Edward Ayoub, V. Mohanty, Yuki Nishida, Tallie Patsilevas, Mahesh Basyal, Russell Pourebrahim, M. Muftuoglu, Ken Chen, G. Issa, M. Andreeff
{"title":"Abstract A41: Single Cell Multiomic Analysis Reveals Association of TP53-mut Loss of Heterozygosity with Primitive Phenotype in Acute Myeloid Leukemia","authors":"Edward Ayoub, V. Mohanty, Yuki Nishida, Tallie Patsilevas, Mahesh Basyal, Russell Pourebrahim, M. Muftuoglu, Ken Chen, G. Issa, M. Andreeff","doi":"10.1158/2643-3249.aml23-a41","DOIUrl":null,"url":null,"abstract":"\n TP53 mutations in acute myeloid leukemia (AML) are associated with copy number abnormalities (CNA), structural variants and high risk of relapse (Döhner et al., 2017; Giacomelli et al., 2018; Bernard et al. 2020). In spite of relatively high remission rates obtained by targeted therapies, TP53 mutant (TP53-mut) clones persist, invariably resulting in relapse (Short et al., 2021; Takahashi et al., 2016). Delineating the clonal architecture and the immunophenotypes of TP53-mut clones during AML therapy may provide a better understanding of the role of TP53-mutations in AML biology. Recent progress in sequencing technologies allows the integration of genotyping and phenotyping at the single cell level. Here, we took advantage of MissionBio Tapestri’s newest platform: single cell DNA + protein for simultaneous genotyping and phenotyping with 45 surface oligo-conjugated antibodies in 10 paired samples from 5 patients with TP53-mut AML before and after therapy. Samples with at least 70% viability were stained with the TotalSeq™-D Human Heme Oncology Cocktail, V1.0. Following surface marker staining, single-cell suspension, encapsulation, and barcoding were performed according to manufacturer’s instruction. For scDNA library preparation, we utilized a validated custom panel (Morita et al. 2020) consisting of 279 amplicons covering recurrent mutations in 37 genes in AML. We sequenced a total of 44,550 cells from 10 samples. We confirmed mutations reported by MD Anderson molecular diagnostic laboratory in the genes covered by the scDNA custom panel. The clonal architecture analysis distinguished between TP53-mut clones with or without loss of heterozygosity (LOH) of the normal TP53 allele. Our data show a primitive immunophenotype in TP53-mut with LOH (LOH+) clones in comparison to TP53-mut LOH- clones. We see 2.4 LOG2FC increase in CD34 and 1.8 LOG2FC increase in CD117 (p<0.001) in TP53-mut LOH+ clones in comparison to TP53-mut LOH- clones. Clonal evolution analysis shows that TP53-mut LOH+ clones are significantly more resistant to therapy than TP53-mut LOH-, consistent with previous publications. On the other hand, TP53-mut LOH- clones showed significantly higher levels of CD2, CD16, CD5, CD3, and CD8 among other lineage markers (LOG2FC= 1.1, 1.2, 1.4, 1.1, and 1.1 respectively; p.value <0.0001) compared to TP53-mut LOH+. This data indicates that TP53-mut LOH- cells can express lymphoid phenotypic markers. Single cell cytokine analysis (IsoPlexis) reveals profound lack of secreted cytokines in T-cells from TP53-mut AML. Further data from bulk-RNA sequencing, ddPCR, and CyTOF will be presented that validates a lymphoid phenotype of TP53-mut LOH-. In summary, we utilize a scDNA+protein multiomic approach to dissect clonal architecture and provide a link between genotype-phenotype in TP53-mut AML. We show that while TP53-mut LOH+ clones are exclusively primitive, TP53-mut LOH- clones retain the capacity to exist outside primitive immunophenotype and might lack differentiation block.\n Citation Format: Edward Ayoub, Vakul Mohanty, Yuki Nishida, Tallie Patsilevas, Mahesh Basyal, Russell Pourebrahim, Muharrem Muftuoglu, Ken Chen, Ghayas C. Issa, Michael Andreeff. Single Cell Multiomic Analysis Reveals Association of TP53-mut Loss of Heterozygosity with Primitive Phenotype in Acute Myeloid Leukemia [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A41.","PeriodicalId":29944,"journal":{"name":"Blood Cancer Discovery","volume":null,"pages":null},"PeriodicalIF":11.5000,"publicationDate":"2023-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Blood Cancer Discovery","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1158/2643-3249.aml23-a41","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"HEMATOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
TP53 mutations in acute myeloid leukemia (AML) are associated with copy number abnormalities (CNA), structural variants and high risk of relapse (Döhner et al., 2017; Giacomelli et al., 2018; Bernard et al. 2020). In spite of relatively high remission rates obtained by targeted therapies, TP53 mutant (TP53-mut) clones persist, invariably resulting in relapse (Short et al., 2021; Takahashi et al., 2016). Delineating the clonal architecture and the immunophenotypes of TP53-mut clones during AML therapy may provide a better understanding of the role of TP53-mutations in AML biology. Recent progress in sequencing technologies allows the integration of genotyping and phenotyping at the single cell level. Here, we took advantage of MissionBio Tapestri’s newest platform: single cell DNA + protein for simultaneous genotyping and phenotyping with 45 surface oligo-conjugated antibodies in 10 paired samples from 5 patients with TP53-mut AML before and after therapy. Samples with at least 70% viability were stained with the TotalSeq™-D Human Heme Oncology Cocktail, V1.0. Following surface marker staining, single-cell suspension, encapsulation, and barcoding were performed according to manufacturer’s instruction. For scDNA library preparation, we utilized a validated custom panel (Morita et al. 2020) consisting of 279 amplicons covering recurrent mutations in 37 genes in AML. We sequenced a total of 44,550 cells from 10 samples. We confirmed mutations reported by MD Anderson molecular diagnostic laboratory in the genes covered by the scDNA custom panel. The clonal architecture analysis distinguished between TP53-mut clones with or without loss of heterozygosity (LOH) of the normal TP53 allele. Our data show a primitive immunophenotype in TP53-mut with LOH (LOH+) clones in comparison to TP53-mut LOH- clones. We see 2.4 LOG2FC increase in CD34 and 1.8 LOG2FC increase in CD117 (p<0.001) in TP53-mut LOH+ clones in comparison to TP53-mut LOH- clones. Clonal evolution analysis shows that TP53-mut LOH+ clones are significantly more resistant to therapy than TP53-mut LOH-, consistent with previous publications. On the other hand, TP53-mut LOH- clones showed significantly higher levels of CD2, CD16, CD5, CD3, and CD8 among other lineage markers (LOG2FC= 1.1, 1.2, 1.4, 1.1, and 1.1 respectively; p.value <0.0001) compared to TP53-mut LOH+. This data indicates that TP53-mut LOH- cells can express lymphoid phenotypic markers. Single cell cytokine analysis (IsoPlexis) reveals profound lack of secreted cytokines in T-cells from TP53-mut AML. Further data from bulk-RNA sequencing, ddPCR, and CyTOF will be presented that validates a lymphoid phenotype of TP53-mut LOH-. In summary, we utilize a scDNA+protein multiomic approach to dissect clonal architecture and provide a link between genotype-phenotype in TP53-mut AML. We show that while TP53-mut LOH+ clones are exclusively primitive, TP53-mut LOH- clones retain the capacity to exist outside primitive immunophenotype and might lack differentiation block.
Citation Format: Edward Ayoub, Vakul Mohanty, Yuki Nishida, Tallie Patsilevas, Mahesh Basyal, Russell Pourebrahim, Muharrem Muftuoglu, Ken Chen, Ghayas C. Issa, Michael Andreeff. Single Cell Multiomic Analysis Reveals Association of TP53-mut Loss of Heterozygosity with Primitive Phenotype in Acute Myeloid Leukemia [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A41.
期刊介绍:
The journal Blood Cancer Discovery publishes high-quality Research Articles and Briefs that focus on major advances in basic, translational, and clinical research of leukemia, lymphoma, myeloma, and associated diseases. The topics covered include molecular and cellular features of pathogenesis, therapy response and relapse, transcriptional circuits, stem cells, differentiation, microenvironment, metabolism, immunity, mutagenesis, and clonal evolution. These subjects are investigated in both animal disease models and high-dimensional clinical data landscapes.
The journal also welcomes submissions on new pharmacological, biological, and living cell therapies, as well as new diagnostic tools. They are interested in prognostic, diagnostic, and pharmacodynamic biomarkers, and computational and machine learning approaches to personalized medicine. The scope of submissions ranges from preclinical proof of concept to clinical trials and real-world evidence.
Blood Cancer Discovery serves as a forum for diverse ideas that shape future research directions in hematooncology. In addition to Research Articles and Briefs, the journal also publishes Reviews, Perspectives, and Commentaries on topics of broad interest in the field.