Summary of single-cell sequencing studies in ALL and lineage-ambiguous leukemia
| Scope . | Single-cell application . | Method . | Disease (n samples) . | Key findings . | Reference . |
|---|---|---|---|---|---|
| Elucidation of transcriptional heterogeneity and lineage plasticity | |||||
| Definition of B-cell developmental states | CITE-seq | Chromium system, 3′v3 (10X Genomics) | Normal mouse B cells | High-risk subtypes have transcriptional signatures of cycling pro-B– and pre-BCR–dependent and pre-BCR–independent stages | 31 |
| Exploring cell of origin in KMT2A-R leukemia | scRNA-seq | Chromium system, 3′v2 chemistry (10X Genomics) | ∼60 000 normal fetal bone marrow cells | Infant KMT2A-R ALL shows a gene expression profile similar to early lymphocyte precursors | 33 |
| Dissection of transcriptional heterogeneity | scRNA-seq | Chromium system, v2 chemistry (10X Genomics) | T-ALL (2) B-ALL (6) | Inverse correlation between predicted leukemic cell developmental state and expression of ribosomal protein genes | 37 |
| Dissection of transcriptional heterogeneity and lineage plasticity | CITE-seq, scATAC-seq | Chromium system, v3 (RNA) and v1 (ATAC) chemistry-seq (10X Genomics) | MPAL (6) | Transcriptional program signatures are similar across immunophenotypically heterogenous cell populations within individual patients; enrichment of RUNX1 motif in MPAL cells | 46 |
| Dissection of transcriptional heterogeneity, lineage plasticity and leukemia cell of origin | scRNA-seq, multiomic scRNA-seq and scATAC-seq | Chromium system, v3 (RNA) and v1 (ATAC) chemistry-seq (10X Genomics) | MPAL (2) AML (1) ETP-ALL (1) | BCL11B gene expression correlated with enrichment for a signature of open chromatin in normal human HSPCs | 18 |
| Developmental origin and leukemic cell heterogeneity of leukemic cells; interactions between leukemic cells and immune cells; cell plasticity following therapy | scRNA-seq, scATAC-seq | Chromium system, v3 (RNA) and v1 (ATAC) chemistry-seq (10X Genomics) | KMT2A-R ALL (25) | Higher plasticity and stem-cell–like blasts in younger patients with KMT2A-R ALL with worse prognosis; the most immature leukemic cells exhibit steroid resistance; stem-cell–like cells contribute to immune evasion in younger patients | 55 |
| Dissection of ALL clonal architecture | |||||
| Elucidation of clonal structure and order of mutation acquisition | scDNA-seq | Fluidigm C1 | B-ALL (6, 5/6 ETV6::RUNX1) | SVs are acquired before SNVs (eg, in KRAS); mutations are driven by APOBEC; clones within the same patient are arrested at varied stages in B-cell development | 63 |
| Elucidation of clonal structure and order of mutation acquisition | scDNA-seq, scRNA-seq | Fluidigm C1; sort in 96-well plates; Chromium system (10X Genomics) | T-ALL (4) | Limited clonal heterogeneity; elucidation of mutation acquisition: (1) mutations in known oncogenes; (2) TR rearrangements, CDKN2A/B deletions, and gene fusions; (3) NOTCH1 mutations; accumulation of somatic mutations in multipotent progenitor cells | 64 |
| Elucidation of clonal structure and order of mutation acquisition | scDNA-seq | Single-cell multiplex quantitative-PCR | T-ALL (3) | NOTCH1 mutations are secondary events in STIL::TAL1 T-ALL | 65 |
| Dissection of heterogeneity and clonality | scDNA-seq | Tapestri (Mission Bio) | B-ALL (12) | ETV6::RUNX1, BCR::ABL1 fusion, BCR::ABL1-like, and IKZF1 N159Y subtypes harbor few additional subclonal mutations in contrast to high hyperdiploidy (1-7 subclones); mutations in signaling pathways are most commonly mutually exclusive | 13 |
| Dissection of heterogeneity and clonality | DAB-seq | Tapestri (Mission Bio) | B-ALL (5) T-ALL (1) | Lineage-related mutations (ETV6, IKZF1, and PAX5) occurred early, as they were present in all leukemia cells, while kinase-related mutations (FLT3, PTPN11, NRAS, KRAS) appeared later and were most frequently mutually exclusive | 66 |
| Elucidation of ALL relapse | |||||
| Establishment of a predictive model of relapse based on the definition of developmental states | scProtein expression | Mass cytometry | B-ALL (60) | Identification of hidden developmentally dependent cell signaling states associated with relapse | 68 |
| Transcriptional heterogeneity at diagnosis and during chemotherapy | scRNA-seq | Chromium system, v2 chemistry (10X Genomics) | B-ALL (6, ETV6::RUNX1) | ETV6::RUNX1 leukemic blasts resemble the pro-B differentiation state of normal B-cells and are heterogenous in cell cycle activity and gene expression; abundance of G1 cell cycle state at diagnosis represents a feature of chemoresistance | 75 |
| Deciphering intratumoral heterogeneity underlying disease progression and treatment resistance | scRNA-seq with clonal tracking barcodes | Chromium system, v2 chemistry (10X Genomics)+SMRT sequencing (Sequel II, Pacific Biosciences) | B-ALL PDX (4) | Cell-intrinsic mechanisms influence tissue homing in PDX | 74 |
| Early detection of relapse and clonal structure after therapy | scDNA-seq | Tapestri (Mission Bio) | T-ALL (8) | Heterogeneity of NOTCH1 mutations at diagnosis; identification of clinically relevant clones at diagnosis and residual leukemic cells at remission | 14 |
| Exploring the interplay between sensitivity to prednisone, cell heterogeneity, and relapse occurrence | scRNA-seq | SORT-seq and Chromium system (10X Genomics) | B-ALL (15, KMT2A-R) | Leukemic cells associated with high relapse risk show basal activation of glucocorticoid response and cell stemness properties | 69 |
| ALL immune microenvironment | |||||
| Role of bone marrow microenvironment in therapy response | scRNA-seq, CITE-seq | Chromium system (10X Genomics) | B-ALL (9) | Overrepresentation of a nonclassic monocytic subpopulation within the B-ALL immune microenvironment; anti-CSF1R therapy enhances targeted treatment of Ph+ B-ALL models in vivo | 86 |
| Analysis of clonal kinetics and transcriptional programs that regulate the fate of CAR T cells after infusion | scRNA-seq, scTCR-seq | Chromium system (10X Genomics) | NHL (2)∗ CLL (2) | Clonal diversity is highest in the infusion products and declines after infusion; clones that expand after infusion may originate from infused clusters with higher expression of cytotoxicity and proliferation genes | 95 |
| Analysis of T-cell composition of B-ALL bone marrow before blinatumomab infusion | scRNA-seq, scTCR-seq | Chromium system, v1 chemistry (10X Genomics) | B-ALL (4) | Response to blinatumomab therapy correlated with a higher proportion of TCF7-expressing stemlike CD4+ T cells, central memory T CD8+ T cells, and TCR diversity | 90 |
| Characterization of bone marrow TME | scRNA-seq | Chromium system (10X Genomics) | B-ALL (7, from Witkowski et al.86) | Establishment of a score-based model with prognostic predictions | 123 |
| Elucidation of heterogeneity of relapsed/refractory ETP-ALL with NOTCH1 mutations before and therapy with Notch inhibitor | scRNA-seq | Smart-Seq2 protocol | ETP-ALL (5) | Functionally distinct stemlike and mature immunomodulatory states coexist in ETP-ALL; immunomodulation of CD8+ T-cell dysfunction is mediated by galectin-9 expression | 116 |
| Characterization of functional T-cell clusters | scRNA-seq | Chromium system (10X Genomics) | B-ALL (3) | Identification of patient-specific and clonally expanded effector-like T-cell subpopulations | 89 |
| Understanding the molecular determinants of CAR T-cell persistence | CITE-seq, scATAC-seq | B-ALL/lymphoma (6) | Gene expression and chromatin accessibility patterns regulated by TCF1 are associated with naive T-cell state and long-term persisting anti-CD19 CAR T-cell products | 91 |
| Scope . | Single-cell application . | Method . | Disease (n samples) . | Key findings . | Reference . |
|---|---|---|---|---|---|
| Elucidation of transcriptional heterogeneity and lineage plasticity | |||||
| Definition of B-cell developmental states | CITE-seq | Chromium system, 3′v3 (10X Genomics) | Normal mouse B cells | High-risk subtypes have transcriptional signatures of cycling pro-B– and pre-BCR–dependent and pre-BCR–independent stages | 31 |
| Exploring cell of origin in KMT2A-R leukemia | scRNA-seq | Chromium system, 3′v2 chemistry (10X Genomics) | ∼60 000 normal fetal bone marrow cells | Infant KMT2A-R ALL shows a gene expression profile similar to early lymphocyte precursors | 33 |
| Dissection of transcriptional heterogeneity | scRNA-seq | Chromium system, v2 chemistry (10X Genomics) | T-ALL (2) B-ALL (6) | Inverse correlation between predicted leukemic cell developmental state and expression of ribosomal protein genes | 37 |
| Dissection of transcriptional heterogeneity and lineage plasticity | CITE-seq, scATAC-seq | Chromium system, v3 (RNA) and v1 (ATAC) chemistry-seq (10X Genomics) | MPAL (6) | Transcriptional program signatures are similar across immunophenotypically heterogenous cell populations within individual patients; enrichment of RUNX1 motif in MPAL cells | 46 |
| Dissection of transcriptional heterogeneity, lineage plasticity and leukemia cell of origin | scRNA-seq, multiomic scRNA-seq and scATAC-seq | Chromium system, v3 (RNA) and v1 (ATAC) chemistry-seq (10X Genomics) | MPAL (2) AML (1) ETP-ALL (1) | BCL11B gene expression correlated with enrichment for a signature of open chromatin in normal human HSPCs | 18 |
| Developmental origin and leukemic cell heterogeneity of leukemic cells; interactions between leukemic cells and immune cells; cell plasticity following therapy | scRNA-seq, scATAC-seq | Chromium system, v3 (RNA) and v1 (ATAC) chemistry-seq (10X Genomics) | KMT2A-R ALL (25) | Higher plasticity and stem-cell–like blasts in younger patients with KMT2A-R ALL with worse prognosis; the most immature leukemic cells exhibit steroid resistance; stem-cell–like cells contribute to immune evasion in younger patients | 55 |
| Dissection of ALL clonal architecture | |||||
| Elucidation of clonal structure and order of mutation acquisition | scDNA-seq | Fluidigm C1 | B-ALL (6, 5/6 ETV6::RUNX1) | SVs are acquired before SNVs (eg, in KRAS); mutations are driven by APOBEC; clones within the same patient are arrested at varied stages in B-cell development | 63 |
| Elucidation of clonal structure and order of mutation acquisition | scDNA-seq, scRNA-seq | Fluidigm C1; sort in 96-well plates; Chromium system (10X Genomics) | T-ALL (4) | Limited clonal heterogeneity; elucidation of mutation acquisition: (1) mutations in known oncogenes; (2) TR rearrangements, CDKN2A/B deletions, and gene fusions; (3) NOTCH1 mutations; accumulation of somatic mutations in multipotent progenitor cells | 64 |
| Elucidation of clonal structure and order of mutation acquisition | scDNA-seq | Single-cell multiplex quantitative-PCR | T-ALL (3) | NOTCH1 mutations are secondary events in STIL::TAL1 T-ALL | 65 |
| Dissection of heterogeneity and clonality | scDNA-seq | Tapestri (Mission Bio) | B-ALL (12) | ETV6::RUNX1, BCR::ABL1 fusion, BCR::ABL1-like, and IKZF1 N159Y subtypes harbor few additional subclonal mutations in contrast to high hyperdiploidy (1-7 subclones); mutations in signaling pathways are most commonly mutually exclusive | 13 |
| Dissection of heterogeneity and clonality | DAB-seq | Tapestri (Mission Bio) | B-ALL (5) T-ALL (1) | Lineage-related mutations (ETV6, IKZF1, and PAX5) occurred early, as they were present in all leukemia cells, while kinase-related mutations (FLT3, PTPN11, NRAS, KRAS) appeared later and were most frequently mutually exclusive | 66 |
| Elucidation of ALL relapse | |||||
| Establishment of a predictive model of relapse based on the definition of developmental states | scProtein expression | Mass cytometry | B-ALL (60) | Identification of hidden developmentally dependent cell signaling states associated with relapse | 68 |
| Transcriptional heterogeneity at diagnosis and during chemotherapy | scRNA-seq | Chromium system, v2 chemistry (10X Genomics) | B-ALL (6, ETV6::RUNX1) | ETV6::RUNX1 leukemic blasts resemble the pro-B differentiation state of normal B-cells and are heterogenous in cell cycle activity and gene expression; abundance of G1 cell cycle state at diagnosis represents a feature of chemoresistance | 75 |
| Deciphering intratumoral heterogeneity underlying disease progression and treatment resistance | scRNA-seq with clonal tracking barcodes | Chromium system, v2 chemistry (10X Genomics)+SMRT sequencing (Sequel II, Pacific Biosciences) | B-ALL PDX (4) | Cell-intrinsic mechanisms influence tissue homing in PDX | 74 |
| Early detection of relapse and clonal structure after therapy | scDNA-seq | Tapestri (Mission Bio) | T-ALL (8) | Heterogeneity of NOTCH1 mutations at diagnosis; identification of clinically relevant clones at diagnosis and residual leukemic cells at remission | 14 |
| Exploring the interplay between sensitivity to prednisone, cell heterogeneity, and relapse occurrence | scRNA-seq | SORT-seq and Chromium system (10X Genomics) | B-ALL (15, KMT2A-R) | Leukemic cells associated with high relapse risk show basal activation of glucocorticoid response and cell stemness properties | 69 |
| ALL immune microenvironment | |||||
| Role of bone marrow microenvironment in therapy response | scRNA-seq, CITE-seq | Chromium system (10X Genomics) | B-ALL (9) | Overrepresentation of a nonclassic monocytic subpopulation within the B-ALL immune microenvironment; anti-CSF1R therapy enhances targeted treatment of Ph+ B-ALL models in vivo | 86 |
| Analysis of clonal kinetics and transcriptional programs that regulate the fate of CAR T cells after infusion | scRNA-seq, scTCR-seq | Chromium system (10X Genomics) | NHL (2)∗ CLL (2) | Clonal diversity is highest in the infusion products and declines after infusion; clones that expand after infusion may originate from infused clusters with higher expression of cytotoxicity and proliferation genes | 95 |
| Analysis of T-cell composition of B-ALL bone marrow before blinatumomab infusion | scRNA-seq, scTCR-seq | Chromium system, v1 chemistry (10X Genomics) | B-ALL (4) | Response to blinatumomab therapy correlated with a higher proportion of TCF7-expressing stemlike CD4+ T cells, central memory T CD8+ T cells, and TCR diversity | 90 |
| Characterization of bone marrow TME | scRNA-seq | Chromium system (10X Genomics) | B-ALL (7, from Witkowski et al.86) | Establishment of a score-based model with prognostic predictions | 123 |
| Elucidation of heterogeneity of relapsed/refractory ETP-ALL with NOTCH1 mutations before and therapy with Notch inhibitor | scRNA-seq | Smart-Seq2 protocol | ETP-ALL (5) | Functionally distinct stemlike and mature immunomodulatory states coexist in ETP-ALL; immunomodulation of CD8+ T-cell dysfunction is mediated by galectin-9 expression | 116 |
| Characterization of functional T-cell clusters | scRNA-seq | Chromium system (10X Genomics) | B-ALL (3) | Identification of patient-specific and clonally expanded effector-like T-cell subpopulations | 89 |
| Understanding the molecular determinants of CAR T-cell persistence | CITE-seq, scATAC-seq | B-ALL/lymphoma (6) | Gene expression and chromatin accessibility patterns regulated by TCF1 are associated with naive T-cell state and long-term persisting anti-CD19 CAR T-cell products | 91 |
CLL, chronic lymphocytic leukemia; DAB-seq: single-cell DNA and antibody sequencing; NHL, non-Hodgkin lymphoma; PCR, polymerase chain reaction; PDMS, polydimethyl siloxane; PDX, patient-derived xenograft; SNVs, single-nucleotide variations; SVs, structural variations; TME, tumor microenvironment; TR, T-cell receptor and epitopes by sequencing.
Initial bulk TCR analysis was performed on B-ALL, CLL, and NHL samples, and subsequent single-cell analysis was performed on CLL and NHL samples.