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Journal Articles
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GSEA analysis and validation of a primary cell line from a Ewing tumor with...
Published: 20 April 2022
FIGURE 1 GSEA analysis and validation of a primary cell line from a Ewing tumor with a BARD1 pathogenic variant. A, Schematic overview of tumor samples associated with analyses in B – F . B, Gene-set enrichment analysis (GSEA) of RNA-seq data comparing the lung relapse of the Ewing tumor with a germline BARD1 pathogenic variant to the original primary/pretreatment biopsy. Genesets significantly impacted (P < 0.05) are included. C, Phase contrast image (400×) of the PSaRC318 Ewing tumor cell line. D, Flow cytometry showing presence of surface CD99 expression in the PSaRC318 cell line. E, Schematic detailing the difference between Type 1 and Type 3 EWS-FLI fusions (top) and Western blot analysis with anti-FLI1 antibody of Ewing sarcoma cell lines with type 3 (PSaRC318) versus type 1 (A673, CHLA9, CHLA10, and TC71) EWS-FL1 fusions. F, Western blot demonstrating BARD1 protein expression in the same Ewing sarcoma cell lines as in E . PSaRC318 cells demonstrate significantly (P < 0.05) less BARD1 expression as compared with other Ewing cell lines. Densitometry values below the blot indicate relative expression values. Experiments in D – F were completed minimally in biological triplicate. More
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Loss of BARD1 enhances Ewing sarcoma cell sensitivity to PARP inhibition.  ...
Published: 20 April 2022
FIGURE 2 Loss of BARD1 enhances Ewing sarcoma cell sensitivity to PARP inhibition. A, Western blot analysis for PARP1 expression in Ewing sarcoma cells. B, p-γH2AX and DAPI immunofluorescence staining of PSaRC318, A673, and CHLA10 cells treated with DMSO or 100 nmol/L talazoparib (Tal). Cells imaged at 630× and p-γH2AX foci were quantified (bottom graphs). C, IncuCyte assay comparing the confluence of PSaRC318 cells treated with DMSO versus 100 nmol/L talazoparib over 1 week. D, qRT-PCR showing BARD1 mRNA expression in A673 or CHLA10 cells treated with control (ctsi) or BARD1 (BARD1si) siRNA. E, Western blot analysis for BARD1 expression in untreated (NT) A673 or CHLA10 cells or cells treated with Ctsi or BARD1si. F, IncuCyte monitoring of cell confluence at increasing concentrations of talazoparib versus DMSO controls in A673 and CHLA10 cells treated with Ctsi versus BARD1si. A673 and CHLA10 cell data is graphed at the 60-hour time point. Normalized expression values from densitometry analyses are included under Western blots. Experiments were completed minimally in biological triplicate. NS, not significant; *, P < 0.01. Error bars, SD. More
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BARD1 loss enhances Ewing sarcoma cell apoptosis in response to niraparib p...
Published: 20 April 2022
FIGURE 3 BARD1 loss enhances Ewing sarcoma cell apoptosis in response to niraparib plus radiation. A, Relative apoptosis (caspase 3/7 activity) data from IncuCyte assays showing the effect of 0.5 μmol/L niraparib (Nir) versus DMSO control plus either 0 or 2 Gy radiation on PSaRC318 cells. B, Confluence data from IncuCyte assays showing the effect of 0.5 μmol/L niraparib versus DMSO control plus either 0 or 2 Gy radiation on PSaRC318 cells. C and D, A673 cells were treated with control (Ctsi) or BARD1 (BARD1si) siRNA, niraparib (at doses indicated) versus DMSO control, and either 0 or 2 Gy radiation and monitored via IncuCyte apoptosis assay ( C ) or confluence assay ( D ). For these experiments, cells were seeded in the presence of niraparib and radiation was performed at 12–15 hours. Relative apoptosis (caspase 3/7 dye activity) is calculated as green fluorescence in μm2 divided by confluence. Experiments were completed minimally in technical and biological triplicates. *, P < 0.01 as determined by ANOVA analysis with Tukey multiple comparisons test. Error bars, SD. More
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Impact of BARD1 loss on the Ewing sarcoma cell transcriptome.  A,  Volcano ...
Published: 20 April 2022
FIGURE 4 Impact of BARD1 loss on the Ewing sarcoma cell transcriptome. A, Volcano plot of genes up-/downregulated upon loss of BARD1 as compared with A673 Ewing sarcoma cells transfected with Ctsi in three biological replicates. Horizontal dashed lines denote P = 0.05 (5 on −log10 scale). Vertical dashed lines denote a value of 1 on the log2 scale. BARD1 is circled in blue and highlighted as to verify that it is significantly downregulated upon RNA-seq analysis. In addition, the inset image demonstrates RT-PCR analysis of BARD1 expression as a second means by which to validate reduction of BARD1 expression in these RNA samples (×3 biological replicates) as compared with Ctsi-treated cells, *, P < 0.05; error bars, SD. B, List of most significantly (Padj) upregulated and downregulated genes with a log2 fold change of 2 or greater when comparing cells treated with BARD1 siRNA versus Ctsi. C, Pathway analysis (C2 and Hallmark genesets) of BARD1 siRNA-treated cells as compared with Ctsi-treated cells. NES, normalized enrichment score; Padj, adjusted P value. More
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Guanylate-binding protein 1 (GBP1) contributes to Ewing cell sensitivity to...
Published: 20 April 2022
FIGURE 5 Guanylate-binding protein 1 (GBP1) contributes to Ewing cell sensitivity to DNA damage noted upon loss of BARD1. A, IHC analysis of GPB1 expression in the PSaRC318 patient tumor and in two additional independent Ewing tumors. Images provided at both low (200×) and high (1,000×) power. B, Western blot for GBP1 in Ewing sarcoma cell lines. Numbers under the blot indicate normalized expression as determined by densitometry analysis. C, Normalized GPB1 mRNA expression of PSaRC318 cells treated with control (ct) or GBP1 siRNA (si). *, P < 0.0001. D, PSaRC318 cells were transfected with control or GBP1 siRNA for 72 hours. Cells were then seeded into 96-well plates in quadruplicate, allowed to adhere and then radiated (dose = 1 Gy). Live-cell IncuCyte monitoring of cell confluence and apoptosis (caspase 3/7 activity) was monitored. The graph displays the relative apoptosis (apoptosis/confluence) in control versus GBP1 siRNA-treated cells over time. *, P < 0.05. E, PSaRC318 cells treated with siRNA and seeded as in D and then treated with DMSO, 0.5 μmol/L niraparib (Nir), or 0.75 μmol/L niraparib. The graph displays the relative apoptosis (apoptosis/confluence) over time. Representative IncuCyte images at 48 hours are included (right). NS, not significant; *, P < 0.05. Experiments completed minimally in biological triplicate. Error bars, SD. More
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HD volunteers have higher T-cell counts, increased CD4/CD8 ratio, and an ex...
Published: 23 March 2022
FIGURE 1 HD volunteers have higher T-cell counts, increased CD4/CD8 ratio, and an expanded naïve T-cell population compared with patients with multiple myeloma. A, Clinical features of HD volunteers and patients with multiple myeloma (MM) used in the study to generate PBMCs, including age and prior treatment regimens. Three main groups were studied: (i) HD volunteers with no underlying disorders and age < 30 (HD), newly diagnosed multiple myeloma patients not yet exposed to any treatment regimen, and relapsed multiple myeloma patients, who have undergone several lines of treatment as described. The third group is subdivided into early relapse (≤3 prior lines of therapy) and late relapse (>3 prior lines of therapy). B, T-cell counts per 10,000 live PBMCs after PBMC isolation from peripheral blood: HD (n = 9), newly diagnosed MM (n = 4), early relapsed MM (n = 9), and late relapsed MM (n = 4). C, CD4/CD8 T-cell ratios from the four groups analyzed above. D, Representative FACS plots describing the T-cell memory phenotype from a HD subject versus a relapsed multiple myeloma patient. Cells were previously gated on live CD3+ T cells and based on the expression of CD45RO and CD62L were characterized into: naïve/stem cell memory (SCM), central memory (CM), and effector memory (EM) T cells. CD45RO and CD62L cells were quantified as effector T cells. Memory phenotype of CD4 ( E ) and CD8 T cells ( F ) present in HD (n = 6), newly diagnosed multiple myeloma (n = 4), and relapsed multiple myeloma patients (n = 8, including early and late rMM patients), quantified by FACS based on CD45RO and CD62L expression. Data represent mean values ± SEM. *P < 0.05; **P < 0.005. Statistical analysis was performed using two-tailed unpaired t test. Abbreviations: Auto, autologous stem cell transplant/autograft; BTD, Bendamustine, Thalidomide, Dexamethasone; CD, Cyclophosphamide, Dexamethasone; CRD, Cyclophosphamide, Lenalidomide, Dexamethasone; CTD, Cyclophosphamide, Thalidomide, Dexamethasone; CVD or VCD, Cyclophosphamide, Velcade, Dexamethasone; Dara, Daratumumab; DVD, Daratumumab, Velcade, Dexamethasone; ESHAP, Etoposide, Solu-medrone, High-dose cytarabine, Cisplatin; KCD, Carfilzomib, Cyclophosphamide, Dexamethasone; Nil, no prior treatment regimen; PAD, Velcade, Adriamycin, Dexamethasone; RD – Lenalidomide, Dexamethasone; RVD, Lenalidomide, Velcade, Dexamethasone; VD, Velcade, Dexamethasone; VMP, Velcade, Melphalan, Prednisolone; VPD, Velcade, Panobinostat, Dexamethasone; VTD, Velcade, Thalidomide, Dexamethasone. More
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HD-derived anti-BCMA CAR T cells have a higher proportion of central memory...
Published: 23 March 2022
FIGURE 2 HD-derived anti-BCMA CAR T cells have a higher proportion of central memory CD8 T cells compared with multiple myeloma–derived anti-BCMA CAR T cells. A, Schematic diagram of the anti-BCMA CAR construct used, which includes an anti-BCMA scFv, a rituximab-based off-switch, CD8-based hinge and transmembrane region, 4–1BB co-stimulatory domain and CD3z intracellular signaling domain. B, Representative dot plots depicting anti-BCMA CAR expression on live CD3+ T cells by FACS after 14 days of anti-BCMA CAR T-cell production. UT cells were generated from the same donor. C, Percentage of anti-BCMA CAR+ T cells at the end of production generated from HD (n = 7), ND MM (n = 2), early rMM (n = 4), and late rMM patients (n = 4). D, Expression of anti-BCMA CAR (MFI) on CAR T cells generated as above. E, CD4/CD8 ratio of anti-BCMA CAR T cells generated as above. Memory phenotype of CD4 ( F ) and CD8 ( G ) anti-BCMA CAR T cells produced from HD (n = 3), newly diagnosed multiple myeloma (ND MM) (n = 2), and relapsed multiple myeloma (rMM) patients (n = 4, including early and late rMM patients). Data represent mean values ± SEM. *P < 0.05; NS, not statistically significant. Statistical analysis was performed using two-tailed unpaired t test. More
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HD-derived anti-BCMA CAR T cells express lower levels of PD-1, TIGIT and LA...
Published: 23 March 2022
FIGURE 3 HD-derived anti-BCMA CAR T cells express lower levels of PD-1, TIGIT and LAG3 compared with multiple myeloma–derived anti-BCMA CAR T cells and show superior cytotoxicity in a rechallenge assay. A, Representative histograms comparing the expression of the T-cell checkpoint inhibitory molecules TIGIT, PD-1, LAG3, and TIM3 on HD-derived (blue) versus multiple myeloma (MM)-derived (red) anti-BCMA CAR T cells, analyzed by FACS at the end of production (day 14, before cryopreservation). B, Quantitation of MFI of TIGIT, PD-1, LAG3, and TIM3 across HD-derived (n = 3) and late rMM-derived (n = 3) anti-BCMA CAR T-cell samples. C, Percentage of TIGIT+, PD-1+, LAG3+, and TIM3+ on anti-BCMA CAR T cells from HD (n = 3) versus late rMM patients (n = 3) at the end of production (day 14). D, Representative density plots depicting the expression of the checkpoint inhibitory molecules PD-1 and LAG3 on HD-derived versus late rMM–derived anti-BCMA CAR T cells, analyzed by FACS and gated on PD-1+LAG3+ double expression. E, Percentage of HD-derived versus late rMM–derived anti-BCMA CAR T cells expressing PD-1+TIGIT+, PD-1+LAG3+, PD-1+TIM3+ and the permanently dysfunctional CD38+CD101+ T-cell population. F, Schematic diagram describing the cytotoxicity rechallenge assay consisting of a prolonged coculture of anti-BCMA CAR T cells and the multiple myeloma cell line U266, using a 1:10 E/T ratio. U266 cells were added to the anti-BCMA CAR T cells twice, on days 1 and 5. The coculture assay wells were analyzed by FACS on day 8. UT cells were used to measure T-cell background killing. G, Percentage of U266 cell lysis (day 8) when cocultured with HD-derived UTs (n = 3) or HD-derived anti-BCMA CAR T cells (n = 3) versus late rMM–derived UTs (n = 3) or late rMM–derived anti-BCMA CAR T cells (n = 3), analyzed by FACS using the viability dye e450. H, T-cell expansion of HD-derived (n = 3) versus late rMM–derived (n = 3) anti-BCMA CAR T cells at the end of the rechallenge assay (analyzed by FACS). Data represent mean values ± SEM. *P < 0.05. Statistical analysis was performed using two-tailed unpaired t test. More
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HD-derived anti-BCMA CAR T cells specifically and efficiently target multip...
Published: 23 March 2022
FIGURE 4 HD-derived anti-BCMA CAR T cells specifically and efficiently target multiple myeloma (MM) primary cells within the BM microenvironment of different patient subgroups. A, Schematic timeline of the ex vivo cytotoxicity (CTL) assay using BM samples from patients with multiple myeloma to test the activity of HD-derived anti-BCMA CAR T cells. The coculture was performed for 4 hours at different E/T ratios. BM samples were always used fresh and the available volume varied from sample to sample, which limited the number of samples used in specific assays. B, Table describing the clinical features of the patients with multiple myeloma used in this assay (n = 11), including patient age, genomic subgroup, disease stage, and prior treatment regimens. BM sample #20 is an age-matched normal plasma cell control with no detectable multiple myeloma cells in the BM. C, Representative dot plots describing the gating strategy used by FACS to identify multiple myeloma cells within the BM sample after staining with the multiple myeloma antibody panel. Multiple myeloma cells are sequentially gated as CD138+/CD38hi, CD38hi/CD45 and CD56+/CD19. D, Specific cancer cell lysis of MM#16 primary cells after coculture with anti-BCMA CAR T cells (continuous line) or UT cells (dotted line) at different E/T ratios. REH cell line and REH-BCMA+ cell line were used as negative and positive controls, respectively. UT cells were used to measure background T-cell killing. E, Specific anti-BCMA CAR T-cell killing (%) against different BM samples (n = 12) after the ex vivo CTL assay at 10:1 E/T ratio. The anti-BCMA CAR T-cell killing percentage was quantified as: [(%multiple myeloma cell lysis cocultured with anti-BCMA CAR T cells − % multiple myeloma cell lysis cocultured with UT cells) / % spontaneous multiple myeloma cell lysis]. REH and REH-BCMA were used as negative and positive controls, respectively. F, Representative FACS plot of a BM sample using the SSC/CD45 gating strategy to identify the different BM cell types present, including: granulocytes, monocytes/macrophages, and T cells. G, Cell viability of monocytes/macrophages, T cells, and granulocytes present within the multiple myeloma BM samples (n = 11) after the ex vivo CTL assay with UT cells or anti-BCMA CAR T cells. H, Percentage of BM microenvironmental cells (T cells, granulocytes, and macrophages/monocytes) present in the multiple myeloma BM samples prior to the ex vivo CTL assay (n = 10). I, Correlation between % BM microenvironmental cells and CAR T-cell cytotoxic activity for each multiple myeloma sample (n = 10). J, Percentage of BM microenvironmental cells (T cells, granulocytes, and macrophages/monocytes) present in samples with lower CAR T-cell killing (below CAR T-cell killing average of 32.9%) versus higher CAR T-cell killing (above CAR T-cell killing average of 32.9%). Data represent mean values ± SEM. NS, not statistically significant; *P < 0.05. Statistical analysis was performed using two-tailed unpaired t test. Abbreviations: Auto, Autologous stem cell transplant/autograft; Cyclo, Cyclophosphamide; Dara, Daratumumab; Dex, Dexamethasone; DVD, Daratumumab, Velcade, Dexamethasone; IRD, Ixazomib, Lenalidomide, Dexamethasone; Len, Lenalidomide; KCD, Carfilzomib, Cyclophosphamide, Dexamethasone; Nil, no prior treatment regimen; VMP, Velcade, Melphalan, Prednisolone; VTD, Velcade, Thalidomide, Dexamethasone. More
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Anti-BCMA CAR T-cell activity can be enhanced by increasing BCMA levels on ...
Published: 23 March 2022
FIGURE 5 Anti-BCMA CAR T-cell activity can be enhanced by increasing BCMA levels on the multiple myeloma (MM) cell surface. A, Histograms showing the BCMA expression of multiple myeloma primary cells from each BM sample, analyzed by FACS. The age-matched normal BM20 control and the MM23 primary cells are BCMA according to a T-cell internal control. All the other multiple myeloma samples express BCMA at varying levels. B, Correlation between specific anti-BCMA CAR T-cell activity and BCMA expression for each BM sample analyzed (n = 12). The relative BCMA expression (mean fluorescence intensity, MFI) was normalized to the BCMA expression on BM endogenous T cells, used as an internal negative control. C, Expression levels of BCMA (MFI) on multiple myeloma primary cells (MM48, MM52, MM55, MM57) after treatment with GSI PF-03084014 for five hours at different concentrations. D, Diagram illustrating the experimental timeline used to combine the treatment of GSI PF-03084014 with anti-BCMA CAR T cells in the ex vivo CTL assay with multiple myeloma BM samples. BM samples were always used fresh and the available volume varied from sample to sample, which limited the number of samples used in specific assays. E, Specific killing of MM48, MM52, and MM57 cancer cells by anti-BCMA CAR T cells or UT cells, with and without GSI treatment. F, Normalized cell killing of anti-BCMA CAR T cells against MM52 and MM57 primary cells (n = 2) with or without 10 nmol/L of GSI treatment. Values normalized to the specific killing of anti-BCMA CAR T cells only. ***P < 0.005. Statistical analysis was performed using one-tailed paired t test. More
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MxIF study design and classification validation. Overview of MxIF workflow ...
Published: 10 February 2022
FIGURE 1 MxIF study design and classification validation. Overview of MxIF workflow ( A ). Visual assessment of classification in QuPath ( B–D ). Virtual H&E of a tumor margin FOV and MxIF images visualizing the lymphocyte, myeloid, tumor, and vasculature components ( B ). Classification mask ... More
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Single-cell composition of metastatic melanoma lymph node by anatomic subre...
Published: 10 February 2022
FIGURE 2 Single-cell composition of metastatic melanoma lymph node by anatomic subregion. Abundance plots of classified cells across all FOVs in tumor core, tumor margin, and normal adjacent lymphoid tissue ( A ). Statistical significance in abundances between cell types in responders and nonresponders (NR; n = 136 R FOVs, n = 193 NR FOVs; B ). More
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Single-cell expression of HLA-I on classified tumor cells. Proportion of cl...
Published: 10 February 2022
FIGURE 3 Single-cell expression of HLA-I on classified tumor cells. Proportion of classified tumor cells expressing HLA-I (bright red) versus no expression of HLA-I (dark red). More
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Cellular criteria required for TCCN definition ( A ). Derivation of study-s...
Published: 10 February 2022
FIGURE 4 Cellular criteria required for TCCN definition ( A ). Derivation of study-specific 50-μm-diameter TCCN size based on number of neighborhoods satisfying cell and nonoverlapping criteria across various radius sizes (0–60 μm; B ). Stepwise derivation of TCCN from tumor margin FOV ( C ) and ... More
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TCCNs by anatomic subregion. Box plots of immune cell subsets populating TC...
Published: 10 February 2022
FIGURE 5 TCCNs by anatomic subregion. Box plots of immune cell subsets populating TCCNs in the tumor core and tumor margin. Comparisons between responder and nonresponder TCCNs were compared using the Spearman correlation statistic ( A ). Virtual H&E and corresponding spatial plots visualizing... More
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TCCNs by HLA-I status of the iTC. Box plots of immune cell subsets populati...
Published: 10 February 2022
FIGURE 6 TCCNs by HLA-I status of the iTC. Box plots of immune cell subsets populating TCCNs with HLA-Ipos iTC ( A ) and HLA-Ineg iTC ( B ) in the tumor core and margin. Comparisons between responder and nonresponder TCCNs were compared using the Spearman correlation statistic. More