PMC10036931_01
Bone marrow specimens. Wright-Giemsa staining of the bone marrow shows an increased number of blast cells (panel A, red arrow), multinucleated erythrocytes (panel B, red arrow), small binucleated megakaryocytes (panel C, red arrow), and basophils (panel D, red arrow) (magnification x 1000). Hematoxylin and eosin staining of a bone marrow biopsy specimen (panel E) shows hypercellularity for the patient's age (80% cells) and a reduced myeloid:erythroid ratio (1:1), with a markedly increased number of megakaryocytes. CD34 (panel F) and CD117 (panel G) immunostaining of the biopsy specimen shows frequent blast cells. CD42b immunostaining shows megakaryocytic hyperplasia, including hypolobated megakaryocytes with separated nuclei forms (panel H) (magnification x 40). Cytogenetics revealed t (5;19) in the bone marrow (panel I, red arrow). Metaphase fluorescence in situ hybridization analysis was negative for PDGFRB (5q32) gene rearrangements in bone marrow cells (panel J; break apart FISH with red-3'PDGFRB, green-5'PDGFRB, and yellow fusion signals). RNA-seq assay showing the deletion in the q arm of chromosome 5 (panel K). Based on the hg19 assembly, the deletion started at position Chr5:149,505,140 in exon 12 of PDGFRB and ended at Chr5:150,422,101 in exon 11 of TNIP1. Gel electrophoresis showing the amplified 540 bp TNIP1::PDGFRB cDNA fragments. Partial sequence chromatograms of the cDNA amplified fragment showing the junction positions of exon 11 of TNIP1 with exon 12 of PDGFRB.
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The bone marrow aspirate demonstrated hypercellularity with 13.5% blasts, marked dysplasia in granulocytes, erythrocytes, and megakaryocytes (Fig. 1, panel A, B, C, D). ASXL1 p.g649 * (variant allele frequency 40%), U2AF1 S34F (42.8%), ETV6 R430I (40%). A karyotypic abnormality, 46, XY, t (5; 19) (q33; p13.2), was detected in 15/20 metaphases (Fig. 1, panel I). However, the PDGFRB break-apart probe did not reveal a PDGFRB rearrangement in metaphase cells (Fig. 1, panel J). The fusion was generated by an 916 kb interstitial deletion in 5q32~33 segment, which retained the catalytic domain (exons 13-21) of PDGFRbeta protein (Fig. 1, panel K).
Platelet and molecular responses. Peripheral blood platelet response to therapy over 16-week duration (panel A). Gel electrophoresis showing the amplified TNIP1::PDGFRB cDNA in bone marrow samples before and 14 weeks after imatinib treatment (panel B). Relative expressions of TNIP1::PDGFRB mRNA before and after treatment indicated a progressive decrease in fusion transcripts. Two-tailed unpaired student t-test was performed between two times. **** P < 0.0001 (panel C). AZA, azacytidine; DEC, decitabine; Mock, water (negative control).
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After six doses of decitabine, the patient achieved complete remission with partial hematologic recovery (WBC 2.46 x 109/L, ANC 1.16 x 109/L, HGB 89 g/L, and PLT 103 x 109/L; Fig. 2, panel A) and complete cytogenetic remission with normal karyotype in 20 metaphases. TNIP1::PDGFRB mRNA was barely detectable (Fig. 2, panel B, C). The allelic burden of pathogenic mutations was also significantly lower than before treatment (ASXL1 G649 *, 8.1%; U2AF1 S34F, 8.9%; ETV6 R430I, 1.1%).