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Markers of Myeloproliferative Diseases in Childhood Polycythemia Vera and Essential Thrombocythemia

Luciana Teofili, Fiorina Giona, Maurizio Martini, Tonia Cenci, Francesco Guidi, Lorenza Torti, Giovanna Palumbo, Angela Amendola, Robin Foà, Luigi M. Larocca

From the Departments of Hematology and Pathology, Catholic University; Division of Hematology, Department of Cellular Biotechnologies and Hematology, La Sapienza University, Rome, Italy

Address reprint requests to Luigi M. Larocca, MD, Department of Pathology, Catholic University, Largo F. Vito 1, I-00168 Rome, Italy; e-mail: llarocca{at}rm.unicatt.it

	ABSTRACT

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Purpose: Polycythemia vera (PV) and essential thrombocythemia (ET) can present in pediatric age as sporadic or familial diseases. To define the biologic profile of childhood PV and ET, we evaluated specific markers in a cohort of pediatric patients affected by PV and ET, including cases with familial occurrence.

Patients and Methods: Thirty-eight children with PV and ET were investigated. The control group included 58 adults with PV and ET. Endogenous erythroid colonies, qualitative reverse transcriptase polymerase chain reaction for polycythemia rubra vera-1 (PRV-1) RNA expression, human androgen receptor assay and allele specific polymerase chain reaction for JAK2 V617F mutation were undertaken in all patients. Thrombopoietin, thrombopoietin receptor (c-mpl), and erythropoietin receptor mutation analysis was performed by direct sequencing in familial cases.

Results: The JAK2 V617F mutation in children with PV was significantly less frequent than in adult PV. The most common myeloproliferative marker found in these patients was PRV-1 RNA overexpression. Children and adults with sporadic ET showed a similar proportion of patients with PRV-1 RNA overexpression, JAK2 V617F mutation, and clonality, while none of the familial ET showed JAK2 V617F mutation and clonality. Also, PRV-1 RNA overexpression was significantly less common. Furthermore, most patients with familial ET exhibited the dominant-positive activating mutation of c-mpl. Finally, children with PV and ET had a significant lower incidence of thrombosis than adults.

Conclusion: This study demonstrates that familial and sporadic ET recognize different pathogenetic mechanisms. Myeloproliferative markers are specific tests for the diagnosis of ET in children with sporadic forms, while a significant proportion of children with PV can prove negative.

	INTRODUCTION

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Philadelphia-negative myeloproliferative disease (MPD) includes polycythemia vera (PV), essential thrombocythemia (ET), and idiopathic myelofibrosis.¹ These diseases share similar biologic features, such as endogenous erythroid colony (EEC) growth,^2,3 clonal hematopoiesis,⁴ or polycythemia rubra vera-1 (PRV-1) RNA overexpression.^5,6 Recently, many authors reported that most patients with PV and about half of those with ET and idiopathic myelofibrosis have a somatic point mutation in a highly conserved residue of the pseudokinase domain of the JAK2 tyrosine kinase, the JAK2 V617F mutation.^7-11 MPD can occur in childhood as well, although less frequently than in adults. The annual incidence of newly diagnosed ET in children is about 60-fold lower than in adults,¹² and only 0.1% of patients with PV present at younger than 20 years.¹³ The low incidence recorded in children suggests that the biology of the disease could be different compared with the adult forms. Furthermore, many children with primary thrombocytosis and erythrocytosis can have a familial form of thrombocythemia and polycythemia.^14,15 Familial ET and PV are very heterogeneous disorders, and specific mutations of the thrombopoietin (TPO) gene,^16-19 TPO receptor gene,²⁰ or erythropoietin (EPO) receptor gene²¹ have been identified only in the minority of familial cases.

To better elucidate the biologic profile of MPD in childhood, we investigated the incidence of EECs’ growth, granulocyte PRV-1 RNA overexpression, JAK2 V617F mutation, and clonal hemopoiesis in a cohort of pediatric patients affected by PV and ET, including cases with a familial occurrence. The results obtained in the pediatric population were then compared to those derived from a series of adult patients affected by PV and ET.

	PATIENTS AND METHODS

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Study Population
The study was carried out in 38 children with a diagnosis of PV (n = 9) and ET (n = 29), consecutively observed between 1980 and 2005 at the Hematology Department of the University La Sapienza of Rome, Italy. Among them, 12 patients had familial MPD (11 familial thrombocythemia, belonging to four families, and one familial polycythemia). The median follow-up was 131 months (range, 3 to 312 months). Twenty-three patients (12 with sporadic ET, eight with PV, two with familial thrombocythemia, and one with familial polycythemia) were on therapy at the time of the study. Treatment included hydroxyurea, interferon alfa, or anagrelide in thrombocythemic patients, and it included phlebotomy or hydroxyurea in polycythemic patients. As a control group, 58 consecutive adult patients (32 with ET and 26 with PV), which were observed at the Hematology Department of the Catholic University of Rome in Rome, Italy, between 2001 and 2005, were investigated. Among them, one female patient had a familial history of thrombocythemia. All patients affected by ET and PV were diagnosed according to the PV Study Group or WHO criteria.^3,22 Tables 1 present the clinical and hematologic features of pediatric and adult patients.

PRV-1 Assay, Clonal Hemopoiesis, and JAK2 Mutation Analyses
The PRV-1 assay was carried out by qualitative reverse transcriptase polymerase chain reaction (RT-PCR), as previously described.^6,23. Clonal hemopoiesis was assessed by the human androgen receptor (HUMARA) polymorphism assay⁶ and by the HUMARA methylation-specific PCR.²² The HUMARA polymorphism assay was carried out in granulocytes isolated from female patients. T lymphocytes were used as a control. One µg of DNA, isolated using DNAzol or TRIzol reagents (Invitrogen Corp, Carlsbad, CA), was incubated with and without 20 units of HpaII (New England Biolabs, Ipswitch, MA) at 37°C for 12 hours in a final volume of 20 µL. After 10 minutes of incubation at 95°C, 3 µL of all samples were amplified with specific primers and following PCR conditions, as previously described.⁶ In patients older than 60 years, only results indicating a polyclonal hemopoiesis were considered to exclude the possibility of age-related skewing of X-chromosome inactivation. Results obtained by HUMARA polymorphism assay were confirmed by HUMARA methylation-specific PCR analysis.²⁴ Briefly, after the treatment of 1 µg of DNA with sodium bisulfite and the purification and desulfonation with sodium hydroxide, DNA was amplified with specific primers for the methylated and unmethylated HUMARA gene.²⁴ PCR amplification and correction of the ratio of peaks for an appropriate X-inactivation pattern were performed using the ABI PRISM 3100-Avant Genetic Analyzer and GeneMapper ID software (Applied Biosystems, Warrington, UK).

The presence of the JAK2 V617F mutation was investigated according to Baxter et al.⁸ Briefly, 80 ng of DNA patients were used to amplify the mutated and unmutated exon 12 of JAK2 in allele-specific PCR. PCR products were separated onto a 3% agarose gel, stained with ethidium bromide, and viewed under UV light. A 203 base pair fragment indicates the presence of 1849G more than T mutation.

The EECs’ growth, PRV-1 RNA, JAK2 V617F mutation, and clonal hemopoiesis analysis were independently carried out by three different investigators (M.M., T.C., and F.G.), blinded to diagnosis and to clinical and laboratory findings of the enrolled patients.

TPO Receptor (c-mpl) and EPO Receptor Mutation Analysis
Mutational analysis of TPO and c-mpl was conducted on the 5'-untranslated region for the TPO gene and on exon 10 for the c-mpl gene, which have been reported to be mutated in hereditary thrombocythemia.^16-20 Approximately 100 ng of DNA were amplified in a total volume of 50 µL containing 50 picomoles of each primer, 1 x polymerase chain reaction (PCR) buffer (10 mmol/L of Tris HCl, pH 8.3; 50 mmol/L of KCl), 1.5 mmol/L of MgCl₂, 200 µmol/L of deoxyribonucleotide triphosphates, 0.3 unit Taq DNA Polymerase (AmpliTaq DNA Polymerase; Perkin-Elmer Roche, Foster City, CA). The specific primers and PCR conditions for TPO were 5'-ACC CTG CCA GGC AGT CTC TTC–3' and reverse 5'-GAG GGG TGG ATT CCC TGG GTT–3'; 37 cycles at 95°C for 1 minute, 60°C for 40 seconds, and 72°C for 40 seconds. The specific primers and PCR conditions for c-mpl were forward 5'-CCG AAG TCT GAC CCT TTT TG–3' and reverse 5'-ACA CAG CGA ACC AAG AAT GC–3'; 37 cycles at 95°C for 40 seconds, 56°C for 30 seconds, and 72°C for 40 seconds. Thereafter, 5 µL of PCR product was treated with ExoSAP-IT (USB Corp, Cleveland, Ohio) following the manufacturer's protocol, amplified with BigDye Terminator version 3.1 cycle sequencing kit (Applied Biosystems) using forward and reverse primers, and sequenced with an ABI PRISM 3100-Avant Genetic Analyzer (Applied Biosystems).

Mutational analysis of EPO receptor was conducted on exon 8 of the gene, according to Kralovics et al.²¹ The PCR product was treated with ExoSAP-IT (USB Corp) following the manufacturer's protocol, amplified with BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems), using forward and reverse primers, and sequenced with an ABI PRISM 3100-Avant Genetic Analyzer (Applied Biosystems).

Statistical Analysis
Statistical comparison of continuous variables was performed by the Kruskal-Wallis or the Mann-Whitney U-test, as appropriate. Comparison of categoric variables was performed by ² statistic, using the Fisher's exact test. P values less than .05 were considered statistically significant.

	RESULTS

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Table 1 presents clinical and hematologic differences between pediatric and adult patients. Table 2 presents the results of the EECs’ assay, PRV-1 RNA overexpression, JAK2 V617F mutation, and clonal hematopoiesis analysis in pediatric as compared with adult patients.

Pediatric ET
Children with ET did not differ from adult patients with respect to sex distribution or hematologic features (Table 1). As observed for the PV group, children with ET had a significantly lower incidence of thrombosis than adults (P = .0009; Table 1). Moreover, the proportion of patients with a familial history of thrombocythemia was significantly higher among children than adults (P = .0008; Table 1). With regard to myeloproliferative markers, children with sporadic ET were similar to adult ET patients for PRV-1 RNA expression, JAK2 V617F mutation, and clonality of hematopoiesis, although the EECs’ growth was less frequent in children than in adults (P = .013; Table 2). On the contrary, children with familial thrombocythemia resulted significantly less frequently EEC-positive (P = .002), PRV-1 RNA–positive (P = .023), and JAK2 mutated (P = .0008) as compared with adult ET (Table 2). It is of interest that, similarly to pediatric patients, the patient with familial thrombocythemia belonging to the adult group was PRV-1 RNA–negative wild type for JAK2 and exhibited polyclonal hematopoiesis.

Finally, although children with sporadic and familial ET had a similar age and sex distribution, follow-up duration, and hematological parameters, they differed significantly for PRV-1 RNA overexpression (P = .001), JAK2 V617F mutation, and clonal hemopoiesis (P = .025 for both assays).

The presence of TPO or c-mpl gene mutation was investigated in five families with ET, including 12 patients (11 belonging to the pediatric series and one to the adult group). We detected the serine 505 to asparagine 505 (Ser505Asn) mutation of the c-mpl gene in nine children belonging to three families and in the adult patient with familial thrombocythemia (Fig 1). This latter patient was a 57-year-old-woman. Her mother, affected by ET, died of cerebral vein thrombosis. The c-mpl mutation was investigated in two of her three children. One of them had a normal platelet count and a wild-type c-mpl, while the children with thrombocytosis carried the Ser505Asn mutation (Fig 1). This genetic defect is transmitted by dominant autosomal inheritance and confers constitutive activation of the TPO receptor.²⁰ Two members of the fifth investigated family showed a wild-type c-mpl gene. Finally, none of the reported mutations of the TPO gene were identified in our patients.

View larger version (11K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 1. Familial trees of the four families affected by the Ser505Asn thrombopoietin receptor mutation. The squares denote men, circles are women, and slash marks are deceased members. Solid blue indicates affected members, solid yellow is for the members with thrombocytosis not investigated for the mutation, and white is for the members with a normal platelet count. (A-C) Pediatric patients; (D) adult patient.

	DISCUSSION

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MPD is quite a rare disease in childhood.^11,12 As a consequence, all biologic information usually obtained from studies on adult series are applied to pediatric patients as well. Recently, Randi et al²⁸ evaluated the incidence of the JAK2 V617F mutation and of clonal hemopoiesis in 20 children with ET. The authors found that childhood ET, compared with adult ET, was more frequently polyclonal and had wild-type JAK2 V617F.²⁸ In the present study, we investigated a large number of pediatric patients with PV and ET for several biologic markers of MPD, in comparison with an adult population of patients with PV and ET. While the hematological findings were similar in children and adult patients, we observed that the incidence of thrombotic complications was significantly lower in pediatric patients than in adults, thus confirming the important role of age in increasing the risk of thrombotic complications during the course of the disease.²⁹ Furthermore, in agreement with previous observations,¹⁵ a familial occurrence was present in a significant proportion of our pediatric patients. The definition of familial MPD is usually used to indicate two distinct hematological diseases: (1) hereditary thrombocytosis resulting from TPO^16-19 or c-mpl²⁰ gene mutations and primary erythrocytosis due to mutations in the EPO receptor gene;²¹ and (2) acquired thrombocytosis and erythrocytosis occurring in two or more members of the same family in the absence of specific underlying known genetic defects and exhibiting, as sporadic MPD, clonal hemopoiesis, or EECs’ growth.³⁰ Accordingly, we investigated in familial cases both MPD markers and reported mutations of the EPO receptor,²¹ TPO^16-19 and c-mpl²⁰ genes.

We found that the pattern of expression of MPD markers differs significantly in childhood and adult PV. Indeed, most adult patients exhibited JAK2 V617F mutation, PRV-1 RNA overexpression, and EEC growth. In contrast, PRV-1 RNA overexpression was the unique assay commonly found positive in children. Surprisingly, the incidence of JAK2 V617F mutation in childhood PV appeared significantly lower than that observed in adult PV, and all JAK2 V617F–mutated patients also had high levels of PRV-1 RNA. Thus, in childhood PV, biologic markers of MPD are clustered only in a minority of patients. Several studies pointing to the definition of the role of JAK2 V617F mutation in the pathogenesis of MPD agree that in PV the presence of the homozygous mutation of JAK2 correlates with disease duration.^7-10,31 It could, therefore, be speculated that children showing a wild-type JAK2 may become JAK2 V617F mutated at a later stage. Alternatively, in childhood PV other genetic defects beside JAK2 V617F mutation may be associated with the development of the disease.

Moreover, our study highlights the different incidences of PRV-1 RNA overexpression, JAK2 V617F mutation, and clonal hemopoiesis between sporadic and familial ET, although these diseases appear indistinguishable on the basis of the commonly used diagnostic criteria for ET.^3,22 Indeed, all patients with familial ET tested negative for JAK2 V617F mutation and clonal hemopoiesis, and they rarely showed PRV-1 RNA overexpression and EEC growth. These findings prompted us to investigate the presence of hereditary genetic defects in these patients, and we found that in four of the five investigated families all affected members exhibited the activating mutation of the c-mpl gene.²⁰ Interestingly, among patients with the Ser505Asn mutation, there was a 57-year-old woman. Her mother, affected by ET, died of cerebral vein thrombosis. The c-mpl mutation was present in one of her children showing thrombocytosis, while the other children having normal platelet counts had wild-type c-mpl. We describe for the first time this autosomal-dominant alteration in an Italian population of patients with ET, and its high frequency in our series of patients suggests that this molecular defect might be more diffuse in our country than previously reported.³² Recently, an interesting French study performed by Bellannè-Chantelot et al³³ evaluated the incidence of JAK2 V617F mutation in 72 families with MPD. Among them, seven families, including 43 patients, tested negative for JAK2 V617F mutation.³³ The authors reported that these patients were significantly younger at diagnosis than the JAK2-mutated patients, and they hypothesized that unknown mutations in other genes could be responsible for the disease. Thus, these observations, together with our findings, strikingly support the need for a careful screening for hereditary genetic defects in young patients with familial evidence of thrombocytosis.

One of the aims of this study was to clarify if a panel of biologic markers of MPD could be potentially useful in the diagnostic screening of children with thrombocytosis and erythrocytosis, as already demonstrated in adult patients.³⁴ In pediatric patients, the availability of specific diagnostic tests on peripheral blood samples would be particularly helpful, allowing to avoid the use of radioactive compounds and reserving bone marrow biopsy to selected cases. Randi et al recently suggested that JAK2 V617F mutation and clonal hemopoiesis are not as frequent as reported in adult ET.²⁸ In contrast, our study based on a large panel of MPD markers, including PRV-1 RNA and EEC evaluation, clearly demonstrated that children with sporadic thrombocythemia behave very similarly to adult ET, showing a similar frequency of JAK2 V617F mutation, PRV-1 RNA overexpression, and clonal hematopoiesis.

In conclusion, this study clearly demonstrated that familial and sporadic thrombocythemia in children recognize different pathogenetic mechanisms due to different molecular defects. Biologic markers of MPD, such as PRV-1 RNA overexpression, JAK2 V617F mutation, and clonal hemopoiesis assay, are useful diagnostic tests in children with sporadic ET, while their diagnostic usefulness in children with PV is lower than in adult patients, considering that a significant proportion of children with PV can prove completely negative.

	AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

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The authors indicated no potential conflicts of interest.

	AUTHOR CONTRIBUTIONS

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Conception and design: Luciana Teofili, Luigi M. Larocca

Financial support: Luciana Teofili, Luigi M. Larocca

Provision of study materials or patients: Fiorina Giona, Maurizio Martini, Tonia Cenci, Francesco Guidi, Lorenza Torti, Robin Foà

Collection and assembly of data: Luciana Teofili, Fiorina Giona, Maurizio Martini, Tonia Cenci, Francesco Guidi, Lorenza Torti, Giovanna Palumbo, Angela Amendola

Data analysis and interpretation: Luciana Teofili, Fiorina Giona, Maurizio Martini, Angela Amendola, Robin Foà, Luigi M. Larocca

Manuscript writing: Luciana Teofili, Robin Foà, Luigi M. Larocca

Final approval of manuscript: Luciana Teofili, Fiorina Giona, Maurizio Martini, Tonia Cenci, Francesco Guidi, Lorenza Torti, Giovanna Palumbo, Angela Amendola, Robin Foà, Luigi M. Larocca

	NOTES

 
Supported in part by Fondi d’Ateneo, Progetti D1 2005 to 2006, Catholic University, Rome, Italy.

Authors’ disclosures of potential conflicts of interest and author contributions are found at the end of this article.

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Submitted August 10, 2006; accepted December 14, 2006.

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