Absence of the JAK2 V617F activating mutation in classical Hodgkin lymphoma and primary mediastinal B-cell lymphoma

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Alterations of JAK/STAT signaling molecules have been reported for several lymphomas including primary mediastinal B-cell lymphoma (PMBL) and classical Hodgkin lymphoma (cHL), where constitutive activation of STATs was found.1, 2 PMBL is a rather rare disease, however, there is increasing evidence that it is closely related to the far more frequent cHL.3 One common characteristic of these two entities is a frequent gain of 9p24 involving JAK2 which consequently has attracted considerable attention as potential trigger in the pathogenesis of these lymphomas. Recent publications revealed a substantial role for a gain of function mutation of JAK2 that results in constitutive activation of the tyrosine kinase, phosphorylation of STAT5, and expression of genetic markers in diverse myeloproliferative disorders, such as polycythemia vera, acute and chronic myeloid leukemia.4, 5, 6, 7 However, it was not found in acute and chronic lymphocytic leukemia.5

The underlying G to T transversion at nucleotide 1849 in exon 12 of JAK2 is located in the JH2 pseudo-kinase domain of JAK2 and leads to a substitution of valine to phenylalanine at position 617 (V617F).7 This region is involved in the auto-inhibition of its kinase activity and results in constitutive JAK2 activation.7 The wild-type sequence of exon 12 provides a specific restriction site for the endonuclease BsaXI, however, the nucleotide exchange of guanine to thymindine abolishes this restriction site preventing cleavage of the amplified exon 12 including sequence. Taking advantage of this, we screened genomic DNA of microdissected Hodgkin/Reed–Sternberg (HRS) cells of 16 cHLs, 20 PMBLs, five cHL (L428, L1236, HDLM-2, KM-H2, and L540) and two PMBL cell lines (MedB-1 and Karpas1106) for presence of the activating V617F mutation in the JAK2 gene by site-specific restriction analysis (Figure 1a).

Figure 1
figure1

Analysis of JAK2 V617F mutation. (a) Upper sequence: detail of JAK2 exon 12 sequence. The codons are highlighted alternately in white and gray and the corresponding protein sequence is denoted. The BsaXI recognition site is boxed with the crucial sequence in bold letters. The cleavage sites of the enzyme are indicated by the arrows. Lower sequence: detail of the mutated JAK2 exon 12 sequence. The G to T transversion in codon 617 abolish the BsaXI restriction site and lead to an amino-acid exchange from valine (V) to phenylalanine (F). (b) JAK2 V617F mutation analysis by site-specific restriction. Gel view of representative samples is given before (−) and after (+) BsaXI digestion. Genomic DNAs of two PMBL lines (MedB-1 and Karpas1106), five cHL lines (L428, L1236, HDLM-2, KM-H2, and L540), 20 PMBLs, and laser-microdissected HRS cells of 16 cHLs were subjected to PCR using JAK2 exon 12 spanning primers. BsaXI digestion completely cleaved the 314 bp PCR products except for the three PVs (polycythaemia vera) used as positive controls for the JAK2 V617F mutation.

Our results demonstrate the absence of the G → T mutation in both alleles of JAK2 since BsaXI completely cleaved the PCR product yielded from DNA of all cell lines, PMBLs, and cHLs (Figure 1b).

Nevertheless, there is evidence for constitutive JAK/STAT activity in PMBL and cHL. We lately found mutations in SOCS-1 (suppressor of cytokine signaling-1), a negative regulator of this pathway. In PMBL lines MedB-1 and Karpas1106, SOCS-1 defects prolonged activity of phospho-JAK2 leading to constitutive STAT5 activation.8, 9 Moreover, in neoplastic cells of cHLs SOCS-1 mutations were equally frequent and were associated with nuclear accumulation of phosph-STAT5 indicating constitutive STAT5 activity.10

Our analysis of JAK2 excluded the V617F mutation in cHL and PMBL. We conclude that this activating mechanism of JAK2 is not operative in these lymphomas. Hence, attention reconcentrates on other JAK/STAT activating mechanisms, for example, mutations or silencing of negative regulators like SOCS-1 and/or protein-tyrosine phosphatases.

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Correspondence to P Möller.

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Melzner, I., Weniger, M., Menz, C. et al. Absence of the JAK2 V617F activating mutation in classical Hodgkin lymphoma and primary mediastinal B-cell lymphoma. Leukemia 20, 157–158 (2006) doi:10.1038/sj.leu.2404036

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