ANGPT2 promoter methylation is strongly associated with gene expression and prognosis in chronic lymphocytic leukemia

Figures & data

Table 1. Clinical and molecular characteristics of the present CLL cohort

Features	Sweden (n = 40)		Italy (n = 48)		p value	All patients (n = 88)
Age, years
Median	61		65		ns	61
Range	32–83		32–85			32–85
Follow up, months
Median	72		46		0.003	59
Range	11–214		0–155			0–214
TTFT, months
Median	9		35		ns	29
Range	0–136		0–138			0–138
	N° patients	%	N° patients	%		N° patients	%
Sex
Male	30/40	75	32/48	67	ns	62/88	71
Female	10/40	25	16/48	33		26/88	29
Binet stage
A	17/30	57	33/46	72	ns	50/76	66
B-C	13/30	43	13/46	28		26/76	34
IGHV mutational status
Mutated (< 98%)	20/40	50	28/48	58	ns	48/88	55
Unmutated (≥ 98%)	20/40	50	20/48	42		40/88	45
CD38 expression
CD38 negative (< 30%)	21/33	64	34/43	79	ns	55/76	72
CD38 positive (≥ 30%)	12/33	36	9/43	21		21/76	28
FISH stratification
Low risk	22/35	63	29/39	74	ns	51/74	69
Intermediate/high risk	13/35	37	10/39	26		23/74	31
Treatment
Yes	11/14	79	19/48	40	0.010	30/62	48
No	3/14	21	29/48	60		32/62	52
Death censored
Yes	27/39	69	10/48	21	< 0.001	37/87	42
No	12/39	31	38/48	79		50/87	58

Low FISH risk, no abnormalities or del(13q); Intermediate/high FISH risk, del(11q), del(17p) or trisomy 12. FISH, fluorescence in situ hybridization; IGHV, immunoglobulin heavy chain variable genes; TTFT, time to first treatment; ns, not statistically significant.

Figure 1. Kaplan-Meier curves for time to first treatment (TTFT) and overall survival (OS) in relation to ANGPT2 mRNA expression. In (A and B), 88 CLL patients were divided according to high and low ANGPT2 expression using the cut-off value 2.967×10⁻⁶ (relative mRNA expression of ANGPT2 to B2M). High ANGPT2 expressing cases show significantly shorter TTFT [(A) median 25 vs. 97 mo, p = 0.046; log-rank test) and OS [(B) median 59 vs 177 mo, p < 0.001; log-rank test] compared with low ANGPT2 expressing cases.

Figure 2. Location of the six CpG sites analyzed by pyrosequencing in relation to transcriptional start site of ANGPT2 gene. The circled region represents the studied CpG island of ANGPT2 gene which was predicted by the site www.urogene.org/methprimer using the following criteria: island size > 100, GC percent > 0.0, obs/exp CpG > 0.6. The start site and the direction of ANGPT2 transcription are indicated by the arrow. The below sequence shows in details the positions of the 6 CpG sites analyzed by pyrosequencing. The CpG site 2 is the same site identified in our previous methylation study.²⁶

Table 2. Methylation percentages of the 6 CpG sites located around the transcription start site of the ANGPT2 gene measured by pyrosequencing in 88 CLL patients

Methylation percentage	CpG site 1	CpG site 2	CpG site 3	CpG site 4	CpG site 5	CpG site 6	Average of 6 CpG sites
Mean ± SD	78 ± 19	49 ± 20	83 ± 25	69 ± 21	40 ± 19	67 ± 15	64 ± 17
Median Range	87 9–100	48 6–100	100 13–100	72 8–100	36 0–100	73 12–89	70 8–94

SD, standard deviation.

Figure 3. Relations between ANGPT2 CpG methylation, ANGPT2 mRNA expression and IGHV mutational status in 88 CLL patients. The percentage of ANGPT2 methylation (average of all 6 CpG sites) is plotted against ANGPT2 mRNA expression (A) and IGHV mutational status (B). The horizontal lines inside boxes indicate the median values. (A) CLL cases are divided in high and low ANGPT2 expression according to cut off value 2.967×10⁻⁶ (relative mRNA expression of ANGPT2 to B2M). High ANGPT2 expressing patients have significantly lower ANGPT2 methylation than low ANGPT2 expressing cases (median 50 vs. 76%; p < 0.001; Mann–Whitney test). (B) IGHV-unmutated patients have significantly lower ANGPT2 methylation than mutated cases (median 50 vs. 76%; p < 0.001; Mann–Whitney test).

Figure 4. Kaplan-Meier curves for time to first treatment (TTFT) and overall survival (OS) in relation to ANGPT2 CpG methylation. In (A and B), 88 CLL patients were divided according to high and low ANGPT2 methylation using the cut-off value 73% (methylation percentage, average of all 6 CpG sites). Low ANGPT2 methylation cases had significantly shorter TTFT [(A) median 23 vs. 97 mo, p = 0.022; log-rank test] and OS [(B) median 79 vs. 177 mo, p = 0.018; log-rank test] compared with cases with high ANGPT2 methylation.

Table 3. Associations between ANGPT2 methylation status and CLL prognostic factors

	Low ANGPT2 methylation (< 73%)	p value
	N° patients (percentage)
Clinical stage
Binet A	25/50 (50%)	ns
Binet B-C	15/26 (58%)
IGHV mutational status
Mutated (< 98%)	15/48 (31%)	< 0.001
Unmutated (≥ 98%)	35/40 (87%)
CD38
CD38 negative (< 30%)	28/55 (51%)	ns
CD38 positive (≥ 30%)	15/21 (71%)
FISH stratification
Low risk	27/51 (53%)	0.039
Intermediate/high risk	18/23 (78%)
ANGPT2 mRNA expression
Low (< 2.967×10^−6)	15/48 (31%)	< 0.001
High (≥ 2.967×10^−6)	35/40 (87%)

Low FISH risk, no abnormalities or del(13q); Intermediate/high FISH risk, del(11q), del(17p) or trisomy 12. FISH, fluorescence in situ hybridization; IGHV, variable region of immunoglobulin heavy chain genes; ns, not statistically significant.

Figure 5. The relative expression of ANGPT2 gene to B2M after 72 h of DAC/TSA treatment in the MEC1 and RAMOS cell lines. Real-time PCR analyses showing re-expression of the methylated ANGPT2 gene in the MEC1 and RAMOS cells after treatment with increasing concentrations of the methyl transferase inhibitor 5-aza-2’-deoxycytidine (DAC) are represented in (A and B). In (A), mean ANGPT2 mRNA levels of MEC1 cells are 0 in control (no treatment), 3.39×10⁻⁶ with 5 μM DAC and 5.68×10⁻⁶ with 10 μM DAC. The last MEC1 sample with highest expression (1.24×10⁻⁵, mean ANGPT2 mRNA level) was treated with a combination of DAC (10 μM) and the histone deacetylase inhibitor, Trichostatin A (TSA). In (B), mean ANGPT2 mRNA levels of RAMOS cells are 0 in the control, 4.87×10⁻⁵ with 5 μM DAC and 7.54×10⁻⁵ with 10 μM DAC. The RAMOS sample with highest expression (1.08×10⁻⁴, mean ANGPT2 mRNA level) was obtained with 10 μM DAC plus TSA treatment. All data are represented in graph as mean ± standard deviation of at least 2 independent experiments.

Kanduri M, Cahill N, Göransson H, Enström C, Ryan F, Isaksson A, et al. Differential genome-wide array-based methylation profiles in prognostic subsets of chronic lymphocytic leukemia. Blood 2010; 115:296 - 305; http://dx.doi.org/10.1182/blood-2009-07-232868; PMID: 19897574

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