IL21R hypomethylation as a biomarker for distinguishing benign and malignant breast tumours

Figures & data

Table 1. Clinical characteristics of BC patients.

Characteristics	Type	n	%
Tumour subtype	Intraductal carcinoma	17	6.1
	Microinvasive ductal carcinoma	28	10.0
	Invasive ductal carcinoma	216	77.4
	Invasive lobular carcinoma	8	2.9
	Others	10	3.6
Tumour stage	Stage 0	17	6.1
	Stage I	104	37.3
	Stage II	125	44.8
	Stage III	31	11.1
	Unknown	2	0.7
Tumour size	Tis	17	6.1
	T1	142	50.9
	T2	113	40.5
	T3&T4	7	2.5
Lymph node involvement	pN0	187	67.0
	pN1	59	21.2
	pN2	19	6.8
	pN3	10	3.6
	Unknown	4	1.4
ER status	ER-negative	67	24.0
	ER-positive	210	75.3
	Unknown	2	0.7
PR status	PR-negative	111	39.8
	PR-positive	166	59.5
	Unknown	2	0.7
HER2 status	HER2-negative	112	40.1
	HER2-positive	164	59.8
	Unknown	3	0.1
Three receptor status	Triple-negative	22	7.9
	Non-triple-negative	255	91.4
	Unknown	2	0.7

Abbreviation: BC, breast cancer; ER, oestrogen receptor; PR, progesterone receptor; HER2, human epidermal growth factor receptor 2; Triple-negative, ER-, PR- and HER2-negative.

Figure 1. Study design and flow chart. The 29 fresh-frozen tissue samples of the discovery study were subjected to Illumina methylation EPIC 850K beadchip array and RNA-Sequencing. Cg04931655 within intron 1 of IL21R gene was selected by comprehensive analysis of binary omics data in a stepwise selection manner. The DNA methylation was well correlated with mRNA expression of IL21R gene in fresh-frozen tissue samples during the discovery study. We conducted a further independent validation study with FFPE tissue samples. Binary logistic regression analysis was performed to analyse the correlation between IL21R methylation and BC and receiver operating characteristic (ROC) curve analysis was used to verify the ability of IL21R methylation level in distinguishing benign and malignant breast tumours.

Abbreviation: BC, breast cancer; FFPE, formalin-fixed and paraffin-embedded; SNP, single nucleotide polymorphism; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight.

Figure 2. IL21R was hypomethylated in BC and negatively correlated with its mRNA expression in BC. (a) Methylation level of IL21R (cg04931655) detected by 850K beadchip array were lower in tissue samples from early-stage BC (stage 0-IIA, n = 11) than in benign breast tumours (n = 18).***p-value < 0.001. (b) mRNA expression levels of the IL21R gene measured by RNA-Sequencing were higher in BC than in benign breast tumours. ***p-value < 0.001. (c) mRNA expression levels of IL21R were negatively correlated with the methylation levels of cg04931655 evaluated by Spearman’s correlation coefficient. (d) Schematic diagram of IL21R amplicon and the locations of the 5 measurable CpG sites (Chr16:27414142 –27,414,328, build GRCh37/hg19, defined by the UCSC Genome Browser). Among them, CpG_2 represented cg04931655.

Table 2. Methylation difference of IL21R between benign breast tumour and BC.

CpG sites	Benign (n = 287)	BC (n = 279)	OR (95% CI)*	p-value*
	Median (IQR)	Median (IQR)	per-10% methylation
CpG_2	0.67 (0.55–0.72)	0.39 (0.27–0.54)	2.03 (1.78–2.32)	1.46E–25
CpG_3	0.84 (0.65–1.00)	0.53 (0.30–0.78)	1.29 (1.21–1.38)	5.69E–14
CpG_4	0.71 (0.61–0.77)	0.52 (0.36–0.68)	1.72 (1.52–1.95)	2.59E–18
CpG_5	0.80 (0.73–0.86)	0.61 (0.43–0.75)	1.89 (1.65–2.17)	9.35E–20
CpG_6	0.79 (0.66–0.86)	0.59 (0.44–0.75)	1.51 (1.36–1.67)	1.65E–15

*Logistic regression, adjusted for age and different batches for the measurements. The bold values indicate p-value <0.05.

Abbreviation: IQR, interquartile range.

Table 3. Correlation between IL21R methylation and the clinical characteristics of BC.

		Median of methylation levels
Clinical characteristics	Group (n)	CpG_2	CpG_3	CpG_4	CpG_5	CpG_6
Tumour subtype	Intraductal carcinoma (17)	0.52	0.63	0.57	0.66	0.61
	Microinvasive ductal carcinoma(28)	0.42	0.60	0.52	0.63	0.65
	Invasive ductal carcinoma (216)	0.39	0.51	0.52	0.60	0.58
	p-value^a	0.449	0.216	0.776	0.453	0.102
Tumour stage	Stage 0 (17)	0.52	0.63	0.57	0.66	0.61
	Stage I & II (229)	0.39	0.53	0.52	0.62	0.59
	Stage III (31)	0.44	0.52	0.53	0.59	0.56
	p-value^a	0.251	0.544	0.919	0.627	0.795
Tumour size	Tis (17)	0.52	0.63	0.57	0.66	0.61
	T1&T2 (255)	0.39	0.53	0.52	0.62	0.59
	T3&T4 (7)	0.44	0.40	0.48	0.40	0.62
	p-value^a	0.293	0.533	0.798	0.077	0.772
Lymph node involvement	pN0 (187)	0.38	0.51	0.51	0.59	0.59
	pN1 (59)	0.45	0.64	0.60	0.70	0.61
	pN2&pN3 (29)	0.44	0.54	0.53	0.60	0.56
	p-value^a	0.287	0.256	0.197	0.184	0.900
ER status	ER-negative (67)	0.34	0.40	0.37	0.45	0.47
	ER-positive (210)	0.41	0.59	0.55	0.66	0.63
	p-value^b	0.026	2.42E–04	2.00E–06	7.53E–08	5.30E–05
PR status	PR-negative (111)	0.37	0.44	0.42	0.50	0.52
	PR-positive (166)	0.42	0.62	0.58	0.67	0.64
	p-value^b	0.037	0.001	4.79E–07	2.24E–09	1.00E–06
HER2 status	HER2-negative (112)	0.40	0.57	0.53	0.66	0.63
	HER2-positive (164)	0.38	0.52	0.51	0.59	0.57
	p-value^b	0.893	0.119	0.409	0.024	0.183
Three receptor status	Triple-negative (22)	0.27	0.35	0.32	0.45	0.41
	Non-triple-negative (255)	0.40	0.57	0.53	0.63	0.60
	p-value^b	0.014	0.001	0.002	0.001	0.002

^aKruskal-Wallis test; ^b Mann-Whitney U test. The bold values indicate p-value < 0.05.

Figure 3. The methylation levels of IL21R in BC with different ER, PR, and HER2 status. IL21R methylation levels were significantly lower in BC the various ER (a), PR (b) and HER2 (c) statuses compared to the benign breast tumour groups. (d) Both triple-negative (ER-, PR- and HER2-negative) BC and non-triple-negative BC showed hypomethylation of the IL21R gene compared to the benign breast tumour groups. The p-values were calculated by logistic regression adjusted for age and batches of measurements. ***p-value < 0.001.

Figure 4. The clinical value of IL21R methylation in distinguishing BC cases from patients with benign breast tumour. The methylation levels of five CpG sites within IL21R gene were generated a prediction probability. (a) ROC curve analyses for the discriminatory power of IL21R methylation to distinguish BC cases from benign breast tumour subjects. (b) ROC curve analyses for the discriminatory power of IL21R methylation to distinguish BC cases at very early stage from benign breast tumour subjects. (c-j) ROC curve analyses for the discriminatory power of IL21R methylation to distinguish ER-negative (c), ER-positive (d), PR-negative (e), PR-positive (f), HER2-negative (g), HER2-positive (h), triple-negative (i) and non-triple-negative BC cases (j) from benign breast tumour subjects. The ROC analyses were calculated by logistic regression adjusted for age and different batches for the measurements. The grey lines represent the line of no discrimination.

Data availability statement

The datasets generated during and/or analysed during the current study are included in this publication and are available from the corresponding author on reasonable request.