JAK2 V617F Gene Mutation in the Laboratory Work-Up of Myeloproliferative Disorders: Experience of a Major Referral Center in Lebanon

Abstract

Aims: JAK2 V617F mutation is gaining more acceptance in laboratory testing as part of the differential diagnosis work-up of myeloproliferative disorders (MPD). This report is the first of its kind from Lebanon that analyzes the distribution of this mutation among a series of referred cases to a major tertiary referral center. Methods: Real-time polymerase chain reaction using JAK2 V617F MutaScreen assay (IPSOGEN Cancer Profiler) was performed on 229 patients. Results: JAK2 V617F mutation was found to be positive in 100% of polycythemia vera cases, 68.29% of essential thrombocythemia cases, and 55.28% of all MPD cases whereas negative in idiopathic erythrocytosis, reactive thrombocytosis, and other non-MPD cases such as acute chronic myeloid leukemias. Conclusion: Our unique study in this sample of Lebanese patients shows extensive similarities of positivity of JAK2 V617F as compared with the international literature and for the same categories of clinical entities. This will constitute a baseline for future clinical studies that would also help determine prognosis of cases based on the absence or presence of this mutation.

Introduction

JAK2 is a cytoplasmic tyrosine kinase responsible for signal transduction. The binding of type 1 cytokine ligands, including erythropoietin, granulocyte macrophage-colony stimulating factor, and thrombopoietin with its receptor activates JAK2 and causes the production of red blood cells, granulocytes/macrophages, and platelets, respectively (Paradis et al., 2010). JAK2 V617F is a single acquired point mutation of the JAK2 gene, where phenylalanine replaces valine at position 617 of the JAK2 protein due to a G-to-T transversion at nucleotide 1849 (Szpurka et al., 2006), in the JH2 pseudokinase domain that negatively regulates the kinase domain (Saharinen et al., 2000). As a result of this mutation, the tyrosine kinase becomes active; and a dysregulation of the autoinhibition function of the JH2 pseudokinase occurs, leading to uncontrolled proliferation of hematopoietic cells, which, in turn, leads to an increase in the hemoglobin level, increase in white blood cells, and may increase the risk of leukemic transformation (Paradis et al., 2010).

JAK2 V617F is mainly associated with myeloproliferative disorders (MPD) in >90% of polycythemia vera (PV) cases, >57% of essential thrombocythemia (ET) cases, >55% of chronic idiopathic myelofibrosis cases, and in <20% of atypical chronic myeloid leukemia (CML) cases (Szpurka et al., 2006; Paradis et al., 2010). This mutation is also found in 9% of patients with chronic myelomonocytic leukemia (CMML), in 4% of patients with refractory anemia with excess blasts/acute myeloid leukemia (AML), 67% of patients with refractory anemia with ringed sideroblasts associated with marked thrombocytosis, and 12% of patients with other forms of myelodysplastic syndrome (MDS)/MPD-unclassified (Szpurka et al., 2006). Cases with secondary AML after MPD may show JAK2 V617F positivity (Fröhling et al., 2006); whereas cases with de novo AML, Ph+ CML, secondary erythrocytosis, and thrombocytosis are negative for the JAK2 V617F mutation (Horn et al., 2006).

Different qualitative and quantitative methods are being used for the detection of the JAK2 V617F mutation: allele-specific polymerase chain reaction (PCR) (limit of detection of 3%) (Szpurka et al., 2006; Wolstencroft et al., 2007), direct DNA sequencing (limit of detection of 10%) (Szpurka et al., 2006), amplification refractory mutation screening PCR (limit of detection of 1-2%) (McLornan et al., 2006; Wolstencroft et al., 2007), allele-specific multiplex PCR with one common reverse and two separate forward primers, nested PCR (Horn et al., 2006), real-time PCR (limit of detection of 0.5%-1%) and DNA-melting curve analysis (limit of detection of 1%-10%) (Steensma, 2006), restriction fragment length polymorphism (limit of detection of 20%), single-stranded conformational polymorphism analysis, denaturing gradient gel electrophoresis, abd denaturing high-performance liquid chromatography (limit of detection of 2%) (Steensma, 2006). However, the sensitivity, specificity, cost, and turnaround time vary from one technique to another and are factors to be considered on selection of any of these techniques for diagnostic application.

This is the first study conducted in a tertiary-care center in Lebanon and aims toward reporting the distribution of JAK2 V617F mutation among patients referred for work-up and differential diagnosis of leukocytosis, erythrocytosis, thrombocytosis, leukemias, and MPD.

Materials and Methods

Samples and DNA extraction

This analysis was conducted at the American University of Beirut Medical Center, which is a major tertiary-care Lebanese center where we report on 229 adult cases. The initial extraction of the DNA material was done using the PEL-FREEZ extraction kit (PEL-FREEZ) and genomic material stored at −80°C for JAK2 testing.

The JAK-2 MutaScreen Kit and quantitative PCR

The JAK2 MutaScreen Kit (Ipsogen Cancer Profiler) was used for the detection of JAK2 V617F mutation in genomic DNA. It is a simple technique with a limit of detection of 1.25%, and the results obtained by this assay are highly precise and sensitive (Cankovic et al., 2009).

The assay followed exploits the real-time quantitative polymerase chain reaction (RQ-PCR) Double Dye hydrolysis oligonucleotide probes principle. Quantitative PCR results can be promptly obtained by real-time detection of fluorescent signals during and/or following the PCR cycling and without any post-PCR processing. The amplification mix includes forward and reverse primers and double dye probes that are labeled with a 5′ reporter and 3′ quencher dyes. The assay followed uses the 5′-3′ exonuclease activity of the Taq polymerase. PCR is used to amplify the portion of the JAK-2 region that has the mutation. The thermocycler program consists of an initial step of 55°C for 2 min, then 95°C for 10 min, followed by 50 cycles of 92°C for 15 s, and 60°C for 1 min. At the end of the amplification run, the LightCycler 2.0 instrument (Roche Diagnostics) was used to obtain the FAM/VIC (530/560) ratio. The mean ratio of the samples tested was simply compared with the reference sample mean ratio value. If the mean ratio of sample is equal or superior to the mean ratio of the reference sample, then the sample is considered mutant. However, if the mean ratio of sample is less than the mean ratio of the reference sample, the sample is considered as wild type.

Results

As per Table 1, out of the 229 referred adult patients, 68 (29.69%) were positive for the JAK2 V617F mutation. All AML, CML, CMML, erythrocytosis, hypereosinophilia, leukocytosis, MDS, and thrombocytosis (reactive) cases were negative for this mutation. Of the unclassified MPD cases, 39.13% were JAK2 V617F positive, whereas 68.29% of the ET cases were positive for this mutation, and all the PV cases were JAK2 V617F positive. Please note that the 69 unclassified patients with MPD refer to those patients who were referred to our diagnostic laboratory for JAK2 testing based on the diagnosis of MPD based on bone marrow aspirate and/or biopsy material and other peripheral blood parameters as assessed by their external treating physicians (data not within our reach). Thus, no further subclassification of these cases was possible at this level. Accounting for all the MPD referred cases (including the unclassified MPD, PV, and ET subgroups), JAK2 V617F was found to be positive in 68 out of 123 referred cases (55.28%). Also interestingly, and accounting for all high platelets referrals (including ET and thrombocytosis), JAK2 V617F mutation was found to be positive in 28 out of 76 referred cases (36.84%).

Table 1.

Distribution of JAK2 V617F Mutation Among All the Referred Cases

		JAK2 V617F positive
Category	Number of patient	n	%
AML	10	0	0.00
CML	5	0	0.00
CMML	3	0	0.00
Erythrocytosis	11	0	0.00
Hypereosinophilia	4	0	0.00
Leukocytosis	8	0	0.00
MDS	30	0	0.00
Unclassified MPD	69	27	39.13
Polycythemia vera	13	13	100.00
Essential thrombocythemia	41	28	68.29
Thrombocytosis	35	0	0.00
Total	229	68	29.69

AML, acute myeloid leukemia; CML, chronic myeloid leukemia; CMML, chronic myelomonocytic leukemia; MDS, myelodysplastic syndrome; MPD, myeloproliferative disorders.

Technically speaking, the FAM/VIC ratio calculated values for the positive samples, the wild-type genotypes and the reference strand controls ranged as 0.73-1.18, 0.55-0.71, and 0.58-0.91, respectively.

Discussion

The diagnosis of MPD entails a variety of clinical and laboratory tests including peripheral blood picture presentation, bone marrow aspirate and biopsy, cytogenetic studies, and, recently molecular diagnostics. It is highly important, though, to eliminate other entities from the differential diagnosis of, for example, PV from idiopathic erythrocytosis (IE) and ET from reactive thrombocytosis.

The diagnosis of IE, a case of increase of red blood cell mass with no defined etiology, is made by ruling out PV, secondary acquired polycythemias, and other types of polycythemias. IE is more frequently found than PV; both cases are characterized by absolute erythrocytosis and they can be differentiated using several tests including serum erythropoietin level, acquired cytogenetic abnormalities, and assay for JAK2 V617F mutation (Finazzi et al., 2006). About 90% of the PV cases are JAK2 V617F positive, whereas IE cases are negative for this mutation. A study done in Spain showed that the JAK2 V617F negative PV cases and the JAK2 V617F negative IE cases can be positive for other JAK2 mutation such as JAK2 exon12 mutations. This type of mutation was detected in 20% of the V617F negative cases, and most of the exon12 positive cases belonged to the patients with IE (Martínez-Avilés et al., 2007).

In patients presenting an elevated platelet count and expected to have ET, it is important to exclude long-lasting reactive thrombocytosis to confirm ET (Brière, 2007). Thrombocytosis is a common finding but it poses a difficult diagnostic challenge. Secondary thrombocytosis can be due to iron deficiency, infection, or chronic autoimmune disease, whereas primary thrombocytosis occurs due to ET (Goldman, 2005; Tefferi and Barbui, 2005). Several methods are used to differentiate between primary and secondary thrombocytosis. The presence of JAK2 V617F mutation rules out secondary thrombocytosis and directs the diagnosis toward primary thrombocytosis and ET given that this mutation is positive in around 55% of ET cases (Goldman, 2005; Tefferi and Barbui, 2005). A study done in Munich, Germany, showed that 73% of the patients with ET were JAK2 V617F positive, whereas all the reactive erythrocytosis and reactive thrombocytosis cases were negative for this mutation (Horn et al., 2006).

In this first report of its kind from Lebanon, we found the distribution of the JAK2 V617F mutation to be within the limits of the internationally published data and did not entail any major differences or inherent properties in our community of MPD patients. For example, and similar to the available international literature, we also found all cases referred for the work-up of PV to be positive for JAK2 V617F mutation, which is an extremely important marker to help in the differential diagnosis of this entity. In addition, and out of all cases referred for MPD diagnosis and/or confirmation of diagnosis, around 55.28% of the cases turned out to be positive for this mutation, which typically falls within the framework of all articles published to date.

The same reasoning applies in the differential diagnosis of referred ET cases whereby all cases with thrombocytosis, and that turned out to be reactive for a variety of other conditions, did not carry the JAK2 V617F mutation and that among all the referred ET cases, 68.29% of the referred patients were positive for this mutation. This rate is on the high side of the reported interval of JAK2 V617F mutation positivity rate among patients with ET (which is up to 70%). It is important and interesting to note, however, that JAK2 testing in the differential diagnosis and work-up of a high platelet count was able to differentiate at least up to 36.84% of the cases studied in this report.

In our institution, JAK2 V617F mutation analysis has become an integral part of the diagnosis algorithm and work-up of patients referred under the category of MPD, especially in narrowing the differential diagnosis in cases of erythrocytosis and thrombocytosis. Future larger clinical studies reflecting the impact of the presence or absence of this mutation among the different subgroups being studied are needed to assess the usefulness of testing for JAK2 V617F mutation in other clinical entities and to monitor the response of patients to treatment depending on the status of this mutation. This first report from our institution will serve as a baseline for any prospective studies in Lebanon.

Footnotes

Disclosure Statement

No competing financial interests exist.

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