Additional file 1 of Single-cell heterogeneity and dynamic evolution of Ph-like acute lymphoblastic leukemia patient with novel TPR-PDGFRB fusion gene
Creators: name: Zhang, Xuehong
name: Hou, Zhijie
name: Huang, Dan
name: Wang, Furong
name: Gao, Beibei
name: Zhang, Chengtao
name: Zhou, Dong
name: Lou, Jiacheng
name: Wang, Haina
name: Gao, Yuan
name: Kang, Zhijie
name: Lu, Ying
name: Liu, Quentin
name: Yan, Jinsong
Publisher: name: figshare
Year: 2024
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 1: Table S1. Clinical information of the one Ph-like ALL patient at different disease stages. Table S2. The fusion gene list of one Ph-like ALL patient at different disease stages. Table S3. Summary of therapeutic regimens for the Ph-like ALL patient. Figure. S1 The clinical diagnosis of one Ph-like ALL patient. (A) Diagram of the whole treatment process of the Ph-like ALL patient. NR: not remission; PR: partial remission; CR: complete molecular remission; MRD: minimal residual disease. (B) Karyotype analysis showed the normal karyotype at diagnosis for the Ph-like ALL patient. (C) Wright stain of bone marrow aspirate smear from four specimens showed the blast cells clearly. (D) Flow cytometry analysis of immunophenotypic markers for Ph-like ALL patient at diagnosis and three relapse timepoints. Figure. S2 Evaluation of Ph-like expression signature and molecular marker potential for patient with novel TPR-PDGFRB fusion gene. (A) Correlation analysis between the TPR-PDGFRB positive Ph-like patient and different subtypes from TARGET-ALL-P2 cohort. Correlation coefficient was calculated between the TPR-PDGFRB positive Ph-like patient and each of the patients in different subtypes using person method based on the expression level of all overlapped genes. (B) Hierarchical clustering analysis between the Case 13 and other B-ALL patients for three cohorts using the genes defined previously. The red and bold line in each dendrogram for three datasets highlights the location of Case 13. (C) Sanger sequencing of the RT-PCR product validated the TPR-PDGFRB fusion junction. (D) The percentage of blasts evaluated by morphology and immuno-phenotyping at thirteen time points. (E) Amplification plot of qRT-PCR for TPR-PDGFRB fusion transcript and controls. qRT-PCR analysis on the standard reference sample with serial 10-fold gradient dilutions of TPR-PDGFRB fusion transcript copies/μl and TPR-PDGFRB fusion patient’s specimen at diagnosis. Additional amplifications include a no template control (NTC) and an unrelated control cDNA came from a pediatric B-ALL specimen that does not involve a TPR-PDGFRB fusion. (F) MRD of detected by qRT-PCR for TPR-PDGFRB fusion transcript. (G) MRD of detected by ddPCR for TPR-PDGFRB fusion transcript. Figure. S3 Clusters and transcriptomic feature of different cell types in Ph-like ALL. (A) tSNE visualization of 10,273 individual cells from the patient with Ph-like ALL (TPR-PDGFRB fusion positive) with matched diagnosis (n = 4,486) and relapse (n = 5,787) bone marrow samples. (B) Unsupervised t-SNE plot displaying 10,273 cells from one Ph-like ALL patient at diagnosis and relapse, color-coded by 17 clusters. (C) Heatmap of the top-five genes marking 17 clusters. (D) Violin plot of selected genes which are differently expressed in classical monocytes and non-classical monocytes. (E) tSNE projections of selected T cell and NK cell genes. (F) Dot plot showing the average expression levels and cell expression proportions of selected MEP and cell cycle genes in the indicated clusters. (G) Violin plots show the expression of cluster-specific markers for memory B cells. Figure. S4 The functional annotation and clinical outcomes of clusters specific for different disease status in Ph-like ALL. (A) Percentage of cell origin within each clusters (n = 17). (B) Cell types overlaid on the tSNE representation and split by samples, color-coded by cell types. For all non-B cells, the corresponding cluster numbers were labeled using the pink text and cells were indicated by the dotted oval. (C) Representative GSEA plots of three primary specific clusters (cluster 4, 6 and 15) comparing with other B cell clusters. The enrichment score (ES) and false discovery rate (FDR) are shown in the graph. (D) Survival analysis of the relapsed B-cell feature (top 10 up-regulated genes) in Figure 2I in the TARGET-ALL-P2 cohort. (E) Expression levels of relapse-specific gene HRK overlaid on the tSNE representation (left panel). Boxplot (right panel) showed that the higher expression of HRK in relapsed patients with CNS-L. Figure. S5 Single-cell transcriptional profiles of Ph-like and Ph+ ALL patients. (A) The tSNE visualization of 25,559 individual cells from one patient with Ph-like ALL (TPR-PDGFRB fusion positive) and two patients with Ph+ ALL with matched bone marrow samples obtained at diagnosis and after relapse. (B) Unsupervised tSNE plot split into Ph-like and Ph+ ALL patients, displaying 25,559 cells color-coded by 23 clusters. (C) Expression levels (x-axis) of cell type-defining genes in each cluster. Violin plots showing the distribution of the normalized expression levels of genes that are color coded on the basis of the cluster, as in (B). (D) Percentage of cell origin within each clusters (n = 23). (E) A volcano plot of the DEGs that were up-regulated (red) or down-regulated (blue) in the Ph-like specific clusters (clusters 1 and 8); the top 10 up-regulated genes are labeled. The p-value is derived using the Wilcoxon rank-sum test. (F) Representative GSEA plots of Ph-like specific clusters (cluster 1, 8) comparing with other B cell clusters. The ES and FDR are shown in the graph. (G) Violin plots showing the distribution of the normalized expression levels of cytotoxic genes (left panel), NK cell genes (middle panel) and exhausted genes (right panel) in T cell (clusters 4, 9, 10, and 16) and NK cell clusters (clusters 15 and 23). (H) Scatter plot for the signature scores about cytotoxicity and NK feature in the T cell clusters. (I) tSNE visualization of cells colored by the expression of cytotoxicity or NK receptor signatures. (J) Violin plot of immune checkpoint genes including CLEC2D and KLRB1.