Pediatric patients with essential thrombocythemia are mostly polyclonal and V617FJAK2 negative

The diagnosis of essential thrombocythemia (ET) is usually made by exclusion criteria because no biologic markers are known to differentiate it either from primitive myeloproliferative disorders (MPDs)¹  or from reactive thrombocytosis.² 

The primitively proliferative nature of ET was first documented by Fialkow et al³  who analyzed female patients for X-chromosome inactivation patterns (XCIPs) of G6PDH. It was found therein that 3 cases showed clonal expansion. However, further studies^4,5  demonstrated that only 20% to 35% of adult ET is monoclonal. Therefore, it is possible that monoclonal and polyclonal patterns define different disorders with a similar phenotype. It is relevant that recently a somatic mutation of Janus kinase 2 (JAK2) was found in about 35% to 50% of adult ET patients.^6,7 

ET in pediatric age is a rare disorder that might also embed a spectrum of different diseases.⁸  Unaware of previous comparative reports, we evaluated the clonal patterns and JAK2 gene mutations in pediatric ET patients.

We studied 15 girls and 5 boys affected by ET in agreement with the Polycythemia Vera Study Group criteria⁹ : ET diagnosed in pediatric age; platelet count continuously more than 800 × 10⁹/L (range, 850-4500 × 10⁹/L); and no known causes of reactive/secondary thrombocytosis. None of the children had any family history of either MPD or thrombocytosis. None had a prothrombotic condition. Moreover, in these patients, serum erythropoietin (EPO) and thrombopoietin (TPO) contents, platelet function, standard cytogenetic analysis, bcr/abl rearrangement, and sequences of TPO and c-MPL genes¹⁰  were evaluated; no abnormality was found. The clinical findings and therapeutic options adopted are summarized in Table 1. Approval for these studies was obtained from the Padua University Department of Medical and Surgical Sciences institutional review board, and informed consent was provided according to the Declaration of Helsinki.

For comparison as adult controls, 36 consecutive women and 11 men, all younger than 50 years (mean age 43 ± 7 years) affected by ET were studied in our department, which is a reference center for thrombo/hemorrhagic conditions.

We also studied, as negative controls, 21 healthy individuals (17 women and 4 men) with normal platelet count and no thrombotic complication or prothrombotic state.

Granulocytes, T lymphocytes, and platelets were isolated as described by Teofili et al.¹¹  DNA was extracted with the Miller et al method.¹²  RNA was extracted from platelets using TRIZOL (Invitrogen, Carlsbad, CA) reagent according to the manufacturer's protocol.

The state of activation of the X-chromosome was determined by using a methylation-sensitive restrictive enzyme (HpaII) or transcriptional polymorphism on the active X-chromosome. XCIPs were assessed using the following: primers flanking the CAG repeats of HUMARA gene¹³ ; “nested” primer pairs flanking the region of BstXI polymorphism of PGK gene¹⁴ ; or primers for allele-specific polymerase chain reaction (PCR) of an exonic polymorphism of MPP1 gene (p55) on platelet RNA.¹⁵ 

To detect the V617FJAK2 mutation in peripheral blood granulocyte DNA, sequence analysis and allele-specific PCR were used. Digestion by BsaXI was used to determine homozygosity of JAK2 mutation.⁷ 

The comparison between variables was performed by chi-square statistics with Yates variables, where P values below .05 were considered statistically significant.

While reactive thrombocytosis is relatively common and some familial cases were reported,¹⁶  sporadic ET is extremely rare in childhood.¹⁷  These cases have clinical and laboratory characteristics different than those of adult ET.⁸  In the present study, we found that pediatric ET differs from adult ET also with respect to the clonality pattern (Table 2); indeed, we show that 10 children exhibit a polyclonal pattern on granulocytes, while only 4 (28.5%) are monoclonal. Because there may be a restriction of the clonal population to cells of the megakaryocyte lineage,¹⁸  we also studied clonality in platelet mRNA from 8 children (6 polyclonal and 2 monoclonal), and the observed patterns were consistent. Clonality studies were interpretable for all but one child, who had a skewing pattern. On the other hand, adults, notwithstanding the relatively young age, were not informative in about half of the cases considered, as reported by others.⁴  Moreover, 45% of the informative patients were monoclonal, a percentage significantly higher than in children. The known increase of clonality incidence with age¹⁹  cannot exclude that our children might develop a clonal pattern over time. At present, however, 4 of the 10 girls with a polyclonal pattern have been followed for more than 8 years, 2 patients being now older than 20 years (Table 1).

The somatic mutation of JAK2, considered a primary event in MPD,²⁰  was reported in most patients with polycythemia vera (PV) and in a subgroup of ET patients. In our study, the V617FJAK2 mutation was significantly less frequent in children (4/20) than in adults (28/47) (P = .009). Our results for adults are consistent with data reported elsewhere.^6,21  It is relevant to note that, in addition to the sequencing method, we used the allele-specific PCR, which is considered a highly sensitive technique to JAK2 mutations.²¹  Neither children nor adults showed homozygous JAK2 mutations.

The association of JAK2 mutations with thrombohemorrhagic events is still a matter of debate.^6,7,20,22  Increased frequency of venous thrombosis in uncommon sites in adults presenting the V617FJAK2 mutation was reported for large cohorts²¹  and confirmed by our cases. Among adults, 7 of the 9 unusual site thromboses occurred in mutated patients, irrespective of their clonality status. It is worthwhile noting that one pediatric case with both JAK2 mutation and clonal disease presented at 9 months with Budd-Chiari syndrome (Table 2). Whether this single case represents a random association or hints at a pathogenetic implication is not clear at present. In fact, another girl showed a monoclonal pattern and the V617FJAK2 mutation but no clinical symptoms. The simultaneous proof of clonal myelopoiesis on XCIP analysis and JAK2 somatic mutation could be suggestive of a primitive MPD that may have a more severe clinical course.⁴ 

JAK2 mutation in ET may imply the existence of a disease closely related to PV. Campbell et al²¹  recently suggested that mutation-positive and mutation-negative ET represent distinct disorders. Our study includes a number of children who have a rare disease, and thus our data indeed confirm the possible existence of different types of ET. Rare cases are characterized by monoclonal pattern and/or JAK2 mutation and possibly by a severe clinical behavior. The other cases may actually have developed a disease different from adult ET as previously hypothesized.⁸  Moreover, the difference between child and adult ET is underlined when, as suggested by Kiladjian et al,²³  we combine the results of clonality assays and JAK2 evaluation: only 30% of children seem to have a clonal proliferation, compared with 72% of adult cases.

Because criteria for the treatment of ET are not specific for children, therapeutic choices are highly heterogeneous (Table 1). Thus, new diagnostic and prognostic classifications might help in shaping improved therapeutic recommendations, with wide implications.²⁴ 

In conclusion, the heterogeneous molecular features found in our large set of pediatric ET cases may help in defining various subgroups, allowing for the clinical course to be predicted, mainly for thrombotic risks. This is necessary to assess which child with ET really deserves cytoreductive therapy, as already defined for adults. Larger samples are obviously needed to confirm our results, thus further prompting the use of biologic markers for diagnosis and treatment of ET.

We thank Dr C. Consarino, Pediatric Hematological Service, Catanzaro Hospital; Dr P. Pierani, Pediatric Clinic, Ancona; Dr K. Tettoni, Pediatric Clinic, Spedali Civili Brescia; and Dr G. M. Fiori, Pediatric Clinic Cagliari for providing samples from some patients.