Publication Date:
2019-11-13
Description:
Little is known about the disease-specific factors that predict responsiveness to CAR-T cell therapy, other the direct presence of the CAR-T target. Clinical outcomes at our center have demonstrated that durable responses to CD19-directed CAR-T therapy in pediatric pre-B-ALL (acute lymphoblastic leukemia) are associated with persistence of CAR-T cells in the peripheral blood, antigenic load (percent CD19-positive cells in marrow prior to CAR-T infusion), and apheresis product T-cell quality (Finney O, et al., 2019). However, in a small number of cases where both antigenic load and T-cell quality predicted a good response, the treatment failed rapidly. This led us to undertake a detailed investigation of the leukemia itself in order to discover potential disease-associated factors that correlate with resistance to CAR-T therapy. We employed advanced exomic, and single-cell genomic and epigenomic analysis techniques to define signatures present in four CD19-CAR-T resistant bone marrow biopsy specimens, in comparison to five specimens from CD19-CAR-T responsive disease, from patients enrolled in a phase I clinical trial at Seattle Children's Hospital (PLAT-02, NCT02028455). Current cytogenic approaches to identify high risks markers (Ph+, Ph-like, MLL) were not informative as to CAR-T susceptibility, as high risk leukemias were CAR-T responsive; while a CAR-T resistant leukemia contained a marker (ETV6-RUNX1 fusion) previously associated with a good prognosis. Thus, we performed bulk whole-exome sequencing and RNAseq, single cell (sc) RNAseq, sc B-cell receptor (BCR)-seq, methylation array, H3K27ac ChIPseq, and ATACseq on these marrow samples. Initial genomic analysis revealed a total of 5 previously described hotspot mutations in ABL1, IKZF1, EP300, and 2 in KRAS. RNAseq analyses identified actionable fusions for ABL1, ETV6, ETV5, and KMT2A. Interestingly, a therapy-sensitive leukemia harbored a KMT2A-AFF1 fusion that was shown to predispose patients to leukemic plasticity and lineage switching when treated with blinatumomab. Importantly, we identified CREBBP-fusions in leukemias that failed to achieve CD19-CAR-T cell induced B cell aplasia. CREBBP perturbations have previously been associated with relapsed and refractory ALL, but not with resistance to CAR-T therapy. Single cell RNAseq and scBCRseq data are being analyzed for the existence of mixed lineage and gene expression-based heterogeneity that may predict clonal selection under CAR-T pressure. RNASeq analysis identified upregulation of JUN and JUND transcripts in CAR-T resistant disease, a finding which is complemented by the hypermethylation of JUND in CAR-T sensitive disease. Similarly, ATACseq and methylation data is being analyzed for lineage specification in CAR-T resistant leukemia. In comparing dysfunctional to functional CAR-T responders by ATACseq, 〉 10,000 unique open chromatin regions were identified in dysfunctional responders, as opposed to
Print ISSN:
0006-4971
Electronic ISSN:
1528-0020
Topics:
Biology
,
Medicine
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