Abstract LB-056: An integrated genomic, epigenetic, proteomic, and single cell analysis of pediatric B cell acute lymphoblastic leukemia to elucidate resistance mechanisms to CD19 CAR T cell therapy
Authors: Masih Katherine E. , Gardner Rebecca , Gryder Berkley E. , Lack Justin , Stanton Benjamin Z. , Wilson Ashley , Finney Olivia , Sindiri Sivasish , Song Young , Rae Zachary , Kelly Michael , Wang Chaoyu , Wen Xinyu , Cheuk Adam , Wei Jun S. , Jensen Michael , Orentas Rimas , Khan Javed 
Published: August 2019

Background

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer with a peak incidence at 3-5 years of age. Despite the improved survival rate of 90% for newly diagnosed children with ALL, the outcome for patients with relapsed disease is poor with a less than 30% overall survival. CD19 CAR T cell therapy has shown impressive response rates in relapsed/refractory disease. However, long-term survival analysis has shown that despite initial response rates exceeding 80%, durable response rates at one year are closer to 40%. Currently, little is known about molecular factors predicting durable response to CAR T therapy. We hypothesized that patients with CD19 CAR T therapy resistant ALL have a molecularly distinct disease compared to patients who respond to therapy, which can be identified in pre-treatment leukemia samples. Utilizing advanced genomic, epigenetic, proteomic, and single-cell techniques, we characterized the bone marrow of patients that were resistant or sensitive to therapy to identify mechanisms of resistance.

Methods

Patients enrolled in a phase I clinical trial at Seattle Children’s Hospital (PLAT-02) were categorized according to the durability of their response to CD19 CAR T therapy. Bone marrow aspirates from patients with leukemias resistant to therapy (4 pre-treatment with 2 paired post-treatment) were analyzed and compared to patients with therapy sensitive leukemias (5 pre-treatment). We performed bulk whole-exome sequencing and RNA-seq, single cell (sc) RNA-seq, scB cell receptor (BCR)-seq, methylation array, H3K27ac ChIP-seq, and ATAC-seq.

Results

Initial genomic analysis revealed a total of 5 previously reported recurrent hotspot mutations in ABL1, 2 x KRAS (Q61H), IKZF1, and EP300. RNA-seq analyses identified actionable fusions in 2 x ABL1, 2 x ETV6, 2 x ETV5, and 1x KMT2A with variable partners. Interestingly, a therapy-sensitive leukemia harbored a KMT2A-AFF1fusion that was previously shown to predispose patients treated with blinatumomab to leukemic plasticity and lineage switching. Additionally, we identified in-frame CREBBP-fusions in all leukemias that failed to achieve CD19 CAR T cell induced B cell aplasia. CREBBP perturbations have previously been associated with relapsed and refractory ALL. Integrated gene expression and epigenetic analyses identified several pathways associated with resistant disease. ATAC-seq and methylation data are being analyzed for lineage specification. Similarly, scRNA- and scBCR-seq data are being analyzed for the existence of mixed lineage and gene expression-based heterogeneity that may predict clonal selection under CAR T pressure.

Conclusions

This study establishes one of the most comprehensive approaches to genomic profiling for leukemia patient samples. Although our analysis is preliminary and sample number is small, in-depth analyses are highlighting crucial differences in leukemia that will allow improved prediction of responsiveness to CAR T therapy.

Citation Format: Katherine E. Masih, Rebecca Gardner, Berkley E. Gryder, Justin Lack, Benjamin Z. Stanton, Ashley Wilson, Olivia Finney, Sivasish Sindiri, Young Song, Zachary Rae, Michael Kelly, Chaoyu Wang, Xinyu Wen, Adam Cheuk, Jun S. Wei, Michael Jensen, Rimas Orentas, Javed Khan. An integrated genomic, epigenetic, proteomic, and single cell analysis of pediatric B cell acute lymphoblastic leukemia to elucidate resistance mechanisms to CD19 CAR T cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-056.