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Keywords:

  • myeloproliferative neoplasm;
  • molecular diagnosis;
  • JAK2;
  • MPL

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Detection of the JAK2 V617F mutation at diagnosis
  5. Supplementary tests for JAK2 V617F negative cases
  6. Acknowledgements
  7. References

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.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Detection of the JAK2 V617F mutation at diagnosis
  5. Supplementary tests for JAK2 V617F negative cases
  6. Acknowledgements
  7. References

The classical BCR-ABL1 negative myeloproliferative neoplasms (MPN) comprise polycythaemia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis (PMF). In 2005, an acquired mutation within JAK2 exon 14 was identified (c.1849G>T), which results in a valine to phenylalanine substitution at codon 617 – p.Val617Phe, usually abbreviated to V617F (Baxter et al, 2005; James et al, 2005; Kralovics et al, 2005; Levine et al, 2005a). This codon lies in the JH2 pseudokinase domain of JAK2 and the mutation is thought to interfere with JH2-mediated autoinhibition leading to constitutive activation of the tyrosine kinase function. This results in activation of a number of downstream pathways including JAK/STAT, PI3K/AKT and MAPK/ERK. The JAK2 V617F mutation has been observed in up to 98% of patients with PV and 50–60% of patients with ET and PMF. With the exception of the syndrome ‘refractory anaemia with ringed sideroblasts associated with marked thrombocytosis’ (RARS-T) where it is observed in approximately one half of patients (Szpurka et al, 2006; Schmitt-Graeff et al, 2008), the JAK2 V617F mutation is uncommon in other myeloid disorders, such as myelodysplastic syndrome, chronic myelomonocytic leukaemia and acute myeloid leukaemia (Jones et al, 2005; Levine et al, 2005b).

In 2007, mutations within exon 12 of JAK2 were described in some JAK2 V617F negative PV patients (Scott et al, 2007a) as well as in patients previously categorized as idiopathic erythrocytosis, raising the suggestion that all patients with PV carry a mutation within JAK2 (Scott et al, 2007b; McMullin, 2008; Wang et al, 2008). At least 17 different mutations have now been described within exon 12 (Passamonti et al, 2011). Although these mutations are not located within the JH2 domain, they are also thought to interfere with JH2-mediated autoinhibition. In 2006, mutations within exon 10 of the thrombopoietin receptor, MPL, were identified in ET and PMF patients (Pardanani et al, 2006) and at least five different pathogenetic mutations have been described (Chaligné et al, 2008; Schnittger et al, 2009a; Boyd et al, 2010) that affect codons S505 or W515. Other variants have also been described (Ma et al, 2011) but their pathogenicity is not known.

The demonstration of an acquired mutation within JAK2 and/or MPL now forms part of the World Health Organization criteria for the diagnosis of MPN (Swerdlow et al, 2008). Patients presenting with erythrocytosis should be assessed for the presence of a JAK2 mutation. The British Committee for Standards in Haematology (BCSH) guidelines state that the presence of a JAK2 mutation (V617F or exon 12) and a raised haematocrit (>0·52 male; >0·48 female) or raised red cell mass (>25% above predicted) is sufficient to make a diagnosis of PV (McMullin et al, 2007; McMullin, 2008). Likewise, patients presenting with a persistent thrombocytosis should be assessed for the presence of JAK2 V617F and, if negative, MPL exon 10 mutations (Harrison et al, 2010). The presence of an acquired pathogenetic mutation (i.e. JAK2 V617F and/or MPL exon 10 mutation) and a sustained thrombocytosis (platelet count > 450 × 109/l) in the absence of evidence for another myeloid malignancy is sufficient to make a diagnosis of ET (Harrison et al, 2010). For PMF, the demonstration of JAK2 V617F and/or MPL exon 10 mutations is a major diagnostic criterion because it confirms the primary nature of the disorder (Swerdlow et al, 2008). The demonstration of a JAK2 V617F mutation in samples from patients not otherwise meeting specific diagnostic criteria for a MPN, for example presenting with unexplained splanchnic vein thrombosis (Dentali et al, 2009), suggests an underlying MPN or, more rarely, another myeloid malignancy.

The aim of these guidelines is to provide information and suggestions for those diagnostic laboratories that perform or wish to perform assays for the detection of JAK2 V617F, for which many different techniques are available. Diagnostic assays are also available for the detection of JAK2 exon 12 and MPL exon 10 mutations and these are also discussed. A strategy for the efficient combination of JAK2 V617F, JAK2 exon 12 and MPL exon 10 mutations detection assays in suspected myeloproliferative neoplasms is discussed. These guidelines are broadly in line with the screening strategy proposed by Tefferi et al (2011). A step-wise algorithm for supplementary JAK2 exon 12 or MPL exon 10 mutation analysis is both cost-effective and an efficient use of available material and may reduce the need for a bone marrow biopsy in some patients. Furthermore, these guidelines highlight the technical issues of relevance for diagnostic laboratories.

Detection of the JAK2 V617F mutation at diagnosis

  1. Top of page
  2. Summary
  3. Introduction
  4. Detection of the JAK2 V617F mutation at diagnosis
  5. Supplementary tests for JAK2 V617F negative cases
  6. Acknowledgements
  7. References

A number of different factors that contribute to the choice of assay for the detection of the JAK2 V617F mutation at diagnosis will be discussed. Quantification of the JAK2 V617F mutation, either at diagnosis for prognostic information or during treatment as a means of minimal residual disease assessment, is not discussed here although the use of such assays is entirely applicable in the diagnostic setting. Many assays for quantification of the JAK2 V617F mutant burden have been developed which differ markedly in their performance with respect to specificity and sensitivity and these have been subject to a comprehensive comparison by the European LeukemiaNet and MPN&MPNr-EuroNet study groups (Jovanovic et al, 2011).

Type of sample

DNA extracted from peripheral blood or bone marrow is acceptable for JAK2 V617F mutation analysis provided the nucleic acid obtained is of acceptable quality for the assay to be performed successfully. In most cases, peripheral blood is the preferred option and EDTA is the usual anticoagulant. Use of other anti-coagulants is acceptable although care should be taken in the case of Lithium heparin tubes to completely remove any anticoagulant during the nucleic acid extraction procedure because its presence may inhibit polymerase chain reaction (PCR) amplification (Yokota et al, 1999). A sample of sufficient volume to obtain a reasonable amount of nucleic acid should be taken (2–10 ml peripheral blood is usually fine although some centres have optimized routine DNA extraction from smaller volumes), although samples from neutropenic patients may yield less nucleic acid. There does not appear to be a major difference in the JAK2 V617F allele burden between whole blood and bone marrow (Larsen et al, 2008). Therefore, a bone marrow aspirate may be assessed (taken into an EDTA tube or cytogenetic culture medium) but is generally not necessary if peripheral blood is available. The sample should be received within 24–48 h after being taken but, in our experience, for DNA analysis samples of up to 1 week old are usually acceptable for non-quantitative tests. Peripheral blood may be frozen until nucleic acid is extracted and it is often possible to extract DNA from stained or (preferably) unstained and unfixed slides if necessary (Jones et al, 2006). The use of DNA derived from plasma for the detection of the JAK2 V617F mutation has been described (Ma et al, 2008). However, this methodology has not been independently validated and does not offer any obvious advantage over DNA derived directly from peripheral blood.

Isolation of peripheral blood granulocytes

An important question often raised is whether isolation of peripheral blood granulocytes is necessary to perform JAK2 V617F mutation detection assays. The JAK2 V617F mutation arises in a haematopoietic progenitor cell but, in most patients, is restricted to the myeloid lineage. In addition, the proportion of myeloid cells carrying the JAK2 V617F mutation can vary widely amongst patients. In general, patients with ET tend to carry a lower overall level of the JAK2 V617F mutation compared to those with PV or PMF (Passamonti & Rumi, 2009) (due to lower proportion of JAK2 V617F positive cells and the presence of a monoallelic mutation in most V617F positive cells). A further confounding factor is that patients who have been treated with hydroxycarbamide may exhibit lower levels of JAK2 V617F (Girodon et al, 2008). Although other studies have found minimal changes in V617F levels on hydroxycarbamide (Antonioli et al, 2010), the possibility of a lower disease burden is worth bearing in mind if JAK2 V617F testing is carried out after cytoreductive treatment. Overall, the quantitative level of the JAK2 V617F mutation is about 15% lower in peripheral blood compared to purified granulocytes (Hermouet et al, 2007) due to the presence of JAK2 V617F negative lymphocytes. If highly sensitive assays are used, there is no difference in detection rate between peripheral blood and granulocytes as a source of material (Hermouet et al, 2007; Goday-Fernandez et al, 2008; Cankovic et al, 2009). However, when a moderately sensitive assay, such as agarose gel-based allele-specific PCR (Baxter et al, 2005; sensitivity approximately 3%) was used, purification of granulocytes increased the detection rate from 92% to 97% for PV and from 57% to 61% for ET (Goday-Fernandez et al, 2008). Hence, isolation of granulocytes should not be required provided the assay is sufficiently sensitive (sensitivity of 1–3% or better). If the assay utilized has a lower sensitivity (see Table 1), then enrichment of granulocytes (Asimakopoulos et al, 1996) may be necessary. An alternative approach is to prepare nucleic acid from erythropoietin (EPO) independent erythroid burst-forming unit (BFU-E) colonies, as such colonies should consist entirely of clonal malignant cells. However, this is time consuming, technically challenging, no quality assurance scheme is available and EPO-independent BFU-E are not observed in all patients.

Table 1. Diagnostic approaches for the detection of the JAK2 V617F mutation
MethodApproximate sensitivitya (%)Example reference(s)
  1. ARMS, amplification-refractory mutation system; dHPLC, denaturing high performance liquid chromatography.

  2. a

    Actual sensitivity will depend on exact protocol.

  3. b

    Sensitivity usually greater when assessed by capillary gel electrophoresis rather than agarose gel electrophoresis.

ARMS/allele-specific PCRb0·1–5Baxter et al (2005); Chen et al (2007); Jones et al (2005); McClure et al (2006); Tan et al (2007)
Real time allele-specific PCR0·01–1Cankovic et al (2009); Denys et al (2010); Kroger et al (2007); Larsen et al (2007); Lippert et al (2006)
Melting curve analysis1–5Cankovic et al (2009); James et al (2006); McClure et al (2006); Wu et al (2011)
High resolution melt analysis1–5Rapado et al (2009); Qian et al (2010)
Restriction enzyme digestionb2–10Campbell et al (2005); Cankovic et al (2009)
Direct sequencing10–20Lippert et al (2009)
Pyrosequencing5Jelinek et al (2005); Jones et al (2005)
dHPLC1–20Albiero et al (2008); Sattler et al (2006); Stevenson et al (2006)

Nucleic acid

Genomic DNA is the preferred choice of nucleic acid due to its stability although assays involving RNA/cDNA are also available. Analysis of RNA/cDNA also allows platelets to be interrogated, but this is not necessary on a routine basis. Commercial DNA purification kits, either manual or automatic, generally give reliable DNA of acceptable quantity and quality, as do many in-house purification methods. It is advisable to process both control samples and the sample under investigation using the same method to reduce variability. DNA concentration should also be calculated using the same method for all samples because different instruments may produce widely varying apparent concentrations (e.g. UV spectrophotometer; Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE, USA); Qubit fluorometer (Life Technologies, Paisley, UK)). These two points are probably more important for ‘comparative’ techniques, such as high resolution melt (HRM) analysis or denaturing high performance liquid chromatography (dHPLC). The amount of template nucleic acid required will depend on the particular assay chosen.

Choice of assay and sensitivity

A large number of different approaches for the detection of the JAK2 V617F mutation have been described (Table 1). For each type of technique, slightly different assays have been designed that vary with instrument, primer and/or probe sequence and detection method. The techniques described broadly fall into two main categories. Firstly, those assays that are designed to specifically target the c.1849G>T mutation (for example, allele-specific PCR) and, secondly, mutation scanning assays that target the region of exon 14 encompassing the c.1849G>T mutation (for example, direct sequencing, HRM analysis). For assays that specifically target the mutant allele, specificity is usually achieved through the use of a mutation-specific primer or probe. Commercial kits are available for detection of JAK2 V617F and these are based on similar approaches.

Two main criteria are important in the choice of an assay. Firstly, it should be specific (i.e. no false negatives or a clearly defined background level such that JAK2 V617F negative and positive cases can be readily distinguished). Secondly, the assay must be sensitive enough to be able to identify a JAK2 V617F mutant allele with a burden as low as 1–3%. This threshold has been shown to be pathogenetically relevant and carry clinical significance (Wang et al, 2008; Mason et al, 2011). Consequently, direct sequencing is not recommended as the method of choice because it only has a sensitivity of 10–20%. Other assays that possess a sensitivity of 3–5%, such as restriction enzyme digestion, agarose gel-based allele-specific PCR and pyrosequencing, may also fail to identify a small number of patients who carry a pathogenetically important low level JAK2 V617F mutation. Use of more sensitive assays does indeed increase the detection rate of JAK2 V617F in both PV and ET patients particularly when unfractionated peripheral blood is assessed (Campbell et al, 2005; Goday-Fernandez et al, 2008; Wang et al, 2008; Cankovic et al, 2009; Lippert et al, 2009). Finally, to achieve a sensitivity of 1–3%, it is necessary to analyse at least 20 ng of genomic DNA, equivalent to 3030 diploid genomes.

False positives

False positive results may also occur due to cross-reactivity of primers or probes (Mason et al, 2011). Hence, particularly with highly sensitive assays, it is critically important to assess the false positive rate using a series of healthy control samples (see below). The assay should also be able to give an indication of the quality/quantity of the DNA to judge whether it carries sufficient sensitivity for each patient. Sample quality may be judged using absolute copy number of a control gene, the CT value for a control gene, the strength of a band on a gel, the peak height of fragment, the height of the (pyro) sequence or other appropriate output. Specific criteria should be laid down to identify samples that are of poor quality. It is important to stress that all results of molecular investigations should be considered in the context of clinical, morphological, haematological and other laboratory findings.

Interpretation of low level JAK2 V617F at diagnosis

A result of <1–3% V617F should be interpreted in the context of clinical, morphological, haematological and other laboratory findings. Such considerations not only mitigate against occasional technical aberrations but it has been claimed that JAK2 V617F may occasionally be found in haematologically normal individuals when highly sensitive assays are used (Sidon et al, 2006; Xu et al, 2007; Nielsen et al, 2011). Assuming the result does not represent a false positive, it is reproducible and the amplification is distinct from appropriate normal controls, such a result may well represent a true low level clone. In patients with other laboratory or clinical criteria suggestive of a MPN, this result provides objective evidence in support of the diagnosis. Low level JAK2 V617F may occur for a number of reasons: (i) prior treatment with cytoreductive therapy may reduce the level of the JAK2 V617F positive clone within the sample (Girodon et al, 2008); (ii) the presence of two MPN clones in the patient, only one of which is JAK2 V617F positive (Beer et al, 2009). Mutation assessment of JAK2 exon 12 or MPL exon 10 may reveal the existence of such second clones.

In a patient with a low level JAK2 V617F mutation but with a normal full blood count, the clinical significance is less clear. Obviously, iron-deficient PV has to be excluded. It is still possible that this may reflect the presence of a chronic MPN and that the relevant blood parameters have risen above the individual's own baseline, but are not yet above the upper limit for the normal range of the appropriate population. Alternatively, a low level JAK2 V617F positive clone may remain stable, or even occasionally disappear with time (for example, if it arises in a short-lived haematopoietic precursor), without significant clinical effects. Such patients may warrant further clinical surveillance. Whatever the cause and clinical situation pertaining to a low level JAK2 V617F mutation, it is prudent to obtain a fresh sample (e.g. within 3–6 months if possible) to enable the assay to be repeated.

False low levels and false negatives

False low levels or even false negatives can occur due to the presence of an additional exon 14 mutation or inherited polymorphism. If these additional changes lie within one of the primer or probe binding sites, they may reduce the efficiency of V617F-specific PCR amplification. Rare instances of additional acquired mutations or constitutional variants have been reported (Table 2). Depending on the assay utilized, these can lead to a false negative result for V617F or apparent low level amplification. Assessment of the c.1849G>T (V617F) mutation by an additional method that utilizes different primer/probe sets may be helpful in situations of apparent low level amplification.

Table 2. Non-V617F variants within JAK2 exon 14
MutationAmino acidReference
1831T>G/1849G>TL611V/V617FCleyrat et al (2010)
1839T>C/1849G>TY613Y/V617FT. Clench (unpublished observations)
1848T>C/1849G>TC616C/V617FWong et al (2007)
1849G>T/1851C>T/1852T>CV617F/C618RWarshawsky et al (2010); A. Goday-Fernandez and A. J. Bench (unpublished observations)
1849G>T/1853G>TV617F/C618FWarshawsky et al (2010)
1860C>AD620ESchnittger et al (2006)
G1849G>T/1860C>AV617F/D620EGrunebach et al (2006)
1849G>AV617IMead et al (2012)

Considerations for validation of a JAK2 V617F detection assay

Prior to introduction and as part of ongoing quality control, the assay should be appropriately validated. Particularly with the more sensitive assays, a large series of healthy control samples should be assessed to determine the false positive rate (Mattocks et al, 2010). Ideally at least 100 should be tested, which gives a lower confidence interval of 97·5% specificity assuming all results are negative (300 samples are necessary to give 99% specificity). These data should demonstrate lack of a ‘positive’ result for the healthy control panel or, as a minimum, identify a cut-off below which the result is defined as ‘not detected’. If false positives are observed, consideration should be given to modification of the assay to reduce or prevent inappropriate amplification. The validation process should also determine the approximate sensitivity of the assay. This is more straightforward for assays that are able to quantify the absolute amount of mutant JAK2 V617F burden through the use of standard curve reagents. In reality, comparison between laboratories is difficult to achieve given the absence of certified reference reagents that could be universally applied. However a suitable dilution series can be prepared using the JAK2 V617F positive cell lines, such as UKE-1 or HEL and used to determine assay sensitivity. Of these, UKE-1 may be preferable because HEL has multiple copies of mutant JAK2 (Quentmeier et al, 2006). As described above, achievement of a sensitivity of 1–3% is desirable and such an assay would be expected to identify the vast majority of patients with a pathogenetically relevant level of JAK2 V617F mutant clone. Whatever the sensitivity achieved, it is important to indicate the assay sensitivity when reporting results.

Ongoing internal quality control should be performed and appropriate controls should be included on each run. Such controls would include a known JAK2 V617F positive (>5% V617F), a JAK2 V617F positive at a level of 1–3% (or other percentage <5% to assess sensitivity) and normal control(s). As described above, a result that appears to be lower than the 1–3% positive control may still be valid but should be interpreted carefully and in the clinical context. Finally, participation in an appropriate external quality assessment programme for JAK2 V617F detection (e.g. www.ukneqasli.org.uk) provides an independent assessment of test quality. Participation in such a programme is required for laboratory accreditation in the United Kingdom. It is noteworthy that a recent international study from the United Kingdom National External Quality Assessment Service for Leucocyte Immunophenotyping found that 20% of testing laboratories failed to detect mutant JAK2 in a sample with 2% V617F and 9% of laboratories failed to detect the mutation at 5% V617F (Clark et al, 2012).

Supplementary tests for JAK2 V617F negative cases

  1. Top of page
  2. Summary
  3. Introduction
  4. Detection of the JAK2 V617F mutation at diagnosis
  5. Supplementary tests for JAK2 V617F negative cases
  6. Acknowledgements
  7. References

As described earlier, JAK2 V617F negative MPN patients may carry mutations at other loci including JAK2 exon 12, MPL, TET2, ASXL1, CBL, SH2B3 (also termed LNK) and EZH2 (Tefferi & Vainchenker, 2011). Diagnostic assays are available for the detection of mutations within JAK2 exon 12 and MPL exon 10. Assessment of the other loci is not currently performed in a diagnostic capacity in the UK. Changing technologies will facilitate more comprehensive and cost effective mutation screening in the near future, however despite the fact that mutations in some genes, e.g. EZH2, have been associated with a poor prognosis (Guglielmelli et al, 2011), it remains uncertain if the routine detection of mutations in these genes is of any real value. As exclusion of chronic myeloid leukaemia (CML) is one of the criteria for the diagnosis of ET and PMF (Swerdlow et al, 2008), many laboratories also screen for the BCR-ABL1 fusion gene by reverse transcription polymerase chain reaction (RT-PCR) or fluorescence in situ hybridization (FISH). Diverse rearrangements of PDGFRA or PDGFRB are generally associated with eosinophilic MPN or MDS/MPN and should not be routinely screened for in patients with classical MPN without eosinophilia (Jones & Cross, 2004; Reiter et al, 2007).

JAK2 exon 12 mutation

Mutations within exon 12 of JAK2 have, so far, only been reported in patients with polycythaemia vera, some of which were classified as idiopathic erythrocytosis (Percy et al, 2007; Passamonti et al, 2011). JAK2 exon 12 mutation positive patients tend to be characterized by isolated erythrocytosis, erythroid hyperplasia and low serum EPO (Percy et al, 2007; Scott et al, 2007a). At least 17 different mutations have been described, often as a result of a six base pair deletion (Cazzola, 2007; Passamonti et al, 2011). These mutations fall into three main groups – those that result in a deletion of glutamic acid at codon 543 (E543del); those that lead to a lysine to leucine substitution at codon 539 (K539L) and duplications that lead to substitution of the phenylalanine at codon 547.

Given that the presence of a JAK2 exon 12 mutation in a patient with erythrocytosis is diagnostic for PV (McMullin et al, 2007), one strategy is to screen all patients presenting with unexplained erythrocytosis who are JAK2 V617F negative for mutations within JAK2 exon 12 (Fig 1). Alternatively, as most cases of JAK2 V617F negative erythrocytosis turn out not to carry an exon 12 mutation (Fig 1), other tests, such as measurement of serum erythropoietin (EPO), isolation of EPO-independent BFU-E colonies or examination of the bone marrow trephine biopsy, could be performed to exclude cases unlikely to be true PV.

image

Figure 1. Molecular diagnostic algorithm for the classical myeloproliferative neoplasms. Approximately 2% of JAK2 V617F negative cases that present with erythrocytosis carry a JAK2 exon 12 mutation and approximately 8% of JAK2 V617F negative cases presenting with thrombocytosis or myelofibrosis carry a MPL exon 10 mutation. *If blood film/count suggestive of chronic myeloid leukaemia (Harrison et al, 2010). Cytogenetic analysis may also be helpful if no molecular genetic abnormality is detected.

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Due to the large number of possible mutations, techniques that target specific mutations, such as allele-specific PCR, are of limited value for the detection of JAK2 exon 12 mutations. Direct sequencing remains an option but the level of disease in peripheral blood is often even lower than for JAK2 V617F mutation due to the erythroid lineage specificity. Direct sequencing would probably require analysis of the bone marrow aspirate or EPO-independent BFU-E colonies to be of sufficient sensitivity (Cazzola, 2007). Highly sensitive mutation scanning methods have been developed for the identification of JAK2 exon 12 mutations. The most commonly used for are HRM analysis (Jones et al, 2008; Rapado et al, 2009; Ugo et al, 2010), melting curve assay (Schnittger et al, 2009b) and dHPLC. The sensitivity of these assays range from 1% to 10% depending on the mutation present. More sensitive assays, such as PCR clamping assays (Laughlin et al, 2010), would enable low level JAK2 exon 12 positive clones to be identified in the peripheral blood.

MPL exon 10 mutations

Mutations within MPL exon 10 have been reported in 5–10% of patients with ET and PMF patients but not in any PV patients (Pardanani et al, 2006, 2011; Beer et al, 2008). At least 5 pathogenetic mutations within MPL exon 10 have been described (Pardanani et al, 2006; Beer et al, 2008; Chaligné et al, 2008; Schnittger et al, 2009a; Boyd et al, 2010) (W515L; W515K; W515R; W515A; S505N). Other mutations within MPL have been observed although the pathogenetic significance of some of these mutations is not clear (Williams et al, 2007; Chaligné et al, 2008; Pardanani et al, 2011).

Because of the positive diagnostic value of demonstration of a MPL exon 10 mutation, especially for patients presenting with unexplained thrombocytosis, screening for MPL exon 10 mutations has been recommended in cases of suspected ET or PMF that are JAK2 V617F negative (Swerdlow et al, 2008; Harrison et al, 2010) (Fig 1). Bone marrow examination to assess megakaryocyte morphology may not be necessary in patients with ET for whom a JAK2 V617F or MPL exon 10 mutation has been demonstrated (Harrison et al, 2010).

In contrast to JAK2 exon 12 mutations, the repertoire of MPL exon 10 mutations is relatively restricted. Therefore, two main approaches have been applied for the detection of MPL exon 10 mutations:

  • An allele-specific PCR approach for each known mutation in a similar fashion to JAK2 V617F mutation detection. As for detection of JAK2 V617F, pyrosequencing (Beer et al, 2008), allele-specific PCR (Beer et al, 2008) or allele-specific real time PCR assays are available (Laurent et al, 2007; Ghaderi et al, 2008; Pancrazzi et al, 2008), with real time PCR assays generally possessing higher sensitivity (up to 0·1%). Real time PCR thus enables detection of low level MPL W515L/K mutations in the peripheral blood as for JAK2 V617F real time PCR assays. The disadvantage of such an approach is that multiple PCR assays are required to detect all possible mutations. Furthermore, allele-specific real time PCR and pyrosequencing assays are only available for detection of W515L and W515K mutations.
  • Whole exon mutation scanning approach. The most frequently applied approaches are HRM (Boyd et al, 2010) and melting curve analysis (Pardanani et al, 2006, 2011; Schnittger et al, 2009a). These approaches offer the advantage of quickly assessing patients for all W515 mutations and S505 mutations. The sensitivity for these assays is approximately 2–5% – i.e. less sensitive than real time allele-specific PCR but substantially better than direct sequencing. Whether low frequency MPL exon 10 mutation positive clones are missed by these assays is not known. However, given that low level JAK2 V617F mutation is common in ET, it would be expected that some ET patients possess low level MPL exon 10 mutations. The combination of mutation scanning methods with PCR methods that preferentially amplify the mutant allele could improve sensitivity.

BCR-ABL1 assessment

Exclusion of CML is a requirement in the diagnostic criteria of both ET and PMF but not PV (Swerdlow et al, 2008). Despite the rare occurrences of JAK2 V617F positive/BCR-ABL1 positive cases (Hussein et al, 2008; Pieri et al, 2011) the demonstration of a BCR-ABL1 fusion in a patient with thrombocytosis or myelofibrosis indicates a diagnosis of CML and excludes a diagnosis of ET or PMF. Guidelines for investigation of thrombocytosis (Harrison et al, 2010) indicate that screening for the BCR-ABL1 fusion gene should only be necessary if atypical features, such as basophilia or left shift of neutrophils, are present within the blood irrespective of the JAK2 V617F status. Whether assessment for the BCR-ABL1 fusion gene needs to be carried out for JAK2 V617F or MPL exon 10 positive PMF is unclear but may be useful if these mutations are not detected to exclude a diagnosis of CML (Swerdlow et al, 2008).

Other causes of erythrocytosis and thrombocytosis

A number of non-malignant causes of erythrocytosis and thrombocytosis may be investigated and an increasing panel of genes have been identified that are implicated in familial erythrocytosis and thrombocytosis. Congenital causes of erythocytosis include mutations in globin genes giving rise to high oxygen affinity haemoglobin, BPGM mutation resulting in bisphosphoglycerate mutase deficiency, mutations in components of EPO signalling pathway (EPOR) and mutations within components of oxygen sensing pathways such as within VHL, EGLN1 (also termed PHD2) and EPAS1 (HIF2A). Especially in younger patients, mutations within such genes may identify the cause of the erythrocytosis (McMullin, 2008). Inherited forms of thrombocythaemia may be caused by mutations within the 5′ untranslated region of THPO (also termed TPO) or within the MPL locus itself, including K39N (MPL-Baltimore), P106L and S505N mutations (Skoda, 2010). Recently, two families with inherited thrombocytosis and activating mutations within JAK2 (V617I and R564Q) have been reported (Etheridge et al, 2011; Mead et al, 2012). By contrast, the V617F mutation itself has not been reported to be inherited in familial cases although family members may acquire the mutation independently (Cario et al, 2005).

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Detection of the JAK2 V617F mutation at diagnosis
  5. Supplementary tests for JAK2 V617F negative cases
  6. Acknowledgements
  7. References

DG and NCPC gratefully acknowledge support from the Minimal Residual Disease Workpackage (WP12) of the European LeukemiaNet. AJB, AGF and TC carried out experiments. AJB, HEW, LF, ALG, GG, SA, AA, IC, SEL, TC, JC, PAE, DG, AS, MFM, ARG, CNH and NCPC wrote the manuscript.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Detection of the JAK2 V617F mutation at diagnosis
  5. Supplementary tests for JAK2 V617F negative cases
  6. Acknowledgements
  7. References
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