By Dr Stephen Langabeer

The myeloproliferative neoplasms of polycythemia vera, essential thrombocythemia and primary myelofibrosis are clonal hematopoietic disorders characterized by an over-production of mature blood cells. Molecular evaluation is a key component of differentiating malignancy from a host of reactive causes with mutations in JAK2, CALR and MPL genes present in the majority of patients. The implementation of technologies such as next-generation sequencing can also provide information on prognosis allowing a more individually tailored approach to therapy.

Myeloproliferative neoplasms

The classical, Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) of polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) are related, clonal, hematopoietic stem cell disorders in which the malignant process leads to an excessive red cell production, platelet production and bone marrow fibrosis. Although uncommon, occurring at an incidence of approximately 3 cases  per 100 000 of the population [1], the molecular diagnosis of an MPN is confounded by the spectrum of other medical conditions that can mimic the characteristic erythrocytosis or thrombocytosis of MPN (Table 1) that results in a considerable inter-centre and intra-centre variation in requesting pattern and redundancy of testing. The main goals of MPN treatment are to improve the quality of life associated with symptom burden, reduce the inherently increased risk of thrombosis and hemorrhage, and to prevent transformation of the disease to an acute leukemia phase [2–4]. The only potential curative modality is hematopoietic allogeneic stem cell transplantation (HASCT), though this option has to be balanced with the associated morbidity and mortality risks of such a procedure. The past 20 years have seen a revolution in the of the molecular understanding of MPN in parallel with the introduction of techniques such as gene expression profiling and next-generation sequencing (NGS) that has led to improvements in molecular diagnostics, prognostication and therapeutic development.

Table 1. Congenital or acquired clinical scenarios that can mimic either polycythemia vera (erythrocytosis) or essential thrombocythemia and primary myelofibrosis (thrombocytosis)
CML, chronic myeloid leukemia; MDS/MPN, myelodysplastic/myeloproliferative syndromes; TEMPI, telangiectasia, erythrocytosis, monoclonal gammopathy, perinephric collections, intrapulmonary shunting.

Figure 1. Driver mutations for polycythaemia vera, essential thrombocythaemia and primary myelofibrosis indicate a common pathogenetic mechanism for these myeloproliferative neoplasms

Driver mutations

The classical MPN share several phenotypic and clinical similarities but it was not until the discovery of the somatically acquired JAK2 V617F mutation in these diseases that a common pathogenetic mechanism was evident. JAK2 is an intracellular molecule essential for the transduction of hematopoietic growth factors (such as erythropoietin and thrombopoietin) that regulate red blood cell, platelet and white blood cell homeostasis. The JAK2 V617F mutation results in downstream, constitutive activation of the JAK-STAT pathway resulting in the MPN phenotypes. This mutation is present in 95% of patients with PV and in 55–65% of patients with ET and PMF. The remaining cases of PV are characterized by the presence of either substitution, deletions, insertions and duplication mutations within JAK2 exon 12 (Fig. 1). Mutation in the MPL gene, which encodes the receptor for the growth factor thrombopoietin, are present in 5–10% of patients with ET and  PMF. Most MPL mutations occur at codon W515 and are usually substitutions that result in downstream activation of the JAK-STAT pathway. A differing mechanism of activation of the JAK-STAT pathway in MPN is caused by insertion or deletion mutations  within exon 9 of CALR that encodes a protein involved in calcium signalling and protein unfolding. CALR exon 9 mutations always result in a frameshift that generates a novel C-terminus of the protein which is then able to interact with MPL receptor [5]. A spectrum of CALR mutations are present in ET and PMF but are considered absent in PV (Fig. 1). The JAK2 V617F, JAK2 exon 12, CALR exon 9 and MPL exon 10 mutations are considered disease driver mutations as they are essential in recapitulating the MPN phenotypes in murine models. A significant minority of ET and PMF patients possess none of the mutation types described and are termed ‘triple-negative’ MPN. However, sequencing of alternative JAK2 and MPL exons has revealed a considerable number of non-canonical mutations within these genes [6].

World Health Organization classification

Molecular diagnostics has become increasingly important in the classifications of MPN, in as much it is now incorporated as a critical criterion in the World Health Organization classification [7]. It must be emphasized that in addition to driver mutation status, the classification of the distinct MPN entities still requires clinical, other laboratory and histo-morphological evidence (Table 2).

Molecular diagnostic algorithm

Given that the MPN driver mutations are generally mutually exclusive, it is possible to adopt a molecular diagnostic algorithm that sequentially evaluates for the presence of the JAK2 V617F, CALR exon 9, MPL exon 10 or JAK2 exon 12 mutations. A number of different methodologies are available for MPN mutation detection and include Sanger sequencing, pyrosequencing,

Table 2. World Health Organization 2016 diagnostic criteria for polycythemia vera, essential thrombocythemia and primary myelofibrosis
BM, bone marrow; CML, chronic myeloid leukemia, Hb, hemoglobin; HCT, hematocrit; LDH, lactate dehydrogenase; MDS, myelodysplastic syndrome; WHO, World Health Organization.

Myeloproliferative neoplasms

The classical, Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) of polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) are related, clonal, hematopoietic stem cell disorders in which the malignant process leads to an excessive red cell production, platelet production and bone marrow fibrosis. Although uncommon, occurring at an incidence of approximately 3 cases per 100 000 of the population [1], the molecular diagnosis of an MPN is confounded by the spectrum of other medical conditions that can mimic the characteristic erythrocytosis or thrombocytosis of MPN (Table 1) that results in a considerable inter-centre and intra-centre variation in requesting pattern and redundancy of testing. The main goals of MPN treatment are to improve the quality of life associated with symptom burden, reduce the inherently increased risk of thrombosis and hemorrhage, and to prevent transformation of the disease to an acute leukemia phase [2–4]. The only potential curative modality is hematopoietic allogeneic stem cell transplantation (HASCT), though this option has to be balanced with the associated morbidity and mortality risks of such a procedure. The past 20 years have seen a revolution in the of the molecular understanding of MPN in parallel with the introduction of techniques such as gene expression profiling and next-generation sequencing (NGS) that has led to improvements in molecular diagnostics, prognostication and therapeutic development.

Driver mutations

The classical MPN share several phenotypic and clinical similarities but it was not until the discovery of the somatically acquired JAK2 V617F mutation in these diseases that a common pathogenetic mechanism was evident. JAK2 is an intracellular molecule essential for the transduction of hematopoietic growth factors (such as erythropoietin and thrombopoietin) that regulate red blood cell, platelet and white blood cell homeostasis. The JAK2 V617F mutation results in downstream, constitutive activation of the JAK-STAT pathway resulting in the MPN phenotypes. This mutation is present in 95% of patients with PV and in 55–65% of patients with ET and PMF. The remaining cases of PV are characterized by the presence of either substitution, deletions, insertions and duplication mutations within JAK2 exon 12 (Fig. 1). Mutation in the MPL gene, which encodes the receptor for the growth factor thrombopoietin, are present in 5–10% of patients with ET and PMF. Most MPL mutations occur at codon W515 and are usually substitutions that result in downstream activation of the JAK-STAT pathway. A differing mechanism of activation of the JAK-STAT pathway in MPN is caused by insertion or deletion mutations within exon 9 of CALR that encodes a protein involved in calcium signalling and protein unfolding. CALR exon 9 mutations always result in a frameshift that generates a novel C-terminus of the protein which is then able to interact with MPL receptor [5]. A spectrum of CALR mutations are present in ET and PMF but are considered absent in PV (Fig. 1). The JAK2 V617F, JAK2 exon 12, CALR exon 9 and MPL exon 10 mutations are considered disease driver mutations as they are essential in recapitulating the MPN phenotypes in murine models. A significant minority of ET and PMF patients possess none of the mutation types described and are termed ‘triple-negative’ MPN. However, sequencing of alternative JAK2 and MPL exons has revealed a considerable number of non-canonical mutations within these genes [6].

World Health Organization classification

Molecular diagnostics has become increasingly important in the classifications of MPN, in as much it is now incorporated as a critical criterion in the World Health Organization classification [7]. It must be emphasized that in addition to driver mutation status, the classification of the distinct MPN entities still requires clinical, other laboratory and histo-morphological evidence (Table 2).

Molecular diagnostic algorithm

Given that the MPN driver mutations are generally mutually exclusive, it is possible to adopt a molecular diagnostic algorithm that sequentially evaluates for the presence of the JAK2 V617F, CALR exon 9, MPL exon 10 or JAK2 exon 12 mutations. A number of different methodologies are available for MPN mutation detection and include Sanger sequencing, pyrosequencing, capillary electrophoresis for insertions or deletions, high resolution melt curve analysis, digital droplet PCR or NGS. Each of these methodologies has its own technical characteristics of sensitivity, specificity and clinical applicability with an understanding of these attributes necessary for the clinical interpretation of results [8,9].

It has become increasingly apparent that in addition to the driver mutations, several other genes are mutated in MPN. These mutations are also evident in other myeloid malignancies such as myelodysplastic syndromes and acute myeloid leukemia and contribute to the perturbation of a number of critical cellular functions (Table 3). Particularly in patients with PMF, these additional mutations appear to contribute to disease prognosis and also influence whether the disease will progress to acute leukemia.
Given the influence of additional mutations in MPN, it seems judicious to utilize NGS approaches that can simultaneously detect mutations in multiple regions of multiple genes simultaneously. Indeed, NGS methodologies are fast becoming the gold-standard approach for the molecular diagnosis and prognosis of an MPN [10].

Not only is establishment of driver gene mutation status an important diagnostic criterion, it also affords to use these mutations as markers of measurable residual disease. Treatment with interferon, JAK inhibitors and HASCT are all therapies in which reduction and/or rate of reduction of mutant allele burden may be of prognostic significance [11].

Summary

In conclusion, establishing the individual molecular landscape of each patient with PV, ET or PMF has diagnostic, prognostic and therapeutic implications. The future of MPN management lies in leveraging these molecular insights into personalized treatment strategies, aiming for a precision medicine approach that will improve outcomes for patients.

Table 3. Classes of additional genes recurrently mutated in myeloproliferative neoplasms that can modify disease course and prognosis

The author

Stephen Langabeer PhD, FRCPath
Cancer Molecular Diagnostics, St. James’s Hospital,
Dublin, D08 W9RT, Ireland

Email: slangabeer@stjames.ie

 References
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