Insights from response to tyrosine kinase inhibitor therapy in a rare myeloproliferative neoplasm with CALR mutation and BCR-ABL1

To the editor:

Calreticulin (CALR) mutations have been reported primarily in the context of JAK2 and MPL wild-type essential thrombocythemia and primary myelofibrosis.^1-5 CALR mutations are exceedingly rare in the setting of t(9;22)/BCR-ABL1,^4,5  with only a single report in the literature describing a case of an atypical myeloproliferative neoplasm (MPN) in which CALR mutation preceded BCR/ABL1 fusion.⁶ 

Here, we describe a patient with CALR mutation seen in the context of an MPN with the Philadelphia chromosome. The patient was a 67-year-old man found to be hypertensive on routine physical examination. His workup revealed an elevated white blood cell (WBC) count of 25 × 10⁹/L (normal, 4 × 10⁹/L) that prompted bone marrow (BM) aspiration and a biopsy at the referring institution, which was interpreted as suggestive of the patient having an MPN. Conventional cytogenetics and real-time reverse transcriptase polymerase chain reaction (RT-PCR) performed at the referring institution were positive for t(9;22) and BCR-ABL1 fusion, respectively. The patient was treated with dasatinib as frontline therapy and was referred to our institution for further evaluation and management. A complete blood count revealed a WBC count of 39.9 × 10⁹/L (Figure 1A), hemoglobin of 14.1 g/dL, and platelet count of 150 × 10⁹/L. A BM aspiration and biopsy revealed a hypercellular (80%-90%) BM with left-shifted granulocytic hyperplasia and mild megakaryocytic hyperplasia, including a mixture of small and large forms and some with hyperchromatic nuclei (Figure 1E-F). No megakaryocytes with classical “dwarf”⁷  morphology were identified. Moderate reticulin fibrosis was present and characterized by a diffuse and dense increase in reticulin fibers forming extensive intersections.⁸  Conventional cytogenetics revealed a Philadelphia chromosome–positive clone, 46,XY,t(9;22)(q34;q11.2)[14], and an additional Philadelphia chromosome–negative clone with a complex karyotype, 46,XY,der(2)t(2;5)(p21;q15),der(5)t(2;5) del(2)(p22p24),add(7)(q22),der(12)t(7;12)(q22;p11.2)[6]. Fluorescence in situ hybridization studies showed BCR/ABL1 fusion signals in 78% of the interphases analyzed. RT-PCR performed on peripheral blood showed a BCR/ABL1 to ABL1 ratio of >100% (Figure 1B-C). A diagnosis of chronic-phase chronic myeloid leukemia was rendered. The patient was continued on dasatinib therapy. His WBC count and BCR/ABL1 fusion transcript continued to decrease (Figure 1A-B). Follow-up BM aspiration and biopsy performed 7 months later showed strikingly distinctive features compared with the initial BM biopsy specimen, characterized by marked atypical megakaryocytic hyperplasia including many large forms with hyperchromatic and hyperlobulated nuclei and frequent clusters with moderate reticulin fibrosis,⁸  and mild osteosclerosis and no substantial granulocytic hyperplasia, in keeping with PMF (Figure 1G-H). RT-PCR was positive for BCR-ABL1 fusion transcripts (decreased ratio of 0.21) and conventional cytogenetics showed a Philadelphia chromosome–negative clone with a complex karyotype identical to the previous sample. The findings prompted additional mutational workup including JAK2 V617 analysis, which was wild-type, and CALR analysis, which revealed a type 1 mutation (52 bp deletion)⁵  with a mutant allele frequency of 68% (Figure 1B). We retrospectively analyzed the peripheral blood sample from the time the patient initially presented; we detected a CALR mutation with a mutant allele frequency of 51% (Figure 1B,D).

Our findings and the genetic trends observed in this patient (Figure 1A-B) further confirm the observations made by Cabagnols and colleagues,⁶  indicating that although the Philadelphia-positive clone is sensitive to dasatinib, the CALR-mutant clone persists throughout the course of therapy. This case is of interest because it shows that the BM morphologic features were dominated by the presence of BCR/ABL1 fusion in the initial sample, despite the fact that the CALR mutation was also present. The CALR mutation morphologically declared itself subsequent to tyrosine-kinase inhibitor therapy, as shown by the emergence of markedly atypical megakaryocytic hyperplasia, supporting the diagnosis of PMF. Of note, the patient never showed thrombocytosis, before or after dasatinib therapy, potentially because of the presence of moderate myelofibrosis at the time of presentation. Notably, the patient reported by Cabagnols and colleagues⁶  also had moderate myelofibrosis at the time of diagnosis.

Given that the CALR mutant allele burden was 51% and fluorescence in situ hybridization studies showed BCR/ABL1 fusion signals in 78% of interphases analyzed in the initial sample, it is reasonable to assume that a BCR/ABL1-positive subclone arose from a dominant clone, with a heterozygous CALR mutation rendering the BCR/ABL1 fusion a secondary genetic event in this case. This observation is similar to that described earlier by Cabagnols et al.⁶ 

This study emphasizes the importance of BM morphology and expanded molecular testing in the workup of patients with an MPN.