Abstract

Essential thrombocythemia (ET) is rare in children, and little or no information is available about clonality or JAK2 mutations. However, the analyses in this work prove useful for the diagnosis of adult myeloproliferative disorders (MPDs). We evaluated the clonality status and V617FJAK2 mutation in 20 children affected by ET and compared them with 47 consecutive adult ET cases. Clonality was evaluated on the DNA of granulocytes and on the RNA of platelets. V617FJAK2 was analyzed by sequencing tests, allele-specific polymerase chain reaction (PCR), and digestion by BsaXI. A monoclonal pattern was found in 4 (28.5%) of 14 children and in 45% of informative adults. Heterozygous V617FJAK2 was found less frequently in children than in adults (P < .009). Only 2 girls showed both the V617FJAK2 mutation and a monoclonal pattern; one of them was the only child presenting a major thrombotic complication. In contrast to adults, most children with ET do not show either a clonal disorder or the V617FJAK2 mutation.

Introduction

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 studies4,5  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.

Study design

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.

Table 1.

Main clinical findings of children with essential thrombocythemia


Patient no.

Sex

Clonal/JAK2

Age at diagnosis, y

Age at analysis, y

Signs and symptoms

Treatment
1   F   Yes/V617F   0, 8   9   Budd-Chiari syndrome and inferior vena cava thrombosis   OLT + warfarin  
2   F   No/WT   2   2   None   None  
3   F   No/WT   3   14   None   ASA  
4   F   No/WT   4   6   None   None  
5   M   WT   4   15   None   ANAGR  
6   F   No/WT   6   7   Epistaxis, headache   ASA  
7   M   WT   6   7   None   ANAGR  
8   M   V617F   6   7   Headache   ASA  
9   F   Yes/WT   7   8   None   ASA + ANAGR  
10   F   No/WT   7   11   Headache   ANAGR  
11   F   No/WT   8   9   None   ANAGR  
12   F   No/WT   8   20   Headache   IFN/ASA  
13   F   No/WT   8   31   None   ASA  
14   M   V617F   9   15   None   ANAGR  
15   F   No/WT   11   15   Headache   ANAGR  
16   F   No/WT   12   12   None   ASA  
17   M   WT   12   24   Headache   ASA  
18   F   Yes/WT   13   16   None   None  
19   F   No/WT   13   18   Headache   ASA  
20
 
F
 
Yes/V617F
 
14
 
15
 
None
 
ANAGR
 

Patient no.

Sex

Clonal/JAK2

Age at diagnosis, y

Age at analysis, y

Signs and symptoms

Treatment
1   F   Yes/V617F   0, 8   9   Budd-Chiari syndrome and inferior vena cava thrombosis   OLT + warfarin  
2   F   No/WT   2   2   None   None  
3   F   No/WT   3   14   None   ASA  
4   F   No/WT   4   6   None   None  
5   M   WT   4   15   None   ANAGR  
6   F   No/WT   6   7   Epistaxis, headache   ASA  
7   M   WT   6   7   None   ANAGR  
8   M   V617F   6   7   Headache   ASA  
9   F   Yes/WT   7   8   None   ASA + ANAGR  
10   F   No/WT   7   11   Headache   ANAGR  
11   F   No/WT   8   9   None   ANAGR  
12   F   No/WT   8   20   Headache   IFN/ASA  
13   F   No/WT   8   31   None   ASA  
14   M   V617F   9   15   None   ANAGR  
15   F   No/WT   11   15   Headache   ANAGR  
16   F   No/WT   12   12   None   ASA  
17   M   WT   12   24   Headache   ASA  
18   F   Yes/WT   13   16   None   None  
19   F   No/WT   13   18   Headache   ASA  
20
 
F
 
Yes/V617F
 
14
 
15
 
None
 
ANAGR
 

The children were followed in 7 different pediatric units. The median clinical follow up was 7.5 years (range, 18 months to 24 years). The age of patients at diagnosis and at the moment of the analyses is reported. Treatments were chosen by the referring physicians, upon local criteria. Patient no. 1 underwent orthotopic liver transplantation (OLT) at 7 years of age for Budd-Chiari syndrome presented at diagnosis. Patient no. 12 was initially treated with interferon alpha (IFN) with partially transitory response and then shifted to low-dose aspirin (ASA). Patients nos. 16 and 20 were treated with anagrelide (ANAGR) for only 6 months each. No relation between age and molecular findings is recognizable

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.

Results and discussion

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).

Table 2.

Results of molecular studies in 20 children with ET, compared with 47 consecutive adult patients




V617Jak2

WTJak2

Total no.
No.
Major thrombotic complications
No.
Major thrombotic complications
Children      
   Girls*  15   —   —   —   —  
      Monoclonal   4   2   1 Budd Chiari   2   0  
      Polyclonal   10   0   0   10   0  
      Skewing   1   —   —   1   0  
   Boys   5   2   0   3   0  
   All children   20   4   1   16   0  
Adults      
   Women  36   20   —   16   —  
      Monoclonal   9   3   1 Budd Chiari; 1 cerebral vein   6   0  
      Polyclonal   11   7   1 Budd Chiari; 1 cerebral vein   4   0  
      Skewing   9   7   1 PA; 1 DVT   2   0  
      Homozygous   7   3   1 Budd Chiari   4   1 portal vein  
   Men   11   8   2 Budd Chiari; 1 MI   3   1 cerebral vein  
   All adults
 
47
 

 

 

 

 



V617Jak2

WTJak2

Total no.
No.
Major thrombotic complications
No.
Major thrombotic complications
Children      
   Girls*  15   —   —   —   —  
      Monoclonal   4   2   1 Budd Chiari   2   0  
      Polyclonal   10   0   0   10   0  
      Skewing   1   —   —   1   0  
   Boys   5   2   0   3   0  
   All children   20   4   1   16   0  
Adults      
   Women  36   20   —   16   —  
      Monoclonal   9   3   1 Budd Chiari; 1 cerebral vein   6   0  
      Polyclonal   11   7   1 Budd Chiari; 1 cerebral vein   4   0  
      Skewing   9   7   1 PA; 1 DVT   2   0  
      Homozygous   7   3   1 Budd Chiari   4   1 portal vein  
   Men   11   8   2 Budd Chiari; 1 MI   3   1 cerebral vein  
   All adults
 
47
 

 

 

 

 

WT indicates wild-type; PA, peripheral artery thrombosis; DVT, deep vein thrombosis; MI, myocardial infarction; and —, not applicable

*

Monoclonal cases made up 29% of informative cases; polyclonal cases, 71%

Monoclonal cases made up 45% of informative cases; polyclonal cases, 55%. Skewing cases made up 25% of all cases, while homozygous cases made up 19% of all cases

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.

Prepublished online as Blood First Edition Paper, July 18, 2006; DOI 10.1182/blood-2006-04-014746.

The authors declare no competing financial interests.

M.L.R. and M.C.P. contributed equally to this study.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

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.

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