PMC9871410_01
(A) Spleen size, hematological, cytogenetic, and molecular data acquired during the follow-up of this patient with myeloproliferative neoplasm with coexisting BCR-ABL1 fusion gene, CALR, and TET2 mutations. BCR-ABL1 transcript levels are shown as the ratio of BCR-ABL1 to ABL1, expressed as a percentage on the international scale (BCR-ABL1-IS). The CALR and TET2 mutations were detected by next-generation sequencing and confirmed by Sanger sequencing. CALR mutation monitoring was performed using polymerase chain reaction followed by capillary electrophoresis. The variant allele frequency (VAF) of the CALR mutation was calculated by dividing the mutant peak area by the sum of the mutant and wild-type peak areas. TET2 mutation monitoring was performed using next-generation sequencing. (B) Bone marrow aspirate smears of this patient at the first diagnosis of CML and 3 months after treatment. The bone marrow aspirate smear at the first diagnosis of CML showed expansion of the neutrophil lineage and increased basophiles. (C) Chromosome karyotypes of this patient at the first diagnosis of CML and 3 months after treatment. At the first diagnosis of CML, the patient had the characteristic t(9;22)(q34;q11) reciprocal translocation which resulted in Ph chromosome, while the cytogenetic analysis revealed the normal karyotype after 3 months of treatment. (D) Fluorescence in situ hybridization (FISH) with dual-color and dual-fusion translocation probes for ABL1 (red) and BCR (green) of this patient at the first diagnosis of CML and 3 months after treatment. BCR-ABL1-positive cells at the first diagnosis of CML showed two fusion (red/green, yellow), one red and one green signals (2F1R1G), while the normal cells showed two red and two green signals (2R2G) after 3 months of treatment. (E) Sanger sequencing of CALR confirmed a frameshift mutation (c.1103_1136del, p.K368fs*51). Fragment length analysis by capillary electrophoresis showed concurrent amplification of the wild-type allele and a mutant allele. Sanger sequencing of TET2 confirmed a nonsense mutation (c.3748G > T, p.E1259Ter). (F) Clonal evolution from the first diagnosis of CML to the last follow-up. Mutant burdens were estimated according to the percentage of BCR-ABL1-positive cells by FISH and the VAF of CALR/TET2 mutations. Fish plot showed that the CALR-mutant, TET2-mutant, and BCR-ABL1-positive subclone disappeared immediately after nilotinib treatment, while the CALR-mutant, TET2-mutant, BCR-ABL1-negative subclone and the initial dominant CALR-mutant, TET2-wild, BCR-ABL1-negative clone persisted even after treatment with a combination of nilotinib and ruxolitinib. (G) Schematic diagram of speculative clonal architecture and the order of alteration acquisition in this MPN patient. WBC, white blood cell; Hb, hemoglobin; PLT, platelet; *, below the costal margin. December 14, first diagnosis of CML; March 15, 3 months of nilotinib treatment; December 16, diagnosis of primary myelofibrosis; December 18, 18 months of combination therapy with nilotinib and ruxolitinib
CCR3-11-e6801-g001.jpg
PMC9871410_01_CCR3-11-e6801-g001.jpg
ccr36801-fig-0001
Although splenomegaly and anemia persisted (6 cm below the costal margin and Hb: 87 g/L after 1 year of nilotinib treatment), the patient achieved a complete cytogenetic response (CCyR) after 3 months of nilotinib treatment and achieved a major molecular response 12 months later (Figure 1A-D). Molecular studies detected a 34-bp deletion in CALR exon 9 (c.1103-1136del, K368fs*51; Figure 1E), which was also observed in a previous study, with a variant allele frequency (VAF) of 51.3% in the absence of JAK2 V617F and MPL mutations. Retrospective analysis of historical samples showed that the CALR mutation had been present since the initial diagnosis of CML with a stable allele burden regardless of the decrease in BCR-ABL1 IS (Figure 1A). E1250Ter; Figure 1E) was detected in all historical samples analyzed, and the VAFs were slightly lower than that of the CALR mutation (Figure 1A). Within 18 months of combination therapy, the VAF of CALR mutation fluctuated from 45.7% to 46.5%, and BCR-ABL1 IS decreased to 0.01% (Figure 1A).