Two of the first published papers on the JAK2 V617F mutation described the absence of the mutation in T-cells within all three Phneg.-CMPDs (Baxter et al, 2005; James et al, 2005), which has later been confirmed. In addition, the absence of JAK2 V617F mutation in the B-cell compartment in one PV patient and two myelofibrosis patients with previous ET and PV respectively, was demonstrated (Lasho et al, 2005). Accordingly, it was concluded, that the JAK2 V617F mutation is restricted to a myeloid precursor cell. The same group has previously shown that both B- and T-cells may be of clonal origin in IMF (Reeder et al, 2003). This observation does not exclude the possibility that the JAK2 V617F mutation is a secondary event in an already existing clone with a possible other genetic marker e.g. the del 20q, as it has been proposed by others (Kralovics et al, 2006). All these data were based on sequencing methods, which are known to have a limited sensitivity (Campbell et al, 2005). Small B- and T-cell JAK2 V617F clones could have been undetectable because of the limited sensitivity. Most recently, strict myeloid lineage involvement of the JAK2 V617F mutation has been challenged by the findings of the JAK2 V617F mutation in both CD19+ B- and CD3+ T cells in one patient and JAK2 V617F clonal B-cells but not T-cells in another patient by qPCR on FACS sorted cells in a group of ten patients with PV (Ishii et al, 2006). A similar pattern of heterogeneous lympho-myeloid JAK2 V617F clonal involvement in addition to involvement of NK-cells in both PV and the majority of IMF patients has been confirmed in a most recent study (Delhommeau et al, 2006).
In the present study we designed a qPCR assay with a very high sensitivity of at least 1:1000. Because of limitations in cell numbers, and hence DNA amount in the CD19+ B-cell and CD14+ monocyte compartments, this high sensitivity could not be reached in all qPCR reactions, and thus was calculated for each qPCR reaction. In addition to the expected findings of the JAK2 V617F mutation in granulocytes and monocytes, the results clearly demonstrated detectable levels of JAK2 V617F alleles in both CD19+ B-lymphocytes and CD3+ T-lymphocytes in a subgroup of patients, in total six (PV2, PV3, PV7, PV8, PV10, IMF1 and IMF2) and nine (PV1, PV2, PV6, PV8, PV9, PV10, IMF1, IMF2 and ET) respectively. Both IMF patients had JAK2 V617F clonal T-cells, one of them (IMF1) had a majority (83%) of mutated alleles, which definitely should be detectable with sequencing techniques, and this is also likely to be the case for patient PV8. In the other patients (PV1, PV2, PV6, PV9, PV10, IMF2 and ET) the T-cell clone would probably have been missed by sequencing because less than 13% mutated alleles were detected. One of the patients (PV2) with B-lymphocyte clonal involvement had 46% JAK2 V617F mutated alleles and should therefore be detectable by sequencing. In the present study, four (PV2, PV8, PV10 and IMF1) out of the 13 patients had JAK2 V617F clonal involvement of all four cell compartments analysed (granulocytes, monocytes, B-lymphocytes and T-lymphocytes). JAK2 V617F clonal involvement of all four cell compartments has previously been demonstrated in IMF patients (Delhommeau et al, 2006). Interestingly the three PV patients (PV2, PV8 and PV10) with detectable JAK2 V617F levels in all four cell compartments had very high proportions of mutated alleles in their monocytes and granulocytes as well as a significantly longer disease duration than the other PV patients in this study. The results of the present study are comparable with previously published data (Delhommeau et al, 2006; Ishii et al, 2006), and provide further evidence that the JAK2 V617F mutation occurs in a lympho-myeloid progenitor within the Phneg.-CMPDs. In the present study though, JAK2 V617F clonal involvement of T-cells were detected in nine out of 13 patients, which is a considerably higher frequency than reported by Delhommeau et al (2006), whereas the proportion of patients with JAK2 V617F clonal B-cells were comparable. The reason for this finding is not clear, but in the present study the detection of potential JAK2 V617F ‘positive’ B-cells could have been missed regardless of the use of a highly sensitive qPCR assay (>0·1%), because of limitations in DNA amount. On the other hand, both Delhommeau et al (2006) and Ishii et al (2006) used another qPCR method with competitive mutation-specific probes and reported a sensitivity of 2% (Delhommeau et al, 2006). Some of the discrepancies between the studies could therefore be related to technical differences. Nevertheless, the proportion of JAK2 V617F alleles seems to be at much lower levels in lymphoid cells than in myeloid cells in the majority of patients. T-cells are long-living cells, but they are produced throughout adult life by periodically importation of haematopoietic stem cells from the bone marrow to the thymus (Schwarz & Bhandoola, 2006). There is some evidence that the haematopoietic stem cells trafficking from the bone marrow to the thymus are progenitor cells with both myeloid and lymphoid differentiation potential (Katsura, 2002), which could explain the more scarce occurrence of T-cell JAK2 V617F clonal involvement as recorded in the present and previous studies (Delhommeau et al, 2006; Ishii et al, 2006). The identification of progenitors capable of producing B-cells and differentiating into cells of the myeloid lineage has further challenged our classical understanding of lineage-specific progenitors (Hou et al, 2005). It is intriguing to consider the possibility that the JAK2 V617F clone has a proliferative advantage in the myeloid lineage. During progression of the disease the percentage of JAK2 V617F cells increases, myeloid cells become homozygous for the JAK2 V617F mutation and in parallel, the possibility of the JAK2 V617F clone to proliferate in lymphoid lineage may increase. The percentage of JAK2 V617F alleles in progenitors seems to increase from ET to PV (Scott et al, 2006), and it is likely that JAK2 V617F positive CMPDs could be considered as a biological continuum from ET over PV to myelofibrosis (Campbell et al, 2005; Wolanskyj et al, 2005). Although the numbers are too small to draw firm conclusions, a trend towards increasing lymphoid involvement in late PV and IMF was recorded in the present study. A similar association has been described most recently (Delhommeau et al, 2006). It is important to notice that the JAK2 V617F clonal involvement is very heterogenous, probably partly reflecting biological variation among patients, although previous and current cytoreductive therapy might contribute. Moreover, it is of interest to note that T-cell involvement was actually observed in all disease entities (ET, PV and IMF), supporting the model of a biological continuum within the JAK2 V617F-CMPDs. In conclusion, this study provides further evidence that the JAK2 V617F mutation in the Phneg.-CMPDs occurs in a lympho-myeloid progenitor cell. Future studies in a large series of untreated patients are needed to further delineate precisely the level in the stem cell hierarchy at which the JAK2 V617F mutation occurs.