Laboratory Practice Guidelines for Detecting and Reporting JAK2 and ...

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The Journal of Molecular Diagnostics, Vol. 15, No. 6, November 2013

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SPECIAL ARTICLE Laboratory Practice Guidelines for Detecting and Reporting JAK2 and MPL Mutations in Myeloproliferative Neoplasms A Report of the Association for Molecular Pathology Jerald Z. Gong,*y James R. Cook,*z Timothy C. Greiner,*x Cyrus Hedvat,*{ Charles E. Hill,*jj Megan S. Lim,*,** Janina A. Longtine,*yy Daniel Sabath,*zz and Y. Lynn Wang*xx From the JAK2 Mutation Working Group,* Clinical Practice Committee, Association for Molecular Pathology, Bethesda, Maryland; the Department of Pathology, Anatomy, and Cell Biology,y Thomas Jefferson University, Philadelphia, Pennsylvania; the Department of Clinical Pathology,z Cleveland Clinic Foundation, Cleveland, Ohio; the Department of Pathology and Microbiology,x University of Nebraska Medical Center, Omaha, Nebraska; the Department of Pathology,{ Memorial Sloan-Kettering Cancer Center, New York, New York; the Department of Pathology and Laboratory Medicine,jj Emory University, Atlanta, Georgia; the Department of PathologyeHematopathology,** University of Michigan, Ann Arbor, Michigan; the Department of Pathology,yy Icahn School of Medicine at Mt. Sinai, New York, New York; the Department of Laboratory Medicine,zz University of Washington, Seattle, Washington; and the Department of Pathology and Laboratory Medicine,xx Weill Cornell Medical College, New York, New York CME Accreditation Statement: This activity (“JMD 2013 CME Program in Molecular Diagnostics”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity (“JMD 2013 CME Program in Molecular Diagnostics”) for a maximum of 48 AMA PRA Category 1 Credit(s)
. Physicians should only claim credit commensurate with the extent of their participation in the activity. CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose.

Accepted for publication July 12, 2013. Address correspondence to Jerald Gong, M.D., Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, 117 S. 11th St., Suite 301, Philadelphia, PA 19107-4949. E-mail: Jerald. [email protected].

Recurrent mutations in JAK2 and MPL genes are genetic hallmarks of BCR-ABL1enegative myeloproliferative neoplasms. Detection of JAK2 and MPL mutations has been incorporated into routine diagnostic algorithms for these diseases. This Special Article summarizes results from a nationwide laboratory survey of JAK2 and MPL mutation analysis. Based on the current practice pattern and the literature, this Special Article provides recommendations and guidelines for laboratory practice for detection of mutations in the JAK2 and MPL genes, including clinical manifestations for prompting the mutation analysis, current and recommended methodologies for testing the mutations, and standardization for reporting the test results. This Special Article also points to future directions for genomic testing in BCR-ABL1enegative myeloproliferative neoplasms. (J Mol Diagn 2013, 15: 733e744; http://dx.doi.org/10.1016/j.jmoldx.2013.07.002)

In 2005, several groups simultaneously described a point mutation in codon 617 of the protein tyrosine kinase gene Janus kinase 2 (JAK2) in BCR-ABL1enegative myeloproliferative neoplasms (MPN). This mutation was found in the vast majority of patients with polycythemia vera (PV) and in approximately half of patients with essential thrombocythemia (ET) or primary myelofibrosis (PMF).1e4 In 2007, additional mutations in exon 12 of JAK2 gene were found in a small percentage of PV patients.5 The exon 12 mutations and the V617F mutation are mutually exclusive; either V617F or Copyright ª 2013 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jmoldx.2013.07.002

Supported by the Association for Molecular Pathology. The JAK2 Mutation Working Group is a working group of the Association for Molecular Pathology (AMP) Clinical Practice Committee. The 2012e2013 AMP Clinical Practice Committee consisted of Matthew J. Bankowski, Milena Cankovic, Jennifer Dunlap, Larissa V. Furtado, Jerald Z. Gong, Cyrus V. Hedvat, Thomas Huard, Linda Jeng, Loren Joseph (Chair, 2013), Marilyn M. Li, Janina Longtine (Chair, 2012), Kathleen T. Montone, Paul Rothberg, and Siby Sebastian. Standard of practice is not defined by this article, and there may be alternatives. See Disclaimer for further details. Current address of Y.L.W., Department of Pathology, University of Chicago, Chicago, IL.

Gong et al exon 12 mutations are present in virtually all cases of PV. In 2006, mutations in a second gene, myeloproliferative leukemia virus oncogene (MPL), were identified in both ET and PMF patients.6 In contrast to JAK2 mutations, MPL mutations were found less frequently in PMF and ET, and may be present in either JAK2 mutation-positive or mutation-negative cases.6e8 Laboratory tests for these mutations have become standard in assessing clinically suspected BCR-ABL1enegative MPN. The diagnostic value of JAK2 and MPL mutations in MPN is well established. The 2008 World Health Organization classification of hematopoietic neoplasms includes JAK2 mutations as diagnostic criteria in PV and JAK2 and MPL mutations in ET and PMF.9e11 A diagnosis of PV can be made when JAK2 V617F or exon 12 mutation is detected, along with increased hemoglobin and low or normal levels of erythropoietin.9 Clinically, patients with exon 12 mutations typically present with isolated erythrocytosis and suppressed erythropoietin, in contrast to the trilineage hyperplasia characteristic of patients with V617F mutation. Bone marrow from patients with exon 12 mutation often exhibits nonspecific morphology, with isolated erythroid proliferation and absence of prominent megakaryocyte atypia and clustering. Demonstration of exon 12 mutation in these patients is particularly helpful for ruling out reactive erythrocytosis.5 In ET and PMF, JAK2 V617F mutations and/or MPL exon 10 mutations are present in approximately 60% to 70% of patients, with no other well-defined, recurrent JAK2 or MPL mutations found in the remaining patients. Although detection of JAK2 V617F and MPL mutations in the appropriate clinical settings confirms the diagnosis of ET or PMF, absence of the mutations does not rule out disease. Rare mutations in TET2, ASXL1, IDH1/IDH2, EZH2, DNMT3A, and CBL have been described, although the precise roles of these mutations in the pathogenesis of ET and PMF are unknown.12e17 Other hematological neoplasms (eg, myelodysplastic syndrome, chronic myelomonocytic leukemia, acute myeloid leukemia, and acute lymphoblastic leukemia) may harbor JAK2 and MPL mutations in low frequencies.18e20 Because JAK2 and MPL mutations are not completely specific for MPN, finding of these mutations in isolation does not warrant a diagnosis of MPN.21 Several methods have been used to detect JAK2 and MPL mutations. Clinical laboratories may choose to use either commercial detection kits or laboratory-developed tests. With the availability of a broad selection of test methods, laboratories may find it challenging to select the methodology and platform most suitable for their practice. Results from different methods may differ with respect to analytical sensitivity, specificity, and clinical relevance.22 There is a lack of wellestablished consensus concerning common analytical issues, such as what specimen type should be tested, whether it is clinically relevant to differentiate heterozygous from homozygous mutations, whether granulocyte enrichment should be performed, how to interpret a low level of a positive result, and whether quantitative results should be used and reported (and,

The JAK2 gene maps to chromosome band 9p24 and encodes a tyrosine kinase protein composed of 1132 amino acids. It contains three critical domains: JH1, JH2, and four-pointone, ezrin, radixin, moesin (FERM) homolog domains. JH1, the catalytic phosphokinase domain, is located at the carboxyl terminus and induces phosphorylation of target proteins. JH2, which resides upstream to JH1, is structurally similar to JH1 but functions as a pseudokinase domain that negatively regulates basal activity of the kinase domain and receptor-induced activation of the catalytic function.23 The FERM domain, which resides at the amino terminus, functions as a binding protein transducing signals from JAK2 to other transmembrane cytokine receptors (Figure 1).24 JAK2 protein kinase activity is activated by phosphorylation of its kinase domain. Activation of JAK2 induces signal transduction from both type 1 and type 2 cytokine receptors. Constitutive activation of JAK2 by either point mutation or fusion protein causes activation of the JAK/ STAT pathway. The activated JAK2 causes phosphorylation of STATs, which then dimerize and translocate to the nucleus, where they regulate gene transcription. Somatic mutations, such as V617F mutation in JH2, disrupt the inhibitory function of the pseudokinase and thus lead to constitutive tyrosine phosphorylation activity that promotes cytokine hypersensitivity (UniProt, http://www.uniprot. org/uniprot/O60674, last accessed November 28, 2012). Although various JAK2 mutations have been reported in exons 12, 13, 14, and 15, in the vast majority of the cases mutations are found in codon 617, resulting in the replacement of the amino acid valine with phenylalanine [V617F, alias JAK2 NM_004972.3:c.1849G>T (p.Val617Phe)]. This mutation is present in approximately 96% of PV cases, 55% of ET cases, and 65% of PMF cases. Exon 12 mutations are far less common, present in only approximately 3% of PV cases. In contrast to the V617F mutation, which involves

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if yes, in which disease type and in what clinical condition). To address these issues from the laboratory perspective, here we present practice guidelines for JAK2 and MPL mutation testing and reporting. The guidelines were developed by an expert panel from the Association for Molecular Pathology (AMP) based on the current literature and practices in the United States and Canada. The guidelines are intended to establish a helpful reference for laboratory professionals in the selection of methodologies, validation of procedures, and interpretation of results in JAK2 and MPL testing. In addition, the guidelines can serve as a reference guide for clinical practitioners and other health care professionals in interpretation and utilization of the results. Practice guidelines also serve to identify questions and suggest areas for future development.

What Are the Currently Known JAK2 and MPL Mutations? JAK2

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JAK2 and MPL Mutation Guidelines

Figure 1

Schematic representation of JAK2 gene and mutation hot spots. Locations of reported mutations are indicated by colored triangles: red triangles for V617F, black triangles for other SNP mutations, and blue triangles for insertions/deletions. Representative codon numbers are marked above exon boxes. FERM, four-point-one, ezrin, radixin, moesin domain; JH1, kinase domain; JH2, pseudokinase domain; SH2-like, SH2-like domain.

most frequent exon 12 mutation involves an in-frame deletion of six nucleotides at codons 542 and 543 (N542_E543del); it is present in approximately 40% of V617F-negative PV. Other relatively common mutations include R541_E543delinsK (approximately 10%), E543_D544del (approximately 10%), F537_K539delinsL (approximately 10%), K539L (approximately 10%), H538_K539delinsL (approximately 5%), and I540_E543delinsMK (approximately 5%).38,48,49 Mutations in exon 13, exon 15, and exon 14 other than V617F are exceptionally rare, and are observed in a broader variety of disease types.50

MPL one amino acid codon, exon 12 mutations affect a larger region, spanning codons 533 to 547. At least 27 clinically verified exon 12 mutations have been identified to date, including amino acid substitution, deletions, and duplications (Table 1). Additional exon 12 mutations have been reported, but their clinical significance is unknown.44,47 The Table 1

The MPL gene maps to chromosome band 1p34 and encodes the thrombopoietin receptor, which binds to thrombopoietin, the primary cytokine that regulates megakaryocyte development and platelet production, as well as hematopoietic stem cell homeostasis. Binding of thrombopoietin to MPL

JAK2 Mutations in Myeloproliferative Neoplasms

Mutation*

Exon

Frequency

Nucleotide positiony

References

V617F V617F,C618R V617F,C618F V617F,D620E L611V,V617F C616C,V617F D620E V536_I546dup V536_F547dup F537I,K539I F537_K539delinsL F537_I546dup10,F547L F537_547dup H538Q,K539L H538D,K539L,I540S H538_K539del H538_K539delinsF H538_K539delinsI H538_K539delinsL K539L K539L,L545V I540T I540_N542delinsS I540_E543delinsMK I540_E543delinsKK R541_E543delinsK R541K,A542_G543del R541_E543delinsK N542_E543del E543_D544del D544G D544_L545del F547_K549delinsL 547insL,I540_F547dup8

14 14 14 14 14 14 14 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12

w96% PV, w55% ET, w65% PMF G c.1630_1635del NA c.1642_1644ins, c.1645_1668dup

1e4 25 25 26 27 28 29 30 31 32 5 30 33 5 31 31 34 35 36,37 5,37 31 32 38 39,40 41 36,39 42 34 5 43 44 31 45 46

*Mutation descriptors are not translated into the standard HGVS nomenclature, to avoid misinterpretation. y Nucleotide positions are determined based on the cited sources. NA, not available in the original publication.

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Figure 2 Schematic representation of MPL gene and mutation hot spots. Locations of reported mutations are indicated by colored triangles: red triangles for W515L and W515K, and black triangles for other SNP variant mutations. Representative codon numbers are marked above exon boxes. CP, cytoplasmic domain; DCR-NRD, distal cytokine receptor domainnegative regulatory domain; PCR-LBR, proximal cytokine receptor domainligand binding region; TM, transmembrane domain.

leads to activation of JAK2, which phosphorylates MPL and initiates a cascade of downstream signaling events that regulate cell survival, proliferation, and differentiation.51 Mutations of the MPL gene occur in BCR-ABL1enegative MPN.6,7 The mutation W515L represents guanosine-tothymidine substitution in MPL at nucleotide 1544, which leads to exchange of leucine for tryptophan. The mutation results in impaired function of the autoinhibitory region and subsequent ligand-independent thrombopoietin receptor activation.52 This then leads to subsequent activation of downstream tyrosine kinases and activation of transcription factors STAT3 and STAT5, which in turn leads to transformation of hematopoietic cells into cytokine-independent clones, resulting in megakaryocytic hyperplasia and marrow fibrosis. The Y252H mutation, which is located in the extracellular domain of MPL, confers hypersensitivity to thrombopoietin and increases the generation of megakaryocyte colonies in vitro and leads to increased thrombopoietin signaling and cell growth and survival.53 The majority of the MPL mutations are found in exon 10 codon 515; W515L is the most common, followed by W515K. Overall, MPL W515L or W515K mutations are present in patients with PMF and ET at a frequency of approximately 5% and 3%, respectively.6e8 Other codon 515 mutations include W515A and W515R.54 Rarely, double MPL mutations occurring in cis of exon 10 have been identified.54 Mutations in other exons include V501A, Y252H, and S204P (Figure 2).36,53,54 In addition to their association with sporadic MPN, MPL mutations are also associated with familial diseases. S505N mutation has been associated with familial autosomal dominant thrombocytosis.55 MPL and JAK2 mutations are not mutually exclusive, and rare cases of concurrent MPL mutation and JAK2 V617F mutation have been reported.7,8 Clinically verified MPL mutations are listed in Table 2.

When Should JAK2 and MPL Mutational Analysis Be Performed? Screening The usual indications for JAK2 mutation testing include unexplained polycythemia, neutrophilia, or thrombocytosis (when secondary causes have been ruled out), because each may represent a MPN57 (Table 3). Other indications for JAK2

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mutation analysis include splanchnic vein thrombosis and the provisional entity refractory anemia with ring sideroblasts and thrombocytosis (RARS-T). Of patients with splanchnic vein thrombosis, 31% were found to have associated JAK2 mutations and often had a latent MPN, with the most common site of thrombosis occurring in the portal venous system.59 Similarly, nearly one third of patients with RARS-T were found to have a JAK2 mutation.60 After secondary causes of polycythemia have been ruled out, JAK2 analysis (first for exon 14 V617F, and then, if findings are negative, for exon 12) is indicated to support the diagnosis of PV. Ideally, JAK2 analysis for investigating the cause of neutrophilia and thrombocytosis would follow a determination of the absence of the BCR-ABL1 translocation t(9;22). In practice, however, clinicians find it easier to order both the BCR-ABL1 and the JAK2 assays at the same time in screening peripheral blood. There is no difference in analytical performance using peripheral blood or bone marrow samples for testing for JAK2 mutations, because granulocytes constitute the predominant population in both specimens.61 MPL mutation analysis is not indicated in the consideration of PV, because no MPL mutations have been described for this entity. The use of MPL analysis is best considered after pathology review of a bone marrow biopsy and aspirate. If a diagnosis of PMF or ET is entertained, then MPL analysis should be performed in the setting of a negative JAK2 V617F assay, if required to meet World Health Organization diagnostic criteria.10,11

Follow-Up Testing The role of follow-up testing for JAK2 and MPL mutations after therapy is not clearly established. Quantitative realTable 2

MPL Mutations in Myeloproliferative Neoplasms

Mutation* W515L or W515K

V501A,W515L W501A,W515R S505C,W515L S505N A506T L510P W515A A519T S204P Y252H

Exon

Frequency

10 w5% PMF w3% ET

Nucleotide positiony

c.1544G>T c.1543T>A c.1544G>A 9, 10 C c.1544G>T 9, 10 C c.1543T>C 10 A c.1544G>T 10 A 10 A 10 C 10 G c.1544G>C 10 A 4 C 5 C

References 6e8

54 54 54 55,56 56 56 54,56 56 36 53

*Mutation descriptors are not translated into the standard Human Genome Variation Society (HGVS) nomenclature, to avoid misinterpretation. y Nucleotide positions are determined based on the cited sources.

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JAK2 and MPL Mutation Guidelines Table 3

Indications for JAK2 and MPL Mutation Testing

Mutation and indications JAK2 V617F Granulocytosis (especially BCReABL1 negative), no active infection or inflammation Erythrocytosis, with normal or low erythropoietin Thrombocytosis, with common reactive etiologies ruled out Increased bone marrow fibrosis, excluding infiltration by lymphoma or carcinoma Splenomegaly, unexplained Splanchnic vein thrombosis Aquagenic pruritus JAK2 exon 12 mutations JAK2 V617F negative, erythrocytosis with normal or low erythropoietin JAK2 V617F negative, post-PV bone marrow fibrosis MPL mutations JAK2 V617F negative, suspected ET JAK2 V617F negative, suspected PMF Adapted from Tefferi et al.58

time PCR (qPCR) testing for monitoring JAK2 V617F allele burden has been limited to patients enrolled in clinical trials such as the peg-IFN-a-2a and MF-TG101348-001 trials.62,63 Although there are small-molecule inhibitors for JAK2, such as ruxolitinib [approved by the U.S. Food and Drug Administration (FDA) for myelofibrosis], these target the JAK2 and often the JAK1 proteins, and thus are not specific for the V617F mutation.64 Although allele burden has been shown to decrease after use of some inhibitors, there is no one-to-one correlation of allele burden with clinical response (eg, reduction in splenomegaly). Highly sensitive qPCR for JAK2 V617F may have value in followup of patients with primary and secondary myelofibrosis after allogeneic stem cell transplantation. Quantitative tests for JAK2 V617F allele have been used to determine the depth of clinical remission and to guide adoptive immunotherapy, such as donor lymphocyte infusion.65 Thus, for routine clinical practice, there is no recommended schedule for follow-up JAK2 testing, whether

qualitative or quantitative. There is a dearth of follow-up information for MPL mutation analysis, likely because of the low number of cases identified to date.

Which Methodologies Are Used to Detect JAK2 and MPL Mutations? Although there are no FDA-cleared tests, a number of methods have been developed for detecting JAK2 V617F mutation. Traditional Sanger sequencing has been used extensively, but it has relatively poor sensitivity. Other methods used to detect V617F include restriction fragment length polymorphism, denaturing high-performance liquid chromatography, high-resolution melting-curve analysis, pyrosequencing, and various allele-specific PCR systems with electrophoretic analysis of the products. Most of these methods typically do not achieve sensitivities of less than 5% of alleles (Table 4). The most widely used methods for the detection of V617F involve allele-specific qPCR. These methods easily achieve analytical sensitivities of 1% mutant alleles. Allele-specific qPCR also allows for quantification of the mutant as a percentage of all of the JAK2 alleles, as an estimate of disease burden. Using standard curves for both the wild-type and mutant forms, it is possible to perform this calculation; however, there is no standardization for measuring quantity. The clinical utility of quantification of V617F has not yet been established. A limitation of allele-specific qPCR is that, in many cases, signal is generated in the mutant PCR reaction at high CT counts even for known negative samples. In this situation, it is necessary for the laboratory to establish the minimal amount of signal needed to confidently call a sample positive. There are no standard methods for detecting the rare JAK2 mutations, nor for MPL mutations. Direct Sanger sequencing or pyrosequencing has been used for assessment of JAK2 and MPL mutations. Some published methods screen for mutations using denaturing high-performance liquid chromatography or high-resolution melting-curve analysis, followed by

Table 4

Methods in Detecting JAK2 Mutations

Method

Sensitivity (%)

Advantage

Disadvantage

References

qPCR (AS, LNA) PCR (AS)

0.10.01 0.11

High sensitivity; quantitative High sensitivity; simple to perform

Detects only target mutations Detects only target mutations; not quantitative Detects target mutation only; moderate to low sensitivity; poor reproducibility in lowþ samples Detects target mutation only; relatively low sensitivity Relatively low sensitivity; requires post-PCR manipulation; unreliable in lowþ samples; not quantitative. Low sensitivity; time-consuming; not quantitative

66e69 4,70,71

Melting curve analysis

510

Simple to perform; semiquantitative; low cost

Pyrosequencing

510

RFLP

110

Simple to perform; quantitative; low cost Low cost

Sanger sequencing

20

Detects all mutations; bidirectional confirmation

22,32,70,72

73 22,74

1e4

AS, allele-specific; LNA, locked nucleic acid; RFLP, restriction fragment length polymorphism.

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Gong et al direct sequencing to confirm that a mutation is present.75,76 Higher analytical sensitivity may be achieved by using clamped PCR, followed by nucleotide sequencing.77 Traditional allele-specific PCR and allele-specific qPCR assays have also been used, but these assays are limited to assessing specific mutations. With less common mutations, it is not clear what level of analytical sensitivity is needed. Intellectual property restrictions may influence the choice of a JAK2 V617F assay, given that Ipsogen (Marseille, France) acquired a worldwide license on testing for this mutation in 2006. (Ipsogen became Qiagen Marseille in 2013.) Limitations of the Ipsogen qualitative assay (MutaScreen assay) were highlighted in a report that described two MPNs containing variant JAK2 mutations, each with the typical codon 617 (1849G>T) mutation, and in one case an additional codon 617 (1851C>T) substitution and a codon 618 (1852T>C) mutation, and in the other case an additional codon 618 (1853G>T) mutation.25 These two cases gave no signal and a wild-type result, respectively, by the Ipsogen assay, but were detected as abnormal by melting curve analysis and subsequent Sanger sequencing. In principle, the presence of additional exon 14 mutations can interfere with binding of primers and probes in allele-specific assays, but such mutations are exceptionally rare (90% of PV cases and in approximately half of ET and PMF cases. This point mutation may also be found in other myeloid malignancies. Genomic DNA was extracted and assayed for the V617F mutation using a TaqMan allelic discrimination assay with a FAM-labeled mutant-specific probe and a VIC-labeled wild-type specific probe. The sensitivity of this assay is 1% JAK2 V617F mutant in a wild-type background.

Methodology

include the tissue source (eg, peripheral blood, bone marrow, paraffin-embedded tissue) and, if available, the clinical indication for testing. The analytic components include the analytic result (mutation detected or not detected) and a brief description of the methodology used. The postanalytic components include interpretive comments, if needed. A sample report for a quantitative JAK2 V617F assay is presented as Table 5. For qualitative JAK2 V617F testing, the report should clearly indicate a final analytic result (eg, JAK2 V617F mutation DETECTED or JAK2 V617F mutation NOT DETECTED). The methodology summary should include source information regarding any commercial primers and probes used, and it must include a statement of assay sensitivity. JAK2 Sanger sequencing assays, such as those for exon 12 mutations, should report any detected mutations using standard Human Genome Variation Society (HGVS) nucleotide and amino acid nomenclature (HGVS, http:// www.hgvs.org/mutnomen, last accessed February 19, 2013). The methodology summary of sequencing assays should clearly state the regions covered by the assay. An interpretive comment should be included, indicating whether the observed mutation has been previously reported in MPNs or other malignancies or whether the mutation appears to be novel. Online databases, such as the Catalogue of Somatic Mutations in Cancer (COSMIC; Catalogue of Somatic Mutations in Cancer, http://cancer.sanger.ac.uk/ cancergenome/projects/cosmic, last accessed February 19, 2013) and the Database of Single Nucleotide Polymorphisms (dbSNP; National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/snp, last accessed February 19, 2013), are particularly useful for determining whether an uncommon abnormality has been previously reported. An estimate of the relative abundance of mutant to wild-type alleles may be provided; this is especially important for novel mutations, which could represent previously unreported germline SNPs. Because most laboratories offer V617F and exon 12 mutations as separate tests, exon 12 mutation may be mistakenly requested as the initial screening test instead of V617F mutation. This error can be easily avoided by restricting exon 12 testing only to a reflex test. The results can be better

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clarified by incorporating a disclaimer in the exon 12 test report: This test does not include testing for the more common JAK2 V617F (exon 14, c.1849G>T) mutation. Reports of quantitative JAK2 V617F assays should include a statement of qualitative results (eg, JAK2 V617F mutation DETECTED), as well as a statement of quantitative allele burden. The allele burden may be expressed as a ratio of mutant (mut) to wild-type (wt) alleles, or more commonly, as a percentage of mutated JAK2 alleles, calculated as (JAK2mut)/(JAK2mut þ JAK2wt) 100. If available, results of prior serial testing may be provided. If a quantitative cutoff is used to define a positive result, this value should be stated as part of the methodology summary, and a limit of sensitivity must also be included. Allele burden may also be reported in semiquantitative fashion (eg, >50% to 75% mutant alleles). The terms heterozygous and homozygous have been used in the literature to describe cases with allele burdens of F mutation in addition to 617V>F. Blood 2010, 115:3175e3176 Grünebach F, Bross-Bach U, Kanz L, Brossart P: Detection of a new JAK2 D620E mutation in addition to V617F in a patient with polycythemia vera. Leukemia 2006, 20:2210e2211 Cleyrat C, Jelinek J, Girodon F, Boissinot M, Ponge T, Harousseau JL, Issa JP, Hermouet S: JAK2 mutation and disease phenotype: a double L611V/V617F in cis mutation of JAK2 is associated with isolated erythrocytosis and increased activation of AKT and ERK1/2 rather than STAT5. Leukemia 2010, 24:1069e1073 Wong CL, Ma ES, Wang CL, Lam HY, Ma SY: JAK2 V617F due to a novel TG e> CT mutation at nucleotides 1848-1849: diagnostic implication. Leukemia 2007, 21:1344e1346 Schnittger S, Bacher U, Kern W, Schröder M, Haferlach T, Schoch C: Report on two novel nucleotide exchanges in the JAK2 pseudokinase domain: D620E and E627E. Leukemia 2006, 20:2195e2197 Pietra D, Li S, Brisci A, Passamonti F, Rumi E, Theocharides A, Ferrari M, Gisslinger H, Kralovics R, Cremonesi L, Skoda R, Cazzola M: Somatic mutations of JAK2 exon 12 in patients with JAK2 (V617F)negative myeloproliferative disorders. Blood 2008, 111:1686e1689 Schnittger S, Bacher U, Haferlach C, Geer T, Müller P, Mittermüller J, Petrides P, Schlag R, Sandner R, Selbach J, Slawik HR, Tessen HW, Wehmeyer J, Kern W, Haferlach T: Detection of JAK2 exon 12 mutations in 15 patients with JAK2V617F negative polycythemia vera. Haematologica 2009, 94:414e418

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