Hematopoietic stem cell involvement in BCR-ABL1- positive ALL as a potential mechanism of resistance to blinatumomab therapy

Abstract

The bispecific T-cell engager blinatumomab targeting CD19 can induce complete remission in relapsed or refractory B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, some patients ultimately relapse with loss of CD19 antigen on leukemic cells, which has been established as a novel mechanism to escape CD19-specific immunotherapies. Here, we provide evidence that CD19-negative (CD19^-) relapse after CD19-directed therapy in BCP-ALL may be a result of the selection of preexisting CD19^- malignant progenitor cells. We present 2 BCR-ABL1 fusion-positive BCP-ALL patients with CD19^- myeloid lineage relapse after blinatumomab therapy and show BCR-ABL1 positivity in their hematopoietic stem cell (HSC)/progenitor/myeloid compartments at initial diagnosis by fluorescence in situ hybridization after cell sorting. By using the same approach with 25 additional diagnostic samples from patients with BCR-ABL1-positive BCP-ALL, we identified HSC involvement in 40% of the patients. Patients (6 of 8) with major BCR-ABL1 transcript encoding P210^BCR-ABL1 mainly showed HSC involvement, whereas in most of the patients (9 of 12) with minor BCR-ABL1 transcript encoding P190^BCR-ABL1, only the CD19⁺ leukemia compartments were BCR-ABL1 positive (P = .02). Our data are of clinical importance, because they indicate that both CD19⁺ cells and CD19^- precursors should be targeted to avoid CD19^- relapses in patients with BCR-ABL1-positive ALL.

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.

Figure 2.

Analysis of relevant immunophenotypic compartments in 27 adult patients with BCR-ABL1–positive BCP-ALL using FISH after FACS. (A) BCR-ABL1 positivity of immunophenotypic compartments in the 2 predominant patterns of BCR-ABL1 occurrence. The green and orange color content of the boxes represents the ratios of BCR-ABL1 fusion–positive and –negative signal constellations observed in the 27 patients. For details of sorting strategies, see supplemental Data. (B) Detailed results of the analysis for each patient and compartment. Green, BCR-ABL1 fusion negative; orange, BCR-ABL1 positive; gray, not analyzable. Overall, 11 of 27 patients showed an MPP pattern, 13 of 27 showed a B-lineage pattern, and 3 of 27 had a pattern between the two (indeterminable [i.d.]). M, major BCR-ABL1 transcript; m, minor BCR-ABL1 transcript; mM, minor and major BCR-ABL1 transcripts identified; n.a., not analyzable.