Figure 1. CFU assays are a valuable... | American Society of Hematology

Figure 1.

$CFU assays are a valuable model to study IFN-α hematologic and molecular responses in patients with MPN. (A) PBMCs of patients with MPN were seeded in CFU assays, supplemented with or without 500 U/mL IFN-α, and were counted after 10 to 14 days without discrimination between different types of colonies. Each data point represents an individual patient. The significance was analyzed using a Wilcoxon matched-pairs signed ranks test. ∗∗∗∗P < .0001. (B) HC samples were treated in the same manner as MPN samples in panel A. Significance was analyzed using a Wilcoxon matched-pairs signed ranks test. (C) The relative numbers of remaining colonies were calculated using untreated samples as control. Samples were grouped into HC and MPN subtypes. The data were analyzed using a Kruskal-Wallis test with Dunn multiple comparison after failing the Shapiro-Wilk test for normality. None of the comparisons reached significance. Blue indicates HC; Red indicates PV; yellow indicates ET; grey indicates MF; (D) DNA from 25 to 30 colonies per patient for each condition was isolated and screened for the presence of the JAK2V617F mutation via genotyping PCR. The ratio of MPN WT, MPN heterozygous, and MPN homozygous colonies was used to calculate the mutant VAF. The mutant VAF was compared between the untreated and treated condition for each patient. Patients who showed a decrease in mutant VAF upon treatment were grouped as molecular responders, patients who showed no change or an increase in VAF were termed non-responders. The ratios of molecular responders to nonresponders in the CFU model were analyzed in the different MPN subtypes. MF included patients with PMF and those with post–PV-MF. (E) Change in the mutant VAF upon in vitro IFN-α treatment was correlated with the mutant VAF calculated from the untreated CFU sample. Correlation was analyzed using Pearson r after passing the Shapiro-Wilk test for normality. (F) Differences in the fraction of colonies in the untreated and treated condition in responders (left) and non-responders (right) were analyzed. WT, het, and hom colonies were compared. Statistical analysis was performed using a Kruskal-Wallis test, followed by Dunn multiple comparison after failing the Shapiro-Wilk test for normal distribution. ∗∗P < .01; ∗∗∗P < .001. (G) The change in VAF was analyzed in patients treated with IFN-α in the clinics and compared with the change in VAF in the CFU assay. Data were calculated relative to the initial VAF. (H) RNA was isolated from individual colonies. Of each patient or HC, a minimum of 2 colonies was used. Complimentary DNA was generated, and the expression of STAT1, STAT2, IFIT2, and IFIT3 was measured using RT-qPCR with normalization to MT-ATP6. The mean of all measured colonies per patient and condition was calculated and the values were subjected to an unsupervised clustering. (I) PBMCs were seeded, with or without 500 U/mL IFN-α, and colonies were picked after 10 days of cultivation. STAT1 expression was analyzed using RT-qPCR in 30 untreated and treated colonies of an HC (top) and PV sample (bottom; PV14) (supplemental Table 1). Each bar represents a single colony. Expression was normalized to MT-ATP6.$

CFU assays are a valuable model to study IFN-α hematologic and molecular responses in patients with MPN. (A) PBMCs of patients with MPN were seeded in CFU assays, supplemented with or without 500 U/mL IFN-α, and were counted after 10 to 14 days without discrimination between different types of colonies. Each data point represents an individual patient. The significance was analyzed using a Wilcoxon matched-pairs signed ranks test. ∗∗∗∗P < .0001. (B) HC samples were treated in the same manner as MPN samples in panel A. Significance was analyzed using a Wilcoxon matched-pairs signed ranks test. (C) The relative numbers of remaining colonies were calculated using untreated samples as control. Samples were grouped into HC and MPN subtypes. The data were analyzed using a Kruskal-Wallis test with Dunn multiple comparison after failing the Shapiro-Wilk test for normality. None of the comparisons reached significance. Blue indicates HC; Red indicates PV; yellow indicates ET; grey indicates MF; (D) DNA from 25 to 30 colonies per patient for each condition was isolated and screened for the presence of the JAK2V617F mutation via genotyping PCR. The ratio of MPN WT, MPN heterozygous, and MPN homozygous colonies was used to calculate the mutant VAF. The mutant VAF was compared between the untreated and treated condition for each patient. Patients who showed a decrease in mutant VAF upon treatment were grouped as molecular responders, patients who showed no change or an increase in VAF were termed non-responders. The ratios of molecular responders to nonresponders in the CFU model were analyzed in the different MPN subtypes. MF included patients with PMF and those with post–PV-MF. (E) Change in the mutant VAF upon in vitro IFN-α treatment was correlated with the mutant VAF calculated from the untreated CFU sample. Correlation was analyzed using Pearson r after passing the Shapiro-Wilk test for normality. (F) Differences in the fraction of colonies in the untreated and treated condition in responders (left) and non-responders (right) were analyzed. WT, het, and hom colonies were compared. Statistical analysis was performed using a Kruskal-Wallis test, followed by Dunn multiple comparison after failing the Shapiro-Wilk test for normal distribution. ∗∗P < .01; ∗∗∗P < .001. (G) The change in VAF was analyzed in patients treated with IFN-α in the clinics and compared with the change in VAF in the CFU assay. Data were calculated relative to the initial VAF. (H) RNA was isolated from individual colonies. Of each patient or HC, a minimum of 2 colonies was used. Complimentary DNA was generated, and the expression of STAT1, STAT2, IFIT2, and IFIT3 was measured using RT-qPCR with normalization to MT-ATP6. The mean of all measured colonies per patient and condition was calculated and the values were subjected to an unsupervised clustering. (I) PBMCs were seeded, with or without 500 U/mL IFN-α, and colonies were picked after 10 days of cultivation. STAT1 expression was analyzed using RT-qPCR in 30 untreated and treated colonies of an HC (top) and PV sample (bottom; PV14) (supplemental Table 1). Each bar represents a single colony. Expression was normalized to MT-ATP6.

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