Biological and clinical relevance of quantitative global methylation of repetitive DNA sequences in chronic lymphocytic leukemia

Figures & data

Table 1. Biological and molecular features of CLL patients included in the study

Parameter
Age (years)
Median	65
Range	27–87
Gender	No. of patients
Female	31
Male	46
Interphase FISH analysis
Normal	14
11q-	11
13q- (as a sole abnormality)	33 (21)
17p-	12
+12	17
Multiple cytogenetic abnormalities	12
ZAP-70 expression
Negative (<30%)	46
Positive (≥30%)	28
CD38 expression
Negative (<30%)	36
Positive (≥30%)	35
IgVH mutational status
Mutated (<98% homology)	28
Unmutated (≥98% homology)	48

Table 2. Global DNA methylation levels in healthy subjects compared with CLL patients

Methylation markers		Healthy donors		CLLs
	n	med [IQR]	n	med [IQR]
Alu (%5mC)	7	25.9 [25.7–26.6]	76	21.4 [18.1–23.6]
p^a				<0.001
LINE-1 (%5mC)	7	85.7 [83.4–87.1]	77	66.8 [60.5–74.9]
p^a				<0.001
SAT-α (%5mC)	7	88.2 [86.5–90.2]	72	84.0 [64.1–86.2]
p^a				<0.001

IQR, interquartile range: Twenty-fifth and seventy-fifth percentiles. aWilcoxon rank sum test for difference between categories.

Figure 1. DNA methylation levels in relation to different cytogenetic groups. Box plot representation of DNA methylation levels. The p values corresponding to each methylation marker are shown.

Table 3. DNA methylation levels related to different biological markers in CLL patients

		ALU			LINE-1			SAT-α
	n	med [IQR]	p^a	n	med [IQR]	p^a	n	med [IQR]	p^a
CD38 expression
Negative (<30%)	36	21.9 [18.2–23.7]	0.71	36	67.6 [60.1–75.4]	0.94	34	84.9 [75.5–86.1]	0.78
Positive (≥30%)	34	22.1 [18.6–23.8]		35	68 [63.8–73.2]		32	84.1 [73.8–86.5]
ZAP-70 expression
Negative (<30%)	45	21.8 [17.9–23.7]	0.72	46	65.9 [59.1–73.4]	0.21	42	84.3 [68.4–86.4]	0.89
Positive (≥30%)	28	22.2 [18.9–23.6]		28	68.7 [64.3–75.2]		27	83.9 [69.5–86.3]
IgVH mutational status
Mutated (<98% homology)	28	21.4 [18.2–22.6]	0.49	28	66.8 [61.3–75.1]	0.40	26	84 [75.8–85.2]	0.51
Unmutated (≥98% homology)	47	22.1 [17.9–24.0]		48	66.8 [59.3–73.7]		45	84.3 [62.0–86.8]

IQR, interquartile range: twenty-fifth and seventy-fifth percentiles. aWilcoxon rank sum test for difference between categories.

Figure 2. Boxplot representation of absolute RNA expression levels of the three DNMTs(DNMT1, DNMT3a and DNMT3b) in healthy subjects and CLL patients as assessed by microarray analysis. A significantly decreased absolute median RNA expression level in CLLs vs. healthy donors was found for DNMT1 (p = 0.0084).

Figure 3. Cox-derived estimated curves according to the SAT-α methylation levels. The curves show the proportional hazard ratio estimate according to SAT-α methylation level below (n = 13, group A) or above (n = 47, group B) the cut-off value (69.0%5mC).

Table 4. Multivariate Cox proportional hazards analysis of Sat-α methylation level along with either CD38 or ZAP-70 expression or IgVH mutational status as predictors of TFS

	HR (95% CI)	p
Sat-α methylation	4.6 [2.0–10.1]	<0.0001
CD38 positive	1.6 [0.8–3.2]	NS
Sat-α methylation	4.5 [1.9–10.2]	<0.0001
ZAP-70 positive	1.3 [0.6–2.7]	NS
Sat-α methylation	4.4 [2.0–9.9]	<0.0001
IgVH unmutated	1.6 [0.7–3.6]	NS
Sat-α methylation	4.6 [1.9–11.1]	0.0006
17p13.1 deletion	1.3 [0.3–4.4]	NS

HR, hazard ratio; CI, confidence interval; NS, not significant.