Detection of an MPLW515 mutation in a case with features of both essential thrombocythemia and refractory anemia with ringed sideroblasts and thrombocytosis

BCR-ABL-negative chronic myeloproliferative diseases (CMPD) and myelodysplastic syndromes (MDS) are both very heterogeneous and in some cases show cytomorphological and clinical overlaps. This is especially true for the subentities chronic myelomonocytic leukemia (CMML), juvenile myelomonocytic leukemia and myelodysplastic/myeloproliferative disease, unclassifiable', which were classified by the WHO as its own category of myelodysplastic/myeloproliferative disorders. Recently, refractory anemia with ringed sideroblasts associated with anemia but marked thrombocytosis >500 × 10⁹/l, and a proportion of at least 15% of ringed sideroblasts in the bone marrow was added as a distinct entity to this category.^{1, 2, 3, 4, 5} Most cases of RARS-T show a normal karyotype.^{1, 2, 3, 4, 5} In addition, this specific subtype was reported to have a high frequency of the JAK2 V617F mutation.^{1, 2, 4} The V617F mutation is primarily associated with the CMPD, being found in nearly all polycythemia vera (PV) and in 50–60% of all essential thrombocytosis (ET) and chronic idiopathic myelofibrosis (CIMF) cases, but was also detected rarely in MDS.⁶ Therefore, the high frequency of JAK2 V617F in RARS-T emphasizes the ambiguous position of this subentity between CMPD and MDS, as do molecular and cytomorphological aspects. However, the genetic characterization of RARS-T is not complete, as 10–50% of all cases did not show a V617F mutation.

Lately, somatic mutations of the MPL gene with its coding function for the thrombopoietin receptor were detected in ∼5% of CIMF and in ∼1% of all ET. So far, two different variants, the W515L and the W515K mutation, were identified. They are localized in the transmembranous domain and thus might be critical for receptor dimerization. They cause single amino-acid exchanges (tryptophan to leucine or lysine, respectively) and confer constitutive cytokine-independent activation of the JAK–STAT pathway as gain-of-function mutations, similar to the V617F.^{7, 8} So far, they were not detected in PV, AML, MDS or CMML.⁷

Here we report on the detection of an MPLW515 exchange in a patient with clinical and cytomorphological features not only of an ET but also of RARS-T who was negative for the JAK2 V617F mutation. This is, to our knowledge, the first description of an MPLW515 mutation in such an overlapping disease with difficult differential diagnosis.

A 63-year-old male patient showed incidentally marked thrombocytosis (1.222 × 10⁹ G/l; WBC 6.4 × 10⁹ G/l, Hb 133 g/l). He was in good physical condition and did not suffer from any clinical symptoms. There was no organomegaly at clinical examination. Past history revealed no abnormalities. Owing to the isolated thrombocytosis, a diagnosis of ET was suspected. Bone marrow cytomorphology showed normal cellularity and a strongly increased megakaryopoiesis. Granulopoiesis showed normal features, and bone marrow blasts were <5%. Erythropoiesis was slightly increased to 45% of nucleated cells in the bone marrow and showed signs of dysplasia in >10% of cells. More surprisingly, iron staining, which was performed owing to marked dysplasia in erythropoiesis (Figure 1a), revealed 65% of ringed sideroblasts (Figure 1b). Cytogenetics showed a normal karyotype (46,XY) in all 20 metaphases. In addition, deletions of 5q31, 5q33–34, 7q31 and 20q12, and trisomy 8 were excluded by interphase fluorescence in situ hybridization (FISH). There was no evidence of the BCR-ABL translocation as assessed by interphase FISH and reverse transcription-PCR. Owing to the combination of thrombocytosis and the diagnosis of ET, and also the presence of ringed sideroblasts, the case was classified as a borderline case between CMPD (ET) and refractory anemia with ringed sideroblasts and thrombocytosis (RARS-T), although no anemia was found at diagnosis. Mutation analysis for the JAK2 V617F mutation was performed as described before⁹ but was negative. In addition, screening for an MPLW515 mutation was performed by a melting curve-based LightCycler assay following PCR with the forward primer IndexTermGTGACCGCTCTGCATCTA, reverse primer IndexTermGGCTGCAGTGTCCCTAA and hybridization probes IndexTermAGGGCATGCCTCAGTCTCCTG-FL and LC-Red- IndexTermGTGTGCAGGAAACTGCCACC-P. This analysis clearly revealed an MPL mutation (Figure 2a). Direct sequencing of the PCR product revealed a t>g exchange at nucleotide position 1544 leading to an MPLW515L mutation (Figure 2b). Treatment was started with hydroxyurea (1 × 1000 mg) and resulted in the normalization of thrombocytes to 286 × 10⁹ G/l within weeks. Three months after the initial diagnosis, the patient was still feeling well without any complications.

Figure 1

Bone marrow cytomorphology showing increased erythropoiesis (May-Giemsa-Gruenwald) (a) and increased ringed sideroblasts (iron staining) (b).

Figure 2

Melting curve analysis (a) and results of sequencing analysis (b) showing a TGG to TTG exchange at MPL codon 515.

So far, the JAK2 V617F mutation was reported in 50% of cases with ET and in 50–90% of all RARS-T cases.^{1, 2, 4} However, this case illustrates that MPLW515 mutations can also add these two phenotypes and provide information in cases with borderline features. In addition, this case shows that MPLW515 mutations are not as specific for clear CIMF or ET as described previously.^{5, 7, 8} With respect to the benign risk profile, peripheral blood parameters, cytomorphological features and the normal karyotype in accordance with most reported cases,³ this case did not differ significantly from other JAK2 V617F positive or negative in CMPD or RARS-T cases. Similarly, in CIMF , ET and RARS-T so far, it had not been possible to define significant differences in the clinical outcome based on the MPLW515 mutational status.⁸

Only molecular screening of a larger number of cases of CMPD also mimicking features of RARS-T for MPLW515 mutations will reveal the frequency in these borderline cases. It further remains to be clarified whether JAK2 and MPL mutations can occur in coincidence in RARS-T as it was occasionally reported in CIMF.⁷ The different phenotypes of MPLW515-associated disorders—ET, CIMF and RARS-T—emphasize that these disorders are probably the result of a more complex genetic network that remains to be clarified by further analysis of positive and negative regulators of the JAK–STAT pathway.⁸