A 55-year-old man was found out to have leukocytosis on his health check-up and was referred to Saiseikai Central Hospital in July 2013. At the onset, he had systemic symptoms such as flushing and night sweats. The findings of physical examination were unremarkable, and his spleen was not palpable. The complete blood count revealed a white blood cell (WBC) count of 26.7 x 109/L with 85.5% neutrophils, 1.0% metamyelocytes and myelocytes, 12.0% lymphocytes and 1.5% eosinophils, along with a hemoglobin of 12.7 g/dl and a platelet count of 216 x 109/L. Neutrophils were characterized by toxic granulation and Dohle bodies. Bone marrow aspiration showed maturing granulopoiesis without an increase in blasts (Fig. 1A). Karyotype analysis showed no clonal abnormalities. The results of blood chemistry were all within normal limits. The serum level of granulocyte-colony stimulating factor was also within normal range (< 19.5 pg/mL). 18F-fluorodeoxy-glucose positron emission tomography/computed tomography demonstrated splenomegaly (19 cm in length) and diffuse 18F-fluorodeoxy-glucose uptake in the spleen and bone marrow (maximum standardized uptake value: 7.67 and 13.2, respectively) was observed.
The patient was tentatively diagnosed as myeloproliferative neoplasm, unclassifiable and was followed conservatively; however, both WBC and platelet counts continued to rise, together with progressing anemia. In March 2014, nine months after the initial exam, the spleen showed a remarkable increase in size (to 24 cm in length), and the complete blood count showed a WBC of 40.5 x 109/L with 87% neutrophils but no blasts, together with a hemoglobin of 8.5 g/dl, and a platelet count of 114 x 109/L. Genomic sequencing for CSF3R gene from exons 14 and 17 confirmed the presence of heterozygous CSF3R-T618I. He was diagnosed as having CNL, and was placed on hydroxyurea.
In November 2014, he started complaining of splenic pain and early satiety due to the continuing enlargement of his spleen (now 27 cm in length). Splenic irradiation also failed to reduce the spleen size; total dose given was 10 Gy. On follow-up 1 year after initiation of hydroxyurea therapy, in March 2015, results of bone marrow biopsy showed slight reticulin fibrosis and secondary myelofibrosis was diagnosed. He was placed on ruxolitinib at the dose of 5 mg twice daily, which subsequently increased to 20 mg twice daily, and hydroxyurea was discontinued. Ruxolitinib was well tolerated, and all clinical symptoms including cachexia were improved. WBC counts were stabilized within 1 month; however, these beneficial responses were not durable.
One year later, in March 2016, the WBC count showed another rapid increase, to 214.5 x 109/L, and this time 21% of WBCs were small to medium sized blasts bone marrow aspiration showed hypercellular marrow with increased blasts complicated with multilineage dysplasia. Blasts possessed a blocky chromatin pattern but negative for myeloperoxidase activity (Fig. 1B). Some myeloid precursors showed hypogranulation. Erythroid cells were markedly reduced in number. Megakaryocytes were adequate in number, but they were dysplastic, with micromegakaryocytes and small megakaryocytes. The immunophenotype was positive for CD13, CD33, CD34, CD117, HLA-DR. and CD7. Genetic analysis revealed no FLT3/ITD mutations. Cytogenetic examination revealed a t(4;12)(q12;p13) translocation in 19 out of 20 metaphases. Thus, the diagnosis of AML with t(4;12)(q12;p13) was made. Fluorescent in situ hybridization (FISH) analysis using break-apart probes for the 4q12 region on cultured peripheral blood cells confirmed CHIC2 or PDGFRA gene rearrangement (presumably translocated to derivative chromosome 12) in 58% of nuclei (Fig. 1C).
The patient was placed on chemotherapy with cytarabine and daunorubicin. Fourteen days after the induction therapy, the peripheral blood contained as much as 60% blasts. FISH for the 4q12 split signal also showed an increase, up to 85%. Bone marrow biopsy showed a reticulin fibrosis (grade 2) without hematopoiesis. The patient underwent two cycles of azacytidine with reduction in the peripheral blood blasts to 7%. In July 2016, the patient underwent haploidentical hematopoietic cell transplantation after conditioning with fludarabine and total body irradiation (12 Gy). Although he achieved full donor chimerism, he died of hepatic veno-occlusive disease 51 days after the transplantation. The autopsy showed hypocellular marrow without blasts and moderate myelofibrosis; both the liver and spleen were free of leukemic cell infiltration.
To assess the sequential molecular events, we studied the mutations of CSF3R, SETBP1, ASXL1, SRSF2 and CBL using genomic sequencing with the stored samples from the peripheral blood both in CNL (March 2014) and AML (April 2016) phases. At the time of molecular studies in CNL, the patient did not receive any therapy. The patient signed informed consent for sample collection in accordance with the Declaration of Helsinki. The samples were polymerase-chain-reaction amplified using the primers described previously and subject to mutational analysis by Sanger sequencing on an Applied Biosystems 3730xl DNA Analyzer with an Applied Biosystems BigDye Terminator v3.1 Cycle Sequencing Kit. In the chronic phase, the evaluation of mutational status identified heterozygous CSF3R-T315I as was noted previously. This heterozygous mutation remained the same at the AML-transformation phase. Additional SETBP1-gene mutation (I871T) with variant allele frequency of 69% was found at AML transformation, after being negative in the chronic phase. Hence, both t(4;12)(q12;p13) translocation as well as SETBP1-gene mutation were observed during the leukemic transformation.