Methods for detecting mutations in the human JAK2 gene.

Mutations in the JAK2 gene are prevalent in the human myeloid malignancies, being present in virtually all cases of polycythemia vera, and a significant proportion of patients with other myeloproliferative disorders. Various methods for the detection of acquired mutations in this gene are available depending on the need for sensitivity, quantification, or the ability to detect many different mutations. We summarize the various methods published and discuss their relative merits for each application. Two commonly used methods, quantitative real-time PCR (QPCR) for the detection of the JAK2 V617F mutation and high resolution melt-curve analysis (HRM) for the detection of multiple mutations within JAK2 exon 12, demonstrate the utility of each method and their limitations. The choice of methodology is dependent on the application; therefore there is no gold standard for detecting mutations in this gene.

Molecular diagnosis of the myeloproliferative neoplasms: UK guidelines for the detection of JAK2 V617F and other relevant mutations.

Molecular genetic assays for the detection of the JAK2 V617F (c.1849G>T) and other pathogenetic mutations within JAK2 exon 12 and MPL exon 10 are part of the routine diagnostic workup for patients presenting with erythrocytosis, thrombocytosis or otherwise suspected to have a myeloproliferative neoplasm. A wide choice of techniques are available for the detection of these mutations, leading to potential difficulties for clinical laboratories in deciding upon the most appropriate assay, which can lead to problems with inter-laboratory standardization. Here, we discuss the most important issues for a clinical diagnostic laboratory in choosing a technique, particularly for detection of the JAK2 V617F mutation at diagnosis. The JAK2 V617F detection assay should be both specific and sensitive enough to detect a mutant allele burden as low as 1-3%. Indeed, the use of sensitive assays increases the detection rate of the JAK2 V617F mutation within myeloproliferative neoplasms. Given their diagnostic relevance, it is also beneficial and relatively straightforward to screen JAK2 V617F negative patients for JAK2 exon 12 mutations (in the case of erythrocytosis) or MPL exon 10 mutations (thrombocytosis or myelofibrosis) using appropriate assays. Molecular results should be considered in the context of clinical findings and other haematological or laboratory results.

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.

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.

Can cytoplasmic nucleophosmin be detected by immunocytochemical staining of cell smears in acute myeloid leukemia?

Mutations in the C-terminal region of nucleophosmin in acute myeloid leukemia (AML) result in aberrant cytoplasmic nucleophosmin (cNPM) in leukemic blast cells which is detectable by immunocytochemistry in bone marrow trephine (BMT) biopsy sections. We tested whether cNPM is detectable by immunocytochemistry in air-dried smears of AML with nucleophosmin1 (NPM1) mutations. An immunoalkaline phosphatase method was developed using the OCI-AML3 cell line, known to have mutated NPM1, and assessed on blood and marrow smears of 60 AML cases. NPM was detectable in all blast cell nucleoli and cNPM in 21 of 31 of NPM1 mutated and 15 of 29 wild-type cases. Paired air-dried smears and BMT biopsies from the same case (mutated and wild-type) gave discrepancies in cNPM expression and there was no correlation in 10 of 22 cases. Due to the high false positive and negative rates for cNPM in cell smears, this method should not be used as a surrogate for NPM1 mutations in AML.

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

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.

Clonal diversity in the myeloproliferative neoplasms: independent origins of genetically distinct clones.

This study looked for clonal diversity in patients with a myeloproliferative neoplasm associated with more than one acquired genetic lesion. A tyrosine kinase mutation and a cytogenetic lesion were present in the same clone in six of seven patients. By contrast, the genetic lesions were present in separate clones in all six patients with two tyrosine kinase pathway mutations. Moreover, in two patients the clones were genetically unrelated by X-chromosome inactivation studies. These data demonstrated clonal diversity in a subset of patients with early stage haematopoietic malignancy and showed, for the first time, that such clones may arise independently.

Methylation of the suppressor of cytokine signaling 3 gene (SOCS3) in myeloproliferative disorders.

BACKGROUND: The JAK2 V617F mutation can be found in patients with polycythemia vera, essential thrombocythemia and idiopathic myelofibrosis. Mutation or methylation of other components of JAK/STAT signaling, such as the negative regulators suppressor of cytokine signaling 1 (SOCS1) and SOCS3, may contribute to the pathogenesis of both JAK2 V617F positive and negative myeloproliferative disorders. DESIGN AND METHODS: A cohort of patients with myeloproliferative disorders was assessed for acquired mutations, aberrant expression and/or CpG island hypermethylation of SOCS1 and SOCS3. RESULTS: No mutations were identified within the coding region of either gene in 73 patients with myeloproliferative disorders. No disease-specific CpG island methylation of SOCS1 was observed. SOCS1 expression was raised in myeloproliferative disorder granulocytes but the level was independent of JAK2 V617F status. Hypermethylation of the SOCS3 promoter was identified in 16 of 50 (32%) patients with idiopathic myelofibrosis but not in patients with essential thrombocythemia, polycythemia vera or myelofibrosis preceded by another myeloproliferative disorder. Confirmation of methylation status was validated by nested polymerase chain reaction and/or bisulphite sequencing. SOCS3 transcript levels were highest in patients with polycythemia vera and other JAK2 V617F positive myeloproliferative disorders, consistent with SOCS3 being a target gene of JAK2/STAT5 signaling. There was a trend towards an association between SOCS3 methylation and lower SOCS3 expression in JAK2 V617F negative patients with idiopathic myelofibrosis but not in JAK2 V617F positive ones. Finally, SOCS3 methylation was not significantly correlated with survival or other clinical variables. CONCLUSIONS: SOCS3 promoter methylation was detected in 32% of patients with idiopathic myelofibrosis suggesting a possible role for SOCS3 methylation in this disorder. The pathogenetic consequences of SOCS3 methylation in idiopathic myelofibrosis remain to be fully elucidated.

MPL mutations in myeloproliferative disorders: analysis of the PT-1 cohort.

Activating mutations of MPL exon 10 have been described in a minority of patients with idiopathic myelofibrosis (IMF) or essential thrombocythemia (ET), but their prevalence and clinical significance are unclear. Here we demonstrate that MPL mutations outside exon 10 are uncommon in platelet cDNA and identify 4 different exon 10 mutations in granulocyte DNA from a retrospective cohort of 200 patients with ET or IMF. Allele-specific polymerase chain reaction was then used to genotype 776 samples from patients with ET entered into the PT-1 studies. MPL mutations were identified in 8.5% of JAK2 V617F(-) patients and a single V617F(+) patient. Patients carrying the W515K allele had a significantly higher allele burden than did those with the W515L allele, suggesting a functional difference between the 2 variants. Compared with V617F(+) ET patients, those with MPL mutations displayed lower hemoglobin and higher platelet levels at diagnosis, higher serum erythropoietin levels, endogenous megakaryocytic but not erythroid colony growth, and reduced bone marrow erythroid and overall cellularity. Compared with V617F(-) patients, those with MPL mutations were older with reduced bone marrow cellularity but could not be identified as a discrete clinicopathologic subgroup. MPL mutations lacked prognostic significance with respect to thrombosis, major hemorrhage, myelofibrotic transformation or survival.

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.

Mutation of JAK2 in the myeloproliferative disorders: timing, clonality studies, cytogenetic associations, and role in leukemic transformation.

The identification of an acquired mutation of JAK2 in patients with myeloproliferative disorders has raised questions about the relationship between mutation-positive and mutation-negative subtypes, timing of the JAK2 mutation, and molecular mechanisms of disease progression. Here we demonstrate that patients with V617F(-) essential thrombocythemia do not commonly progress to become V617F(+). Consistent with the concept of distinct pathogenetic mechanisms, we show that patients with and without the JAK2 mutation have different patterns of cytogenetic abnormality, with virtually all patients carrying the 20q deletion or trisomy 9 being V617F(+). We also investigated the existence of a "pre-JAK2" phase by comparing the proportion of clonally derived granulocytes, estimated from X-chromosome inactivation patterns (XCIPs), with the proportion of V617F(+) granulocytes. Our results demonstrate that inherent XCIP variability between granulocytes and T cells produces a systematically biased pattern of results that may be misinterpreted as evidence for an excess of clonally derived granulocytes, an observation that limits the utility of XCIP analysis in this context. Lastly, we studied 4 patients with V617F(+) myeloproliferative disorders who subsequently developed acute myeloid leukemia. In 3 patients the leukemic cells were V617F(-), suggesting that in these patients the leukemia arose in a V617F(-) cell.

Size matters: the prognostic implications of large and small deletions of the derivative 9 chromosome in chronic myeloid leukemia.

Deletions of the derivative 9 chromosome (der(9)) are associated with poor prognosis in chronic myeloid leukemia (CML). Several models have been proposed to account for this association. To distinguish between the various models we mapped the deletion in 69 Philadelphia-positive CML patients carrying a der(9) deletion and compared the size of the deletion with the patients' outcome. Our results demonstrate that patients with large deletions had a significantly worse survival than those with small deletions whereas the outcome for patients with small deletions was similar to that of patients lacking a deletion. These results support the tumor suppressor gene model for the pathogenesis of der(9) deletions, argue against alternative models and provide insight into candidate gene location.

Methods for the detection of the JAK2 V617F mutation in human myeloproliferative disorders.

A single acquired mutation in the JAK2 gene has recently been described in human myeloproliferative disorders, including most patients with polycythemia vera and about half of those with essential thrombocythemia and idiopathic myelofibrosis. Reliable and easily implemented methods for detection of this V617F mutation promise to revolutionize the way these disorders are diagnosed and classified, and may in the future have implications for targeted therapeutics. Two polymerase chain reaction-based methods for detection of the mutation are described here. One method is based on allele-specific amplification of the mutant band, and the other on elimination of a restriction enzyme recognition sequence by the mutation. Both methods are significantly more sensitive than conventional sequencing techniques, and could be readily implemented in a molecular diagnostic laboratory.

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 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.

Chromosomal abnormalities and molecular markers in myeloproliferative disorders.

The first possibly causative molecular aberration in patients with myeloproliferative disorders has recently been described. A point mutation in the Janus kinase 2 exchanging a valine for a phenylalanine at position 617 (JAK2 V617F) was found in 65% to 97% of polycythemia vera (PV) patients, as well as in approximately 50% of essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF) patients. In addition, a growing set of molecular and genetic markers, some possibly contributing to disease development, some more likely epiphenomena, has been characterized in these patients over the last few years. Compiling and synthesizing the increasing knowledge on the genetic changes observed in myeloproliferative disorder (MPD) patients will allow us to generate testable hypotheses on the molecular etiology of disease development. Therefore, this review will summarize the current knowledge on chromosomal aberrations, molecular markers, and gene expression studies in MPD patients. From these data, a model depicting our current understanding of the interplay between these markers is presented.

L3mbtl, the mouse orthologue of the imprinted L3MBTL, displays a complex pattern of alternative splicing and escapes genomic imprinting.

L3mbtl encodes a member of the Polycomb group of proteins, which function as transcriptional repressors in large protein complexes. The Drosophila D-l(3)mbt protein is considered a tumor suppressor since its inactivation results in brain tumors. The human L3MBTL gene lies in a region of chromosome 20 frequently deleted in patients with myeloid malignancies and has been proposed as a candidate 20q tumor suppressor gene. Recently we have shown that L3MBTL undergoes monoallelic methylation in hematopoietic tissues and is transcribed from the paternally derived allele. The mouse L3mbtl gene is located on chromosome 2, a region of syntenic homology with human chromosome 20, and in a region containing a number of genes subject to epigenetic regulation. Here we analyze the genomic structure and alternative splicing of L3mbtl and assess its imprinting status in mouse. L3mbtl displays a complex pattern of alternative splicing involving both 5' noncoding and coding exons and is transcribed from two promoters. Unlike its human counterpart, L3mbtl escapes imprinting and there is no differential methylation of its CpG island.

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.

Characterization of the imprinted polycomb gene L3MBTL, a candidate 20q tumour suppressor gene, in patients with myeloid malignancies.

Chromosome 20q deletion is a recurrent chromosomal abnormality associated with myeloid malignancies. L3MBTL represents a strong candidate tumour suppressor gene since it lies within the common deleted region, is a member of the Polycomb-like family, encodes the human homologue of a Drosophila tumour suppressor and is expressed within haematopoietic progenitor cells. We describe the structure of L3MBTL, identify two putative promoters each associated with two CpG islands and characterize a complex pattern of alternative splicing events. Mutation analysis of the gene in patients with and without a 20q deletion identified several polymorphisms but no acquired mutations. The two CpG islands spanning promoter 2 undergo monoallelic methylation in normal haematopoietic cells consistent with imprinting of L3MBTL. Samples from patients with a 20q deletion retained either the methylated or unmethylated allele but retention of the methylated allele did not correlate with reduction in L3MBTL mRNA levels. The absence of a correlation between L3MBTL methylation and transcription could be shown to reflect loss of imprinting in one patient. In addition, our results demonstrate that inactivation of L3MBTL is not a common occurrence in patients with a 20q deletion or in cytogenetically normal patients with polycythaemia vera.

Imprinting of the human L3MBTL gene, a polycomb family member located in a region of chromosome 20 deleted in human myeloid malignancies.

L3MBTL encodes a member of the Polycomb family of proteins, which, together with Trithorax group proteins, is responsible for the coordinated regulation of patterns of gene activity. Members of the Polycomb family also regulate self renewal of normal and malignant hematopoietic stem cells. L3MBTL lies in a region of chromosome 20, deletion of which is associated with myeloid malignancies and represents a good candidate for a 20q target gene. However, mutations of L3MBTL have not been identified in patients with 20q deletions or in cytogenetically normal patients. Here we demonstrate that monoallelic methylation of two CpG islands correlates with transcriptional silencing of L3MBTL, and that L3MBTL transcription occurs from the paternally derived allele in five individuals from two families. Expression of the paternally derived allele was observed in multiple hematopoietic cell types as well as in bone marrow derived mesenchymal cells. Deletions of 20q associated with myeloid malignancies resulted in loss of either the unmethylated or methylated allele. Our results demonstrate that L3MBTL represents a previously undescribed imprinted locus, a vertebrate Polycomb group gene shown to be regulated by this mechanism, and has implications for the pathogenesis of myeloid malignancies associated with 20q deletions.