Eradication of JAK2 V617F mutation after allogeneic transplantation in a patient with myelofibrosis with myeloid metaplasia

Myelofibrosis with myeloid metaplasia (MMM) is the chronic myeloproliferative disorder associated with the poorest prognosis with a median survival of 4 years, and actually allogeneic hemopoietic stem cell transplantation (SCT) represents the only curative option. Recently, the detection of JAK2 V617F mutation in MMM, as in other Philadelphia chromosome-negative myeloproliferative diseases, has revived the prospect of targeted therapeutics as well as molecular monitoring of treatment response. This point mutation results in a substitution of valine for phenylalanine at position 617 in the JH2 domain and leads to constitutive tyrosine phosphorylation and cytokine hypersensitivity; approximately 45% of MMM cases (range 35–50%) are known to possess the JAK2 V617F mutation, although the pathogenesis of this disease still remains unclear. JAK2 V617F-positive MMM patients have been associated to a significantly higher white blood cell (WBC) count and neutrophil count than V617F-negative patients, suggesting that the mutation is associated with expansion of the myeloid lineage; positive patients also have a worse overall survival and have less requirement for transfusion than the negative cases.

Here, we describe a case of a patient with MMM in whom the JAK2 V617F mutation was shown to be eradicated after allogeneic SCT.

A 54-year-old male patient was diagnosed with MMM according to ICSG (Italian Cooperative Study Group) criteria1 in February 2003. At presentation, both Lille2 and Barosi3, 4 scores were 0. Cytogenetic analysis showed the presence of trisomy 8. Bone marrow biopsy revealed a diffuse pattern of fibrosis. No treatment was administered. In December 2003, MMM progression was noted with Lille score 1 (WBC 40 G/l) and severe splenomegaly. Hydroxyurea at the dosage of 1 g/day was added.

In April 2006, clinical conditions worsened with further splenomegaly (interpole diameter 21 cm below the costal margin), progressive pancytopenia and red cell transfusion dependence (a red cell unit (uRBC) every 2 days). CD34+ cell percentage in peripheral blood (PB) increased to 10.7. Bone marrow aspirate revealed 15% of blasts and the biopsy showed marked fibrosis and dysplastic megakaryocytes with 8% of CD34+ blast cells.

The patient was found to have a human leukocyte antigen-identical brother and he was then referred for an allogeneic SCT, which was performed in May 2006. Reduced-intensity conditioning (RIC) or non-myeloablative regimen included 30 mg/m2 fludarabine for 5 days and 140 mg/m2 melphalan for 2 days with cyclosporine and short course of methotrexate for graft-versus-host disease prophylaxis.5 CD34+ cells (7.9 × 106/kg) from PB were infused. Engraftment was achieved on day +14 post-transplantation. Post-transplantation course was unremarkable, except for cytomegalovirus and Epstein–Barr virus reactivation. Clinical improvement, according to International Working Group on MMM,6 at current follow-up on day + 90 post-transplantation has been documented. Bone marrow biopsy of the patient revealed grade 1 of fibrosis; spleen volume reduced to 3 cm below the costal margin and bone marrow aspirate did not show increment of blasts; the patient also reduced his transfusion requirement (1 uRBC/every 10 days).

JAK2 V617F mutation analysis, when allele-specific PCR method7, 8, 9, 10 became available, was retrospectively performed on genomic DNA sample obtained at diagnosis. The mutation was present in samples obtained at diagnosis and just before transplantation. The absence of the mutation was assessed on PB sample from the donor. Quantitative PCR of JAK2 V617F was not available either at diagnosis or before transplantation. JAK2 V617F mutation analysis was then reassessed on day +14, +20, +60 and +80 post-transplantation on bone marrow. No mutation was present leading to apparent JAK2 V617F eradication by allogeneic PBSCT as reported in Figure 1a. Chimerism analysis was also performed using short tandem repeat analysis at the same time intervals of JAK2 analysis, before and after SCT, showing 100% donor chimerism with no traces of DNA recipient (Figure 1b). We could assess, thus, that JAK2 V617F mutation in this patient has apparently been eradicated by a non-myeloablative conditioning regimen followed by transplantation.

Figure 1

Methods for molecular and engraftment monitoring in MMM with JAK2 V617F mutation after allogeneic SCT. (a) Allele-specific PCR for the JAK2 V617F mutation. Lane 1, patient sample before transplant; lane 2, donor; lanes 3–5, patient sample on day +14, +20 and +60 post-transplantation. (b) Chimerism analysis using STR method: pattern at locus D18 of recipient pre-transplantation (upper trace), donor (middle trace), recipient on day +14 post-transplantation (bottom trace).

Standard-dose conditioning regimens followed by allogeneic transplantation are associated with a relatively high transplant-related mortality in MMM. Introduction of RIC regimens has substantially reduced treatment-related mortality, expanding the access of fragile patients with MMM and achieving a 1-year survival rate of 90%.11 Although the optimal conditioning regimen is currently unknown, our observation indicates that JAK2 V617F mutation can be eradicated shortly after RIC regimen and SCT. Molecular monitoring of treatment response in MMM is applicable to JAK2 V617F-positive cases and major molecular response could be defined as the absence of the mutation in samples of previously positive cases; however, whether eradication of mutation reflects also MMM disease resolution or a mere return to a pre-JAK2 V617F state is still unknown.


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