CALR-mutated essential thrombocythemia evolving to chronic myeloid leukemia with coexistent CALR mutation and BCR-ABL translocation

To the editor:

The understanding of the genetic basis of the myeloproliferative neoplasms has increased tremendously in the last few years.¹  In essential thrombocythemia (ET), 50% to 65% of the patients carry the JAK2 V617F mutation, whereas only ∼5% of patients carry MPL exon 10 mutations. More recently, CALR exon 9 mutations were identified in ∼20% to 25% of ET patients.^2,3 CALR mutations have been found almost exclusively in ET and primary myelofibrosis, suggesting that CALR mutations primarily affect the biology of megakaryocytes. Interestingly, CALR-mutant ET seems to be a distinctive entity within myeloid neoplasms with distinguishing clinical features. It affects relatively young individuals and is characterized by markedly elevated platelet count but relatively low thrombotic risk and no progression to polycythemia vera.⁴  Here we report a case of BCR-ABL⁺ chronic myeloid leukemia (CML) presenting 3 years after the diagnosis of a JAK2^–MPL^– ET, both lesions carrying the same CALR mutation. The case occurred in a 26-year-old man who was noted in 2011 to have new-onset thrombocytosis (platelets [PLTs], 1159 × 10⁹/L) with normal hemoglobin and white blood cell (WBC) count. The bone marrow (BM) biopsy was normocellular with a normal myeloid:erythroid ratio but an increased amount of megakaryocytes without fibrosis. The megakaryocytes showed a predominance of hyperlobated “staghorn” forms often present in loose clusters (Figure 1A-C). Molecular studies at the time of diagnosis showed wild-type JAK2 and MPL genes. A diagnosis of JAK2^–MPL^– ET was rendered. The patient was treated with interferon 135 μg once per week and then with a 12-day interval with good response (Figure 1G).

In May 2014, the patient was noted to have new-onset leukocytosis (WBC, 14.8 × 10⁹/L) with normal PLT count. Three months later, the patient’s WBC count increased to 25.7 × 10⁹/L. Molecular studies revealed a BCR-ABL (e14a2) fusion transcript in peripheral blood (PB). A BM biopsy revealed hypercellularity with an elevated myeloid:erythroid ratio. Megakaryocytes were predominantly small and hypolobated (Figure 1D-E). Immunohistochemical analysis with CD61 highlighted the increased number of dwarf megakaryocytes (Figure 1F). Fluorescent in situ hybridization analysis using a BCR-ABL dual color, dual fusion translocation probe (Zytolight, Zytomed) confirmed a BCR-ABL fusion in 99 of 100 cells, indicative of the t(9:22)(q34;q11.2) translocation (Figure 1D, insert). Retrospectively, molecular studies and fluorescent in situ hybridization analysis for the BCR-ABL fusion transcript were performed in the 2011 BM biopsy, both of which rendered negative results. Fragment length analysis with subsequent Sanger sequencing of CALR exon 9 gene demonstrated an identical 5-bp insertion (mutation type 2; c.1154_1155insTTGTC, p.K385fs*47) in both biopsies (Figure 1H-I). Next-generation sequencing performed in purified PB granulocytes revealed a mutant CALR allele burden of 44%, which is in agreement with the heterozygous mutation found with fragment length analysis. To rule out the possibility of a CALR germ line mutation, a hair shaft probe was analyzed that revealed a CALR wild-type sequence confirming the presence of a somatic mutation in the hematopoietic cells.^5,6  The interferon therapy was switched to the tyrosine kinase inhibitor nilotinib (150 mg). After 3 months, the patient has had a good hematologic response but has not achieved complete molecular response (BCR-ABL International Standard 3.23), whereas PLT counts have steadily increased, and no change in the CALR allele burden has been observed (Figure 1G).

This is the first description of a CALR exon 9 mutated ET with characteristic clinical findings (young adult with very high PTL count at presentation) that acquired a t(9;22)(q34;q11.2) translocation and changed the morphology from ET to CML. The high allele burden of mutant CALR concurrent with BCR-ABL translocation in almost 100% of the BM cells is a strong argument in favor of a common clone that harbors both genetic alterations. Our findings suggest that a subclone of the preexisting CALR-mutated heterozygous clone acquired a BCR-ABL translocation, conferring an additional growth advantage to double-mutant progenitors and shifted the morphology from ET to CML. Treatment with nilotinib is causing disappearance of the double-mutant clone and favoring re-emergence of the CALR-mutant–only clone manifested by the constant allele burden of mutant CALR with increased PLTs in PB, returning to the original phenotype. Similar findings have been described in cases with simultaneous BCR-ABL translocation and JAK2 V617F mutations.^7-9  Our case highlights how the clinical and morphologic appearance of myeloproliferative neoplasms is governed by their mutational profile.