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Asxl1 truncation promotes the expansion of myeloid-biased stem cells. (A) Representative hematoxylin and eosin (H&E)–stained tibia cross-sections of mice 1 year post-Cre induction (n = 3 per group). (B) Schematic of serial transplant of all 4 genotypes and timeline of bone marrow characterization by flow cytometry. (C) Flow cytometry analysis of the long-term hematopoietic stem, LT-HSC CD41+/CD41− ratio, and GMP progenitor cells from mice bone marrow at 4 months, 1 year after Cre induction and after both serial transplants (n = 3-4 per group). (D) Quantification of colony-forming unit assays at 1 year post-Cre induction and after both serial transplants using 1 × 104 cells per well of whole bone marrow cells (mean ± standard error of the mean [SEM]; n = 3 per group in triplicate). Significance was evaluated with a 2-way analysis of variance (ANOVA) with Tukey multiple comparison test. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001.

Asxl1 truncation promotes the expansion of myeloid-biased stem cells. (A)...

in Dual ASXL1 and CSF3R mutations drive myeloid-biased stem cell expansion and enhance neutrophil differentiation > Blood Advances
Published: 2025
Figure 2. Asxl1 truncation promotes the expansion of myeloid-biased stem cells. (A) Representative hematoxylin and eosin (H&E)–stained tibia cross-sections of mice 1 year post-Cre induction (n = 3 per group). (B) Schematic of serial transplant of all 4 genotypes and timeline of bone marrow... More about this image found in Asxl1 truncation promotes the expansion of myeloid-biased stem cells. (A)...
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Csf3r mutation induces an aberrant neutrophil phenotype. (A) Comparison of peripheral blood cell counts between 8 weeks and 92 weeks post-Cre induction of female mice (mean ± SEM; n = 9-12 per group). Significance was evaluated with a mixed-effect analysis with Sidak multiple comparison test. (B) Quantification of white blood cells in peripheral blood smears of mice 1 year after Cre induction (mean ± SEM; n = 2 per group in duplicate). Significance was evaluated with a 2-way ANOVA with Tukey multiple comparison test. (C) Peripheral blood neutrophil counts of female mice in 92 weeks. Dotted lines represent normal count limits as described by the Charles River Laboratories for C57BL/6 mice in their North American Colonies for mice aged 8 to 10 weeks. (D) Survival of female mice from each genotype is illustrated in a Kaplan-Meier plot. No significance was found by the Mantel-Cox test. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001.

Csf3r mutation induces an aberrant neutrophil phenotype. (A) Comparison o...

in Dual ASXL1 and CSF3R mutations drive myeloid-biased stem cell expansion and enhance neutrophil differentiation > Blood Advances
Published: 2025
Figure 3. Csf3r mutation induces an aberrant neutrophil phenotype. (A) Comparison of peripheral blood cell counts between 8 weeks and 92 weeks post-Cre induction of female mice (mean ± SEM; n = 9-12 per group). Significance was evaluated with a mixed-effect analysis with Sidak multiple compari... More about this image found in Csf3r mutation induces an aberrant neutrophil phenotype. (A) Comparison o...
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Asxl1 mutation enhances CSF3R-driven differentiation. (A) Representative flow cytometric plots of CD11b and GR1 48 hours after estrogen withdrawal in HoxB8 cells transduced with plasmids containing Asxl1MT2-GFP and/or CSF3RT618I-mCherry and/or their corresponding empty controls producing all following 4 groups: control, Asxl1MT2, CSF3RT618I, and double mutant or Asxl1MT2/CSF3RT618I. Quantification of transduced mCherry/GFP–positive cells (mean ± SEM; n = 3 per group). Significance was evaluated with a 2-way ANOVA with a Tukey multiple comparison test. (B) RNA sequencing was performed 24 hours after estrogen withdrawal with all 4 groups (n = 3 per group). Heat maps represent the normalized, row-scaled (gene-based z-score scaled) counts of the most significantly differentially expressed genes (Padj < .05) between the control and double-mutant cells, and genes are clustered by similar expression patterns. Each cluster has a corresponding Gene Ontology (GO) tree of the top 15 most significantly enriched GO terms and their associated number of gene hits and adjusted P values. (C) Gene Set Enrichment Analysis (GSEA) between Asxl1MT2/CSF3RT618I and control cells. (D) GSEA between Asxl1MT2/CSF3RT618I and Asxl1MT2 cells. (E) GSEA between Asxl1MT2/CSF3RT618I and CSF3RT618I cells. Gene ranks from GSEA are represented as line charts for the top 5 most significant hallmark gene sets from the Molecular Signatures Database (MSigDB). ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001. NES, normalized enrichment score; Padj, adjusted P value.

Asxl1 mutation enhances CSF3R -driven differentiation. (A) Representativ...

in Dual ASXL1 and CSF3R mutations drive myeloid-biased stem cell expansion and enhance neutrophil differentiation > Blood Advances
Published: 2025
Figure 4. Asxl1 mutation enhances CSF3R -driven differentiation. (A) Representative flow cytometric plots of CD11b and GR1 48 hours after estrogen withdrawal in HoxB8 cells transduced with plasmids containing Asxl1 MT 2 -GFP and/or CSF3R T618I -mCherry and/or their corresponding empty co... More about this image found in Asxl1 mutation enhances CSF3R -driven differentiation. (A) Representativ...
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MYC overexpression reduces myeloid differentiation of Asxl1- and CSF3R-mutant cells. (A) Heat maps representing the normalized, row-scaled counts of the top 50 most significantly differentially expressed genes (Padj < .05) between the control and double-mutant cells from the gene subset, Hallmark MYC targets. (B) Quantitative PCR (qPCR) for Myc before and after estrogen withdrawal (mean ± SEM; n = 3 per group). Significance was evaluated with a 2-way ANOVA with Sidak multiple comparison test. (C) qPCR for Myc expression in transduced double-mutant cells with a Tet-on MYC plasmid (mean ± SEM; n = 3 per group). Significance was evaluated with a 2-way ANOVA with Sidak multiple comparison test. (D) Representative flow cytometry plots of CD11b and GR1 at 48 hours post estrogen withdrawal of the double-mutant cells with a Tet-on MYC plasmid. These cells were treated with or without doxycycline 24 hours before estrogen withdrawal. Quantification of CSF3R-mCherry/Asxl1-GFP–positive cells reveals significant repression of CD11b+/GR1+ (mean ± SEM; n = 3 per group). Significance was evaluated with a 2-way ANOVA with Sidak multiple comparison test. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001.

MYC overexpression reduces myeloid differentiation of Asxl1- and CSF3R ...

in Dual ASXL1 and CSF3R mutations drive myeloid-biased stem cell expansion and enhance neutrophil differentiation > Blood Advances
Published: 2025
Figure 5. MYC overexpression reduces myeloid differentiation of Asxl1- and CSF3R -mutant cells. (A) Heat maps representing the normalized, row-scaled counts of the top 50 most significantly differentially expressed genes ( P adj < .05) between the control and double-mutant cells from the... More about this image found in MYC overexpression reduces myeloid differentiation of Asxl1- and CSF3R ...
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Figure 6.

Untitled image

in Dual ASXL1 and CSF3R mutations drive myeloid-biased stem cell expansion and enhance neutrophil differentiation > Blood Advances
Published: 2025
Figure 6. More about this image found in Untitled image
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Asxl1 truncation changes the epigenetic landscape to favor CSF3R-driven differentiation. (A) Representative flow cytometry plots of CD11b and GR1 at 48 hours after estrogen withdrawal in HoxB8 CRISPR-Cas9 modified for Asxl1 truncation and transduced with CSF3RT618I or their corresponding control plasmid producing the following 4 groups: control, Asxl1sgB2, CSF3RT618I, and double mutant or Asxl1sgB2/CSF3RT618I. Quantification of the percentage of CSF3R-mCherry–positive cells (mean ± SEM; n = 3 per group). Significance was evaluated with a 2-way ANOVA with a Tukey multiple comparison test. (B) ATAC sequencing was performed 48 hours after estrogen withdrawal with all 4 groups (n = 3 per group). GREAT analysis using differential peaks between Asxl1sgB2/CSF3RT618I and control groups reveal enrichment of immune system activation and differentiation in the double-mutant mice. (C) HOMER motif enrichment results from the ATAC sequencing differential peaks between CSF3RT618I and control. The orange trend line in the scatterplot is used to compare natural log-transformed motif P values between the sample groups. Residual values in the bar plot represent the difference between the trend line and transformed motif P values. (D) CUT&amp;Tag was performed 48 hours after estrogen withdrawal with all 4 groups (n = 3 per group) with H3K4me1 antibody. Profile plot of mean H3K4me1 activity at regions spanning its consensus peak centers ± 3 kb. (E) Heat map displaying row-scaled (region-based z-score scaled), normalized read counts for each sample condition (averaged across replicates) in regions where H3K4me1 is uniquely active. The top 6 significant enriched motifs from HOMER are revealed for region clusters 1, 5, and 6. (F) HOMER motif enrichment results from the ATAC sequencing differential peaks between Asxl1sgB2 and control. The orange trend line in the scatterplot is used to compare natural log-transformed motif P values between the sample groups. Residual values in the bar plot represent the difference between the trend line and transformed motif P values. (G) CUT&amp;Tag was performed 48 hours after estrogen withdrawal with all 4 groups (n = 3 per group) with H2AK119ub antibody. Profile plot of mean H2AK119ub activity at regions spanning its consensus peak centers ± 3 kb. (H) Heat map displaying row-scaled (region-based z-score scaled) normalized read counts for each sample condition (averaged across replicates) in regions where H2AK119ub is uniquely active. Top 6 significant enriched motifs from HOMER are revealed for region cluster 1. CTCF and BORIS are not specific to cluster 1 as they are top 1 and 2 in all but 1 cluster. ∗P &lt; .05, ∗∗P &lt; .01, ∗∗∗P &lt; .001, ∗∗∗∗P &lt; .0001.

Asxl1 truncation changes the epigenetic landscape to favor CSF3R-driven di...

in Dual ASXL1 and CSF3R mutations drive myeloid-biased stem cell expansion and enhance neutrophil differentiation > Blood Advances
Published: 2025
Asxl1 truncation changes the epigenetic landscape to favor CSF3R-driven differentiation. (A) Representative flow cytometry plots of CD11b and GR1 at 48 hours after estrogen withdrawal in HoxB8 CRISPR-Cas9 modified for Asxl1 truncation and transduced with CSF3R T618I or their corresponding c... More about this image found in Asxl1 truncation changes the epigenetic landscape to favor CSF3R-driven di...
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CONSORT diagram showing the number of patients depending on their remission status. CR defined as &lt;5% blasts in bone marrow. AML-MR, acute myeloid leukemia-myelodysplasia related; CT, chemotherapy; MR, molecular relapse; PIF, primary induction failure.

CONSORT diagram showing the number of patients depending on their remission...

in Pretransplant MRD does not seem to affect survival in NPM1 -mutated AML undergoing allogeneic stem cell transplantation > Blood Advances
Published: 2025
Figure 1. CONSORT diagram showing the number of patients depending on their remission status. CR defined as <5% blasts in bone marrow. AML-MR, acute myeloid leukemia-myelodysplasia related; CT, chemotherapy; MR, molecular relapse; PIF, primary induction failure. More about this image found in CONSORT diagram showing the number of patients depending on their remission...
Images
Figure 2.

Untitled image

in Pretransplant MRD does not seem to affect survival in NPM1 -mutated AML undergoing allogeneic stem cell transplantation > Blood Advances
Published: 2025
Figure 2. More about this image found in Untitled image
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Impact of pre–allo-SCT remission status on clinical outcomes. Kaplan-Meier curves of (A) OS and (B) LFS. Cumulative incidences (CIs) of (C) NRM and (D) relapse. Forest plot showing the HRs of (E) OS and (F) relapse, estimated by univariate regression outcome analysis. CIF, cumulative incidence function; HCT-CI, hematopoietic cell transplantation–specific comorbidity index; MAC, myeloablative conditioning; MMUD, mismatched unrelated donor; MRC, Medical Research Council; MSD, matched sibling donor; NRM, nonrelapse mortality; PIF, primary induction failure.

Impact of pre–allo-SCT remission status on clinical outcomes. Kaplan-Meier...

in Pretransplant MRD does not seem to affect survival in NPM1 -mutated AML undergoing allogeneic stem cell transplantation > Blood Advances
Published: 2025
Impact of pre–allo-SCT remission status on clinical outcomes. Kaplan-Meier curves of (A) OS and (B) LFS. Cumulative incidences (CIs) of (C) NRM and (D) relapse. Forest plot showing the HRs of (E) OS and (F) relapse, estimated by univariate regression outcome analysis. CIF, cumulative incidence func... More about this image found in Impact of pre–allo-SCT remission status on clinical outcomes. Kaplan-Meier...
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Box plots depicting BM NPM1mut kinetics before allo-SCT according to remission status. Nonremission patients in gray, patients in MRD+ CR in blue, and patients in MRD− CR in light blue. NPM1mut transcript levels are reported as the normalized values of NPM1mut copy number per ABL1 copy number.

Box plots depicting BM NPM1 mut kinetics before allo-SCT according to ...

in Pretransplant MRD does not seem to affect survival in NPM1 -mutated AML undergoing allogeneic stem cell transplantation > Blood Advances
Published: 2025
Figure 3. Box plots depicting BM NPM1 mut kinetics before allo-SCT according to remission status. Nonremission patients in gray, patients in MRD + CR in blue, and patients in MRD − CR in light blue. NPM1 mut transcript levels are reported as the normalized values of NPM1 mut cop... More about this image found in Box plots depicting BM NPM1 mut kinetics before allo-SCT according to ...
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Differential Ab responses between responders and nonresponders to EBVST immunotherapy. The volcano plots show the difference in IgA and IgG levels (SSI) between responders (n = 36) and non-responders (n = 20) to EBVST immunotherapy at preinfusion and 2-week (2 wk), 4-week (4 wk), and 3-month (3 mo) postinfusion time points. The x-axis represents the fold change (ratio of SSI for responders vs nonresponders), whereas the y-axis shows the corresponding t test log 10 (P values.) IgA Abs are shown in red, and IgG Abs are shown in blue. Abs with a coefficient of variation (CV) &lt;30% are included in the analysis (IgA = 202; IgG = 74). The dashed lines represent the nominally significant P value threshold.

Differential Ab responses between responders and nonresponders to EBVST imm...

in Differential antibody response to EBV proteome following EBVST immunotherapy in EBV-associated lymphomas > Blood Advances
Published: 2025
Figure 1. Differential Ab responses between responders and nonresponders to EBVST immunotherapy. The volcano plots show the difference in IgA and IgG levels (SSI) between responders (n = 36) and non-responders (n = 20) to EBVST immunotherapy at preinfusion and 2-week (2 wk), 4-week (4 wk), and 3... More about this image found in Differential Ab responses between responders and nonresponders to EBVST imm...
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Multiway variable importance plot of IgA and IgG responses at 3 months after infusion (mean decrease accuracy and mean decrease Gini). This figure illustrates the importance of individual Abs in differentiating between responders and nonresponders, as assessed using the random forest model. The x-axis represents the mean decrease in accuracy, and the y-axis represents the mean decrease in Gini. Higher values on both axes indicate greater importance in the model. Abs in the upper right of the plot are identified as important by both metrics, indicating more substantial contributions to model performance. Notably, Abs that were nominally significant at the 3-month time point (P &lt; .05) in the logistic regression models are also among the most important variables identified by the random forest as the most important variables, demonstrating robust agreement across methods.

Multiway variable importance plot of IgA and IgG responses at 3 months afte...

in Differential antibody response to EBV proteome following EBVST immunotherapy in EBV-associated lymphomas > Blood Advances
Published: 2025
Figure 2. Multiway variable importance plot of IgA and IgG responses at 3 months after infusion (mean decrease accuracy and mean decrease Gini). This figure illustrates the importance of individual Abs in differentiating between responders and nonresponders, as assessed using the random forest m... More about this image found in Multiway variable importance plot of IgA and IgG responses at 3 months afte...
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Change in Ab response between preinfusion and 3 months postinfusion. Ribbon plots showing the mean change (with 25th to 75th percentile range) in Ab response (SSI) for EBV-specific antibody markers in responders (n = 36; yellow) and nonresponders (n = 20; purple). Only antibodies with nominal P value of &lt; .05 in adjusted logistic regression models are shown.

Change in Ab response between preinfusion and 3 months postinfusion. Ribbo...

in Differential antibody response to EBV proteome following EBVST immunotherapy in EBV-associated lymphomas > Blood Advances
Published: 2025
Figure 3. Change in Ab response between preinfusion and 3 months postinfusion. Ribbon plots showing the mean change (with 25th to 75th percentile range) in Ab response (SSI) for EBV-specific antibody markers in responders (n = 36; yellow) and nonresponders (n = 20; purple). Only antibodies with ... More about this image found in Change in Ab response between preinfusion and 3 months postinfusion. Ribbo...
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Process development for generating 19-28z CAR EBV-VSTs. Cryopreserved EBV-VSTs (cultured for 28-35 days before cryopreservation) are thawed and stimulated weekly with irradiated B-LCL (days 0, +7, +14). T cells are transduced with 19-28z CAR at a cell density of 1 × 106 cells per mL and 0.5 × 106 cells per mL, respectively, on days +16 and +17. Cells are then fed and evaluated on days +18, +21, and +23 and then harvested and frozen on day +24.

Process development for generating 19-28z CAR EBV-VSTs. Cryopreserved EBV-...

in Allogeneic off-the-shelf CAR T-cell therapy for relapsed or refractory B-cell malignancies > Blood Advances
Published: 2025
Figure 1. Process development for generating 19-28z CAR EBV-VSTs. Cryopreserved EBV-VSTs (cultured for 28-35 days before cryopreservation) are thawed and stimulated weekly with irradiated B-LCL (days 0, +7, +14). T cells are transduced with 19-28z CAR at a cell density of 1 × 10 6 cells per mL ... More about this image found in Process development for generating 19-28z CAR EBV-VSTs. Cryopreserved EBV-...
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19-28z CAR EBV-VST clinical trial design. (A) Patients in cohort 1 were treated with 1 to 3 cycles of 19-28z CAR EBV-VSTs after relapse after transplant, and patients in cohorts 2 and 3 were treated with 1 to 3 cycles of 19-28z CAR EBV-VSTs as consolidation with hematopoietic cell transplant. (B) Schema showing patients in each cohort along with their diagnosis and transplant donor type. B-NHL, B-cell non-Hodgkin lymphoma; CLL, chronic lymphocytic leukemia.

19-28z CAR EBV-VST clinical trial design. (A) Patients in cohort 1 were tr...

in Allogeneic off-the-shelf CAR T-cell therapy for relapsed or refractory B-cell malignancies > Blood Advances
Published: 2025
Figure 2. 19-28z CAR EBV-VST clinical trial design. (A) Patients in cohort 1 were treated with 1 to 3 cycles of 19-28z CAR EBV-VSTs after relapse after transplant, and patients in cohorts 2 and 3 were treated with 1 to 3 cycles of 19-28z CAR EBV-VSTs as consolidation with hematopoietic cell tran... More about this image found in 19-28z CAR EBV-VST clinical trial design. (A) Patients in cohort 1 were tr...
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Long-term outcomes of patients treated with 19-28z CAR EBV-VSTs. (A) OS of patients with B-ALL vs NHL. (B) OS of patients with CAR EBV-VST products from their transplant donor vs a third-party donor. (C) EFS of patients with B-ALL vs NHL. (D) EFS of patients with CAR EBV-VST products from their transplant donor vs a third-party donor. Note: patients with no initial response to treatment are included with event time of 0. NHL, non-Hodgkin lymphoma.

Long-term outcomes of patients treated with 19-28z CAR EBV-VSTs. (A) OS of...

in Allogeneic off-the-shelf CAR T-cell therapy for relapsed or refractory B-cell malignancies > Blood Advances
Published: 2025
Figure 3. Long-term outcomes of patients treated with 19-28z CAR EBV-VSTs. (A) OS of patients with B-ALL vs NHL. (B) OS of patients with CAR EBV-VST products from their transplant donor vs a third-party donor. (C) EFS of patients with B-ALL vs NHL. (D) EFS of patients with CAR EBV-VST products f... More about this image found in Long-term outcomes of patients treated with 19-28z CAR EBV-VSTs. (A) OS of...
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Figure 4.

Untitled image

in Allogeneic off-the-shelf CAR T-cell therapy for relapsed or refractory B-cell malignancies > Blood Advances
Published: 2025
Figure 4. More about this image found in Untitled image
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T-cell phenotypic analysis of 19-28z CAR EBV-VSTs. (A) Dot plots comparing CD3+ T-cell subsets of paired parental EBV-VSTs with 19-28z CAR EBV-VSTs. (B) Dot plots comparing CD3+ T-cell subsets of 19-28z CAR EBV-VSTs with 19-28z CAR T cells. (C) Dot plots comparing CD3+ CAR+ T-cell subsets of 19-28z CAR EBV-VSTs vs 19-28z CAR T cells. (D) Ring graphs comparing exhaustion marker (PD1, LAG3, ICOS, and CTLA4) expression of CD3+ EBV-VSTs with 19-28z CAR EBV-VSTs. (E) Ring graphs comparing exhaustion marker (PD1, LAG3, ICOS, and CTLA4) expression of CD3+ 19-28z CAR EBV-VSTs with 19-28z CAR T cells. (F) Ring graphs comparing exhaustion marker (PD1, LAG3, ICOS, and CTLA4) expression of CD3+ CAR+ 19-28z CAR EBV-VSTs with 19-28z CAR T cells. (G) Percent expression of individual exhaustion markers (PD1, LAG3, ICOS, and CTLA4) on T-cell subsets of EBV-VSTs vs 19-28z CAR EBV-VSTs. (H) Percent expression of individual exhaustion markers (PD1, LAG3, ICOS, and CTLA4) on T-cell subsets of 19-28z CAR EBV-VSTs vs 19-28z CAR T cells. P values were determined by Mann-Whitney-Wilcoxon tests.

T-cell phenotypic analysis of 19-28z CAR EBV-VSTs. (A) Dot plots comparing...

in Allogeneic off-the-shelf CAR T-cell therapy for relapsed or refractory B-cell malignancies > Blood Advances
Published: 2025
T-cell phenotypic analysis of 19-28z CAR EBV-VSTs. (A) Dot plots comparing CD3 + T-cell subsets of paired parental EBV-VSTs with 19-28z CAR EBV-VSTs. (B) Dot plots comparing CD3 + T-cell subsets of 19-28z CAR EBV-VSTs with 19-28z CAR T cells. (C) Dot plots comparing CD3 + CAR+ T-cell subsets of ... More about this image found in T-cell phenotypic analysis of 19-28z CAR EBV-VSTs. (A) Dot plots comparing...
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Of the 6 indirect measures of iron status measured—ferritin, hemoglobin, MCV, transferrin, transferrin saturation, and transferrin receptor—serum ferritin had the greatest accuracy in identifying iron deficiency.

Of the 6 indirect measures of iron status measured—ferritin, hemoglobin, MC...

in Best but not perfect: indirect measure of low iron stores > Blood Advances
Published: 2025
Of the 6 indirect measures of iron status measured—ferritin, hemoglobin, MCV, transferrin, transferrin saturation, and transferrin receptor—serum ferritin had the greatest accuracy in identifying iron deficiency. More about this image found in Of the 6 indirect measures of iron status measured—ferritin, hemoglobin, MC...
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Kaplan-Meier curves of secondary (time-dependent) end points. (A) PFS. (B) DOR. (C) TTNT. (D) OS. DoR, duration of response.

Kaplan-Meier curves of secondary (time-dependent) end points. (A) PFS. (B)...

in Six-month rituximab-lenalidomide regimen in advanced untreated follicular lymphoma: SAKK 35/10 trial 10-year update > Blood Advances
Published: 2025
Figure 1. Kaplan-Meier curves of secondary (time-dependent) end points. (A) PFS. (B) DOR. (C) TTNT. (D) OS. DoR, duration of response. More about this image found in Kaplan-Meier curves of secondary (time-dependent) end points. (A) PFS. (B)...