the microarray compared with established criteria were 93·9% (495/527 patients) for peripheral blood and 97·6% (201/206 patients) for bone marrow aspirates, showing that the extensive phenotype alone was frequently able to classify the disease when the leukaemic clone was the dominant cell population present. Immunophenotypes for neoplastic cells were distinguishable from normal cells when the leukaemic cell count was at least 5 × 109 cells/l in peripheral blood, or 20% of cells obtained from bone marrow aspirates. This technique may be a useful adjunct to flow cytometry and other methods when an extensive phenotype of the leukaemia cell is desired for clinical trials, research and prognostic factor analysis.

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

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.

KLF2 mutation is the most frequent somatic change in splenic marginal zone lymphoma and identifies a subset with distinct genotype

To characterise the genetics of splenic marginal zone lymphoma (SMZL), we performed whole exome sequencing of 16 cases and identified novel recurrent inactivating mutations in Kruppel-like factor 2 (KLF2), a gene whose deficiency was previously shown to cause splenic marginal zone hyperplasia in mice. KLF2 mutation was found in 40 (42%) of 96 SMZLs, but rarely in other B-cell lymphomas. The majority of KLF2 mutations were frameshift indels or nonsense changes, with missense mutations clustered in the C-terminal zinc finger domains. Functional assays showed that these mutations inactivated the ability of KLF2 to suppress NF-κB activation by TLR, BCR, BAFFR and TNFR signalling. Further extensive investigations revealed common and distinct genetic changes between SMZL with and without KLF2 mutation. IGHV1-2 rearrangement and 7q deletion were primarily seen in SMZL with KLF2 mutation, while MYD88 and TP53 mutations were nearly exclusively found in those without KLF2 mutation. NOTCH2, TRAF3, TNFAIP3 and CARD11 mutations were observed in SMZL both with and without KLF2 mutation. Taken together, KLF2 mutation is the most common genetic change in SMZL and identifies a subset with a distinct genotype characterised by multi-genetic changes. These different genetic changes may deregulate various signalling pathways and generate cooperative oncogenic properties, thereby contributing to lymphomagenesis.