All Publications

  • Single-cell RNA sequencing distinctly characterizes the wide heterogeneity in pediatric mixed phenotype acute leukemia. Genome medicine Mumme, H. L., Raikar, S. S., Bhasin, S. S., Thomas, B. E., Lawrence, T., Weinzierl, E. P., Pang, Y., DeRyckere, D., Gawad, C., Wechsler, D. S., Porter, C. C., Castellino, S. M., Graham, D. K., Bhasin, M. 2023; 15 (1): 83


    Mixed phenotype acute leukemia (MPAL), a rare subgroup of leukemia characterized by blast cells with myeloid and lymphoid lineage features, is difficult to diagnose and treat. A better characterization of MPAL is essential to understand the subtype heterogeneity and how it compares with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Therefore, we performed single-cell RNA sequencing (scRNAseq) on pediatric MPAL bone marrow (BM) samples to develop a granular map of the MPAL blasts and microenvironment landscape.We analyzed over 40,000 cells from nine pediatric MPAL BM samples to generate a single-cell transcriptomic landscape of B/myeloid (B/My) and T/myeloid (T/My) MPAL. Cells were clustered using unsupervised single-cell methods, and malignant blast and immune clusters were annotated. Differential expression analysis was performed to identify B/My and T/My MPAL blast-specific signatures by comparing transcriptome profiles of MPAL with normal BM, AML, and ALL. Gene set enrichment analysis (GSEA) was performed, and significantly enriched pathways were compared in MPAL subtypes.B/My and T/My MPAL blasts displayed distinct blast signatures. Transcriptomic analysis revealed that B/My MPAL profile overlaps with B-ALL and AML samples. Similarly, T/My MPAL exhibited overlap with T-ALL and AML samples. Genes overexpressed in both MPAL subtypes' blast cells compared to AML, ALL, and healthy BM included MAP2K2 and CD81. Subtype-specific genes included HBEGF for B/My and PTEN for T/My. These marker sets segregated bulk RNA-seq AML, ALL, and MPAL samples based on expression profiles. Analysis comparing T/My MPAL to ETP, near-ETP, and non-ETP T-ALL, showed that T/My MPAL had greater overlap with ETP-ALL cases. Comparisons among MPAL subtypes between adult and pediatric samples showed analogous transcriptomic landscapes of corresponding subtypes. Transcriptomic differences were observed in the MPAL samples based on response to induction chemotherapy, including selective upregulation of the IL-16 pathway in relapsed samples.We have for the first time described the single-cell transcriptomic landscape of pediatric MPAL and demonstrated that B/My and T/My MPAL have distinct scRNAseq profiles from each other, AML, and ALL. Differences in transcriptomic profiles were seen based on response to therapy, but larger studies will be needed to validate these findings.

    View details for DOI 10.1186/s13073-023-01241-z

    View details for PubMedID 37845689

    View details for PubMedCentralID PMC10577904

  • Simultaneous monitoring of disease and microbe dynamics through plasma DNA sequencing in pediatric patients with acute lymphoblastic leukemia. Science advances Barsan, V., Xia, Y., Klein, D., Gonzalez-Pena, V., Youssef, S., Inaba, Y., Mahmud, O., Natarajan, S., Agarwal, V., Pang, Y., Autry, R., Pui, C. H., Inaba, H., Evans, W., Gawad, C. 2022; 8 (16): eabj1360


    Treatment of acute lymphoblastic leukemia (ALL) necessitates continuous risk assessment of leukemic disease burden and infections that arise in the setting of immunosuppression. This study was performed to assess the feasibility of a hybrid capture next-generation sequencing panel to longitudinally measure molecular leukemic disease clearance and microbial species abundance in 20 pediatric patients with ALL throughout induction chemotherapy. This proof of concept helps establish a technical and conceptual framework that we anticipate will be expanded and applied to additional patients with leukemia, as well as extended to additional cancer types. Molecular monitoring can help accelerate the attainment of insights into the temporal biology of host-microbe-leukemia interactions, including how those changes correlate with and alter anticancer therapy efficacy. We also anticipate that fewer invasive bone marrow examinations will be required, as these methods improve with standardization and are validated for clinical use.

    View details for DOI 10.1126/sciadv.abj1360

    View details for PubMedID 35442732

  • Accurate genomic variant detection in single cells with primary template-directed amplification. Proceedings of the National Academy of Sciences of the United States of America Gonzalez-Pena, V., Natarajan, S., Xia, Y., Klein, D., Carter, R., Pang, Y., Shaner, B., Annu, K., Putnam, D., Chen, W., Connelly, J., Pruett-Miller, S., Chen, X., Easton, J., Gawad, C. 2021; 118 (24)


    Improvements in whole genome amplification (WGA) would enable new types of basic and applied biomedical research, including studies of intratissue genetic diversity that require more accurate single-cell genotyping. Here, we present primary template-directed amplification (PTA), an isothermal WGA method that reproducibly captures >95% of the genomes of single cells in a more uniform and accurate manner than existing approaches, resulting in significantly improved variant calling sensitivity and precision. To illustrate the types of studies that are enabled by PTA, we developed direct measurement of environmental mutagenicity (DMEM), a tool for mapping genome-wide interactions of mutagens with single living human cells at base-pair resolution. In addition, we utilized PTA for genome-wide off-target indel and structural variant detection in cells that had undergone CRISPR-mediated genome editing, establishing the feasibility for performing single-cell evaluations of biopsies from edited tissues. The improved precision and accuracy of variant detection with PTA overcomes the current limitations of accurate WGA, which is the major obstacle to studying genetic diversity and evolution at cellular resolution.

    View details for DOI 10.1073/pnas.2024176118

    View details for PubMedID 34099548