Single-cell profiling identifies pre-existing CD19-negative subclones in a B-ALL patient with CD19-negative relapse after CAR-T therapy
Chimeric antigen receptor T cell (CAR-T) targeting the CD19 antigen represents an innovative therapeutic approach to improve the outcome of relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL). Yet, despite a high initial remission rate, CAR-T therapy ultimately fails for some patients. Notably, around half of relapsing patients develop CD19 negative (CD19neg) B-ALL allowing leukemic cells to evade CD19-targeted therapy. Herein, we investigate leukemic cells of a relapsing B-ALL patient, at two-time points: before (T1) and after (T2) anti-CD19 CAR-T treatment. We show that at T2, the B-ALL relapse is CD19 negative due to the expression of a non-functional CD19 transcript retaining intron 2. Then, using single-cell RNA sequencing (scRNAseq) approach, we demonstrate that CD19neg leukemic cells were present before CAR-T cell therapy and thus that the relapse results from the selection of these rare CD19neg B-ALL clones. In conclusion, our study shows that scRNAseq profiling can reveal pre-existing CD19neg subclones, raising the possibility to assess the risk of targeted therapy failure.
CellRanger Metadata
| Preview | Accession | Estimated No. of Cells | Mean Reads per Cell | Median Genes per Cell | No. of Reads | % Valid Barcodes | % sequencing Saturation | % Q30 Bases in Barcode | % Q30 Bases in UMI | % Reads Mapped to Genome | % Reads Mapped Confidently to Genome | % Reads Mapped Confidently to Intergenic Regions | % Reads Mapped Confidently to Intronic Regions | % Reads Mapped Confidently to Exonic Regions | % Reads Mapped Confidently to Transcriptome | % Reads Mapped Antisense to Gene | % Fraction Reads in Cells | Total Genes Detected | Median UMI Counts Per Cell |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Preview | SRR12135757 | 655 | 43776 | 1 | 28673024 | 97.4 | 30.0 | 92.7 | 91.1 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 100.0 | 1 | 385 |
| Preview | SRR12135758 | 7719 | 58636 | 1835 | 452614277 | 82.2 | 72.2 | 93.8 | 92.4 | 88.5 | 71.8 | 9.5 | 12.1 | 50.2 | 52.0 | 10.0 | 84.6 | 1835 | 28346 |
CAGE Transcription Start Site (CTSS) File Download
The CTSS positions were taken from dataset BAM files, downloadable here.
Top 10 Marker Genes per Cluster
| Seurat Cluster | Gene Symbol | P Value | Average Log | Pct 1 | Pct 2 | Adjusted P Value |
|---|---|---|---|---|---|---|
| 0 | LYZ | 1.50E-153 | 6.896382 | None | None | 3.62E-149 |
| 0 | S100A6 | 2.13E-142 | 4.98101 | None | None | 1.28E-138 |
| 0 | IFI30 | 2.08E-131 | 5.397628 | None | None | 8.37E-128 |
| 0 | TYROBP | 6.19E-131 | 4.75333 | None | None | 2.14E-127 |
| 0 | S100A9 | 6.01E-150 | 7.3070903 | None | None | 4.84E-146 |
| 0 | S100A4 | 1.43E-130 | 4.081134 | None | None | 4.32E-127 |
| 0 | CST3 | 2.03E-129 | 4.726351 | None | None | 5.44E-126 |
| 0 | S100A8 | 5.85E-151 | 7.345547 | None | None | 7.08E-147 |
| 0 | S100A12 | 7.14E-128 | 6.827904 | None | None | 1.73E-124 |
| 0 | FCN1 | 8.79E-142 | 5.8570004 | None | None | 4.25E-138 |
| 1 | IGLL1 | 2.05E-106 | 4.7282543 | None | None | 8.25E-103 |
| 1 | CD74 | 4.69E-109 | 2.4710486 | None | None | 2.27E-105 |
| 1 | HLA-DRA | 1.62E-88 | 2.4634314 | None | None | 3.91E-85 |
| 1 | MME | 2.35E-99 | 3.8136103 | None | None | 7.11E-96 |
| 1 | CD99 | 3.12E-104 | 3.0626006 | None | None | 1.08E-100 |
| 1 | DNTT | 1.72E-137 | 5.6191196 | None | None | 4.16E-133 |
| 1 | VPREB1 | 1.07E-122 | 5.4283524 | None | None | 1.29E-118 |
| 1 | CD79B | 1.02E-119 | 4.6737022 | None | None | 8.23E-116 |
| 1 | CD9 | 8.84E-89 | 4.041407 | None | None | 2.37E-85 |
| 1 | VPREB3 | 1.61E-115 | 4.897855 | None | None | 9.72E-112 |
| 2 | NKG7 | 1.74E-85 | 6.4847207 | None | None | 7.00E-82 |
| 2 | CFLAR | 3.35E-74 | 3.5875132 | None | None | 9.00E-71 |
| 2 | IFITM1 | 1.11E-74 | 5.2103424 | None | None | 3.35E-71 |
| 2 | CTSW | 1.84E-90 | 6.0179176 | None | None | 1.11E-86 |
| 2 | CD247 | 1.58E-97 | 8.000586 | None | None | 1.27E-93 |
| 2 | GNLY | 4.23E-116 | 8.857956 | None | None | 1.02E-111 |
| 2 | SYTL3 | 2.93E-73 | 3.825518 | None | None | 7.08E-70 |
| 2 | KLRD1 | 1.86E-98 | 7.9139304 | None | None | 2.25E-94 |
| 2 | CD7 | 5.13E-80 | 6.709143 | None | None | 1.77E-76 |
| 2 | CCL5 | 7.25E-66 | 6.8315206 | None | None | 1.35E-62 |
| 3 | Z93241.1 | 1.83E-73 | 3.9325461 | None | None | 7.36E-70 |
| 3 | TEX14 | 4.69E-89 | 3.9665923 | None | None | 1.13E-84 |
| 3 | AL021155.5 | 1.41E-74 | 3.7113712 | None | None | 8.54E-71 |
| 3 | UBC | 3.95E-73 | 1.7043523 | None | None | 1.36E-69 |
| 3 | WDR74 | 5.01E-74 | 3.1111808 | None | None | 2.42E-70 |
| 3 | LINC00910 | 3.55E-76 | 3.4178846 | None | None | 2.86E-72 |
| 3 | TSC22D3 | 1.04E-69 | 2.3409865 | None | None | 2.81E-66 |
| 3 | AC007952.4 | 3.23E-72 | 3.0361028 | None | None | 9.77E-69 |
| 3 | PTCH2 | 2.09E-68 | 3.8293283 | None | None | 4.59E-65 |
| 3 | CCDC200 | 4.04E-80 | 4.0698485 | None | None | 4.88E-76 |
| 4 | RPS23 | 3.47E-63 | 1.579475 | None | None | 1.20E-59 |
| 4 | GAS5 | 7.34E-66 | 2.7521605 | None | None | 3.55E-62 |
| 4 | RPS6 | 4.18E-66 | 2.136714 | None | None | 2.53E-62 |
| 4 | RPS18 | 1.27E-62 | 1.8696109 | None | None | 3.42E-59 |
| 4 | SPINK2 | 8.68E-71 | 6.325777 | None | None | 2.10E-66 |
| 4 | SNHG29 | 3.58E-62 | 2.3767183 | None | None | 8.66E-59 |
| 4 | CRHBP | 1.98E-68 | 8.537857 | None | None | 2.39E-64 |
| 4 | HINT1 | 5.93E-68 | 2.2530556 | None | None | 4.78E-64 |
| 4 | RPS3A | 9.91E-63 | 1.6720243 | None | None | 3.00E-59 |
| 4 | RPS4X | 1.81E-65 | 2.0428183 | None | None | 7.29E-62 |
| 5 | NIBAN3 | 1.72E-53 | 3.3108535 | None | None | 4.63E-50 |
| 5 | AKAP13 | 1.17E-59 | 2.9041889 | None | None | 5.64E-56 |
| 5 | AL079338.1 | 3.71E-53 | 3.9530306 | None | None | 8.97E-50 |
| 5 | SPTBN1 | 1.93E-63 | 3.2673824 | None | None | 1.16E-59 |
| 5 | SRSF11 | 1.28E-54 | 2.6030817 | None | None | 4.44E-51 |
| 5 | AFF3 | 1.31E-67 | 3.684493 | None | None | 3.18E-63 |
| 5 | EBF1 | 2.06E-54 | 3.4777377 | None | None | 6.23E-51 |
| 5 | MIR181A1HG | 3.95E-65 | 3.6019864 | None | None | 4.77E-61 |
| 5 | JMJD1C | 1.42E-57 | 2.616106 | None | None | 5.71E-54 |
| 5 | CLTC | 1.89E-63 | 3.441232 | None | None | 1.16E-59 |
| 6 | PTMA | 2.76E-30 | 1.111324 | None | None | 1.11E-26 |
| 6 | RPL22 | 3.48E-32 | 1.4691601 | None | None | 1.68E-28 |
| 6 | H3F3A | 5.43E-30 | 1.1474571 | None | None | 1.88E-26 |
| 6 | RPS12 | 1.66E-28 | 1.1876 | None | None | 4.46E-25 |
| 6 | PDLIM1 | 1.17E-29 | 2.7140713 | None | None | 3.55E-26 |
| 6 | TCL1A | 8.77E-27 | 3.0234673 | None | None | 2.12E-23 |
| 6 | RPLP0 | 1.30E-35 | 1.5728091 | None | None | 7.88E-32 |
| 6 | PRDX1 | 6.89E-38 | 2.6663465 | None | None | 1.67E-33 |
| 6 | CD24 | 1.03E-35 | 2.8254085 | None | None | 7.88E-32 |
| 6 | TCL1B | 1.73E-36 | 3.6973605 | None | None | 2.10E-32 |
| 7 | TEX14 | 1.68E-40 | 3.152386 | None | None | 6.76E-37 |
| 7 | CCDC200 | 5.61E-36 | 3.3898752 | None | None | 1.51E-32 |
| 7 | AFF1 | 8.76E-42 | 2.9517171 | None | None | 4.23E-38 |
| 7 | WDFY4 | 6.56E-35 | 2.8640008 | None | None | 1.59E-31 |
| 7 | PMAIP1 | 2.78E-42 | 3.209357 | None | None | 1.68E-38 |
| 7 | AC007952.4 | 3.37E-39 | 2.7775474 | None | None | 1.16E-35 |
| 7 | BANK1 | 1.25E-38 | 3.7433197 | None | None | 3.78E-35 |
| 7 | AL079338.1 | 9.42E-44 | 3.45639 | None | None | 2.28E-39 |
| 7 | SMC6 | 6.69E-43 | 3.6110432 | None | None | 5.73E-39 |
| 7 | LINC00910 | 7.11E-43 | 3.1491418 | None | None | 5.73E-39 |
| 8 | SAT1 | 3.71E-38 | 3.3286984 | None | None | 9.96E-35 |
| 8 | IFI30 | 2.24E-42 | 4.7422667 | None | None | 1.81E-38 |
| 8 | MTSS1 | 1.23E-38 | 3.5000365 | None | None | 3.70E-35 |
| 8 | MS4A7 | 4.81E-37 | 4.876444 | None | None | 1.16E-33 |
| 8 | PECAM1 | 1.42E-39 | 3.475113 | None | None | 6.89E-36 |
| 8 | SERPINA1 | 8.38E-40 | 4.5346394 | None | None | 5.06E-36 |
| 8 | LILRB2 | 7.05E-39 | 4.036588 | None | None | 2.43E-35 |
| 8 | VSIR | 6.84E-47 | 3.5805743 | None | None | 1.65E-42 |
| 8 | SMIM25 | 5.25E-44 | 5.086824 | None | None | 6.35E-40 |
| 8 | RNF144B | 6.55E-39 | 4.094445 | None | None | 2.43E-35 |
| 9 | JARID2 | 1.01E-43 | 4.61446 | None | None | 8.15E-40 |
| 9 | PTK2B | 3.18E-47 | 4.5683966 | None | None | 3.84E-43 |
| 9 | FKBP5 | 4.21E-41 | 4.208271 | None | None | 1.45E-37 |
| 9 | ZBTB16 | 5.23E-41 | 4.1288013 | None | None | 1.58E-37 |
| 9 | SAT1 | 7.48E-37 | 3.9141107 | None | None | 1.51E-33 |
| 9 | NEAT1 | 7.06E-48 | 4.0996284 | None | None | 1.71E-43 |
| 9 | TOP1 | 5.25E-43 | 3.9484606 | None | None | 3.17E-39 |
| 9 | PIK3R5 | 1.96E-42 | 4.9562416 | None | None | 9.48E-39 |
| 9 | ARHGAP26 | 2.24E-40 | 4.5587645 | None | None | 6.02E-37 |
| 9 | ZEB2 | 2.42E-41 | 3.2553878 | None | None | 9.76E-38 |
| 10 | H2AFZ | 4.76E-39 | 4.109373 | None | None | 1.92E-35 |
| 10 | TUBA1B | 2.27E-42 | 4.270097 | None | None | 1.37E-38 |
| 10 | HMGN2 | 2.55E-44 | 3.5242922 | None | None | 3.08E-40 |
| 10 | HMGB1 | 8.63E-40 | 2.5868847 | None | None | 4.17E-36 |
| 10 | STMN1 | 2.72E-46 | 4.531771 | None | None | 6.57E-42 |
| 10 | TUBB | 6.77E-38 | 3.8694403 | None | None | 2.34E-34 |
| 10 | NUSAP1 | 8.36E-29 | 6.2973566 | None | None | 2.02E-25 |
| 10 | HMGB2 | 1.55E-42 | 3.977716 | None | None | 1.25E-38 |
| 10 | MCM7 | 3.36E-35 | 5.0199156 | None | None | 1.02E-31 |
| 10 | SMC4 | 4.18E-30 | 4.026527 | None | None | 1.12E-26 |
| 11 | TCL1A | 1.24E-33 | 5.27913 | None | None | 2.99E-29 |
| 11 | BACH2 | 2.50E-30 | 3.9848154 | None | None | 3.03E-26 |
| 11 | AFF3 | 1.02E-25 | 3.278242 | None | None | 3.07E-22 |
| 11 | CD74 | 5.22E-25 | 2.2816353 | None | None | 1.40E-21 |
| 11 | CD79B | 2.03E-27 | 4.109533 | None | None | 9.80E-24 |
| 11 | NIBAN3 | 2.06E-24 | 3.435418 | None | None | 4.99E-21 |
| 11 | CD37 | 8.19E-26 | 2.3658814 | None | None | 2.83E-22 |
| 11 | RUBCNL | 1.78E-28 | 5.327365 | None | None | 1.08E-24 |
| 11 | ACSM3 | 7.51E-30 | 5.831275 | None | None | 6.05E-26 |
| 11 | SYK | 2.70E-26 | 3.148172 | None | None | 1.09E-22 |
| 12 | RPL36 | 8.83E-12 | 1.4432925 | None | None | 3.05E-08 |
| 12 | RPS27A | 3.64E-11 | 1.3157569 | None | None | 7.99E-08 |
| 12 | RPS15 | 1.44E-11 | 1.3617282 | None | None | 4.34E-08 |
| 12 | RPS14 | 6.78E-13 | 1.855227 | None | None | 4.10E-09 |
| 12 | RPS26 | 3.55E-11 | 1.1745187 | None | None | 7.99E-08 |
| 12 | RPL34 | 1.93E-11 | 1.4321003 | None | None | 5.18E-08 |
| 12 | TXNIP | 9.20E-14 | 2.7412422 | None | None | 9.57E-10 |
| 12 | RPL41 | 7.38E-12 | 1.3554273 | None | None | 2.97E-08 |
| 12 | RPL14 | 1.98E-12 | 1.4539199 | None | None | 9.59E-09 |
| 12 | FXYD5 | 1.19E-13 | 1.8744624 | None | None | 9.57E-10 |
| 13 | PGD | 9.45E-17 | 3.0290604 | None | None | 2.54E-13 |
| 13 | NAMPT | 4.18E-20 | 4.336645 | None | None | 5.06E-16 |
| 13 | PYGL | 6.49E-17 | 5.219649 | None | None | 1.96E-13 |
| 13 | CDK14 | 1.59E-13 | 3.413295 | None | None | 8.94E-11 |
| 13 | LYN | 1.66E-16 | 3.3903646 | None | None | 3.09E-13 |
| 13 | FNDC3B | 3.94E-15 | 3.795569 | None | None | 5.01E-12 |
| 13 | PLEK | 2.87E-18 | 3.7863946 | None | None | 1.60E-14 |
| 13 | SRGN | 1.57E-18 | 3.8368897 | None | None | 1.27E-14 |
| 13 | PLAUR | 3.30E-18 | 5.3817134 | None | None | 1.60E-14 |
| 13 | CXCL8 | 1.13E-16 | 5.4783516 | None | None | 2.74E-13 |
| 14 | FTH1 | 5.30E-09 | 2.6405525 | None | None | 1.89E-05 |
| 14 | MCTP2 | 1.95E-08 | 3.2066867 | None | None | 3.96E-05 |
| 14 | SIK3 | 2.13E-08 | 3.4215863 | None | None | 3.96E-05 |
| 14 | GLUL | 6.43E-12 | 3.5213683 | None | None | 1.55E-07 |
| 14 | STAM | 1.11E-08 | 4.200745 | None | None | 2.98E-05 |
| 14 | SRGN | 3.63E-09 | 3.5855346 | None | None | 1.76E-05 |
| 14 | LRRFIP2 | 2.79E-10 | 3.6975598 | None | None | 3.37E-06 |
| 14 | RBM39 | 1.31E-08 | 1.8200392 | None | None | 3.18E-05 |
| 14 | ATP2B1 | 5.46E-09 | 3.5515006 | None | None | 1.89E-05 |
| 14 | PDE3B | 4.59E-10 | 4.218157 | None | None | 3.70E-06 |
| 15 | FCHSD2 | 9.90E-11 | 4.4213204 | None | None | 7.98E-07 |
| 15 | IGKC | 6.33E-09 | 6.998283 | None | None | 2.19E-05 |
| 15 | PLD4 | 5.09E-11 | 6.208099 | None | None | 6.15E-07 |
| 15 | CCDC50 | 3.66E-08 | 4.3103466 | None | None | 9.83E-05 |
| 15 | UGCG | 9.20E-09 | 5.73704 | None | None | 2.78E-05 |
| 15 | IRF4 | 1.01E-11 | 5.6498294 | None | None | 2.45E-07 |
| 15 | SEC61B | 1.06E-09 | 3.278941 | None | None | 5.14E-06 |
| 15 | LDLRAD4 | 4.19E-08 | 2.9054196 | None | None | 0.00010116 |
| 15 | HSP90B1 | 2.06E-09 | 2.9445198 | None | None | 8.31E-06 |
| 15 | JCHAIN | 6.09E-10 | 6.9395804 | None | None | 3.68E-06 |
| 16 | ALAS2 | 8.82E-07 | 15.554054 | None | None | 0.001888548 |
| 16 | HBM | 8.85E-07 | 14.305544 | None | None | 0.001888548 |
| 16 | HBA1 | 8.83E-07 | 16.630383 | None | None | 0.001888548 |
| 16 | AHSP | 8.82E-07 | 14.52481 | None | None | 0.001888548 |
| 16 | SLC25A37 | 5.50E-08 | 7.2864165 | None | None | 0.000332646 |
| 16 | SNCA | 5.01E-08 | 8.070384 | None | None | 0.000332646 |
| 16 | HBA2 | 4.68E-08 | 17.524292 | None | None | 0.000332646 |
| 16 | HBB | 4.68E-08 | 16.406883 | None | None | 0.000332646 |
| 16 | ANK1 | 9.03E-07 | 10.04747 | None | None | 0.001888548 |
| 16 | SOX6 | 9.37E-07 | 9.828221 | None | None | 0.001888548 |