Figure 1.
Figure 1. Leukemic involvement and evolution of BCR-ABL1–positive blasts in patient 21. (A) Blast morphology and flow cytometric dot plots of blasts before blinatumomab (first row) and blasts after blinatumomab (second row). Cytomorphology images were acquired using a 63×/1.40 numeric aperture oil objective in a Zeiss Axioplan 2 microscope (Zeiss, Jena, Germany) after Pappenheim's staining (panoptic staining). Blasts at initial diagnosis were positive for CD19, CD10, cyCD79a, CD34, TdT, cyCD22*, HLA-DR*, and cyIgM*; showed aberrant expression of CD13; and were negative for CD117, CD33*, and MPO*. The expression profile did not fulfill World Health Organization criteria for classification as mixed-phenotype leukemia. At relapse after blinatumomab therapy, blasts were negative for CD19, CD10, cyCD79a, CD34, TdT, HLA-DR*, and MPO* and expressed myeloid antigens CD117, CD13, and CD33* (* indicates respective antigen expression not shown in the dot blots). (B) Hypothetical model of clonal evolution and selection of different subclones based on BCR-ABL1 and immunoglobulin heavy chain (IGH) and T-cell receptor β (TRB) gene rearrangement patterns (figure not to scale). Patients with leukemia were screened at initial diagnosis for clonal IG and TR gene rearrangements. Two clonal IGH gene rearrangements (VH3-23-DH2-2-JH6 and VH6-1-DH3-22-JH4) and 1 clonal cross-lineage TRB gene rearrangement (DB2-JB2.7) were detected, and clone-specific real-time quantitative polymerase chain reaction (RT-qPCR) assays were established on the basis of sequence information. RT-qPCR and BCR-ABL1 FISH showed dominance of the IGH R/R, TRB R/G, and BCR-ABL1–rearranged clone (R, rearranged; G, germ line). At first relapse, the IGH R/G TRB G/G clone was dominant, but the second IGH rearrangement was detected at a level of only 0.1% and TRB only at a level below the quantitative range of 0.1%. At second relapse, the leukemic bulk did not show an IG/TR gene rearrangement, but only the BCR-ABL1 translocation RT-qPCR revealed a subclonal IGH gene rearrangement (0.3%). A clonal evolution of the leukemic bulk with occurrence of a new dominant IGH/TRB gene rearrangement was excluded by IGH/TRB multiplex PCR, which has a sensitivity of about 1% to 5%. (C) Subclonal architecture of BCR-ABL1 fusion and monosomy 7 in immunophenotypic compartments of patient 21 at initial diagnosis analyzed by FISH after FACS. Left: FISH results of each compartment. Orange circle, aberrant signal constellation; green circle, normal signal constellation; asterisk (*) indicates being within the range of the FISH and sorting purity cutoff. Right: Representative interphase nuclei showing the 2 different aberrant signal constellations in a false color display using MetaSystems software. FISH images were acquired using a 63×/1.40 numeric aperture oil objective in a Zeiss Axioskop 2 fluorescence microscope (Axioskop 2). The meaning of signals is as follows: isolated red, ABL1; isolated green, BCR; red-green fusion signal, BCR-ABL1 fusion; blue, centromere 7. APC, allophycocyanin; B, mature B cells; CEP7, centromere 7 signal; F, BCR-ABL1 fusion signal; FITC, fluorescein isothiocyanate; leukemia-associated immunophenotype 20− (LAIP 20−), leukemic bulk without CD20 coexpression; LAIP 20+, leukemic bulk with CD20 coexpression; M1, early myeloid compartment; M2, late myeloid compartment; M3, mature myeloid compartment; MPP, multipotent progenitor cells; n.d., not determined; neg., negative (not detected); PE, phycoerythrin; pos, positive; PRO, myeloid and lymphoid progenitors; SCT, stem cell transplantation; T, mature T cells.

Leukemic involvement and evolution of BCR-ABL1–positive blasts in patient 21. (A) Blast morphology and flow cytometric dot plots of blasts before blinatumomab (first row) and blasts after blinatumomab (second row). Cytomorphology images were acquired using a 63×/1.40 numeric aperture oil objective in a Zeiss Axioplan 2 microscope (Zeiss, Jena, Germany) after Pappenheim's staining (panoptic staining). Blasts at initial diagnosis were positive for CD19, CD10, cyCD79a, CD34, TdT, cyCD22*, HLA-DR*, and cyIgM*; showed aberrant expression of CD13; and were negative for CD117, CD33*, and MPO*. The expression profile did not fulfill World Health Organization criteria for classification as mixed-phenotype leukemia. At relapse after blinatumomab therapy, blasts were negative for CD19, CD10, cyCD79a, CD34, TdT, HLA-DR*, and MPO* and expressed myeloid antigens CD117, CD13, and CD33* (* indicates respective antigen expression not shown in the dot blots). (B) Hypothetical model of clonal evolution and selection of different subclones based on BCR-ABL1 and immunoglobulin heavy chain (IGH) and T-cell receptor β (TRB) gene rearrangement patterns (figure not to scale). Patients with leukemia were screened at initial diagnosis for clonal IG and TR gene rearrangements. Two clonal IGH gene rearrangements (VH3-23-DH2-2-JH6 and VH6-1-DH3-22-JH4) and 1 clonal cross-lineage TRB gene rearrangement (DB2-JB2.7) were detected, and clone-specific real-time quantitative polymerase chain reaction (RT-qPCR) assays were established on the basis of sequence information. RT-qPCR and BCR-ABL1 FISH showed dominance of the IGH R/R, TRB R/G, and BCR-ABL1–rearranged clone (R, rearranged; G, germ line). At first relapse, the IGH R/G TRB G/G clone was dominant, but the second IGH rearrangement was detected at a level of only 0.1% and TRB only at a level below the quantitative range of 0.1%. At second relapse, the leukemic bulk did not show an IG/TR gene rearrangement, but only the BCR-ABL1 translocation RT-qPCR revealed a subclonal IGH gene rearrangement (0.3%). A clonal evolution of the leukemic bulk with occurrence of a new dominant IGH/TRB gene rearrangement was excluded by IGH/TRB multiplex PCR, which has a sensitivity of about 1% to 5%. (C) Subclonal architecture of BCR-ABL1 fusion and monosomy 7 in immunophenotypic compartments of patient 21 at initial diagnosis analyzed by FISH after FACS. Left: FISH results of each compartment. Orange circle, aberrant signal constellation; green circle, normal signal constellation; asterisk (*) indicates being within the range of the FISH and sorting purity cutoff. Right: Representative interphase nuclei showing the 2 different aberrant signal constellations in a false color display using MetaSystems software. FISH images were acquired using a 63×/1.40 numeric aperture oil objective in a Zeiss Axioskop 2 fluorescence microscope (Axioskop 2). The meaning of signals is as follows: isolated red, ABL1; isolated green, BCR; red-green fusion signal, BCR-ABL1 fusion; blue, centromere 7. APC, allophycocyanin; B, mature B cells; CEP7, centromere 7 signal; F, BCR-ABL1 fusion signal; FITC, fluorescein isothiocyanate; leukemia-associated immunophenotype 20− (LAIP 20−), leukemic bulk without CD20 coexpression; LAIP 20+, leukemic bulk with CD20 coexpression; M1, early myeloid compartment; M2, late myeloid compartment; M3, mature myeloid compartment; MPP, multipotent progenitor cells; n.d., not determined; neg., negative (not detected); PE, phycoerythrin; pos, positive; PRO, myeloid and lymphoid progenitors; SCT, stem cell transplantation; T, mature T cells.

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