Biomarkers for detection and prognosis of breast cancer identified by a functional hypermethylome screen

Figures & data

Table 1. Baseline characteristics of breast cancer patient cohorts

		Hopkins training cohort	Hopkins extended training cohort	Hopkins validation cohort	MDxHealth cohort
		n = 30	n = 97	n = 89	n = 132
Age, y	Median	53	54	54	57
	Interquartile range	45–61	46–63	44–63	48–66
Stage, no. (%)	DCIS	6 (20)	22 (21)	15 (17)	21 (16)
	Stage 1	6 (20)	31 (34)	24 (27)	19 (14)
	Stage 2	6 (20)	30 (31)	20 (23)	32 (24)
	Stage 3	6 (20)	6 (6)	24 (27)	51 (39)
	Stage 4	6 (20)	7 (7)	5 (6)	9 (7)
Grade, no. (%)	High	17 (57)	47 (49)	21 (24)
	Low to intermediate	13 (43)	42 (43)	32 (36)
	Unknown		8 (8)	36 (40)
Lymphovascular invasion, no. (%)	Present	9 (30)	20 (21)	27 (30)
	Absent	21 (70)	49 (50)	38 (43)
	Unknown		28 (29)	24 (27)
Perinodal invasion, no. (%)	Present	9 (30)	9 (9)	17 (19)
	Absent	21 (70)	25 (26)	8 (9)
	Unknown		63 (65)	64 (72)
Lymphnodes, no. (%)	Positive	17 (57)	24 (25)	41 (46)
	Negative	13 (43)	59 (60)	38 (43)
	Unknown		14 (15)	10 (11)
ER/PR receptor, no. (%)	Positive	21 (70)	45 (46)	46 (52)
	Negative	6 (20)	35 (36)	16 (18)
	Unknown	3 (10)	17 (18)	27 (30)
HER2 amplification, no. (%)	Positive	6 (20)	18 (19)	13 (15)
	Negative	18 (60)	50 (51)	55 (62)
	Unknown	6 (20)	29 (30)	21 (23)
Ki67 index, no. (%)	≥ 30%	11 (37)	33 (34)	34 (38)
	< 30%	10 (33)	34 (35)	30 (34)
	Unknown	9 (30)	30 (31)	25 (28)

Figure 1. Methylation frequencies of cancer specific methylated genes in primary BCs. Bar graph showing methylation frequencies (%) of 33 cancer specific methylated genes in the Hopkins training cohort (n = 30).

Table 2. Combinatorial beacon marker analysis for detection of breast cancers

(A) MDx Health (stages 0 to 4)
	#Cancer	#Normal	#Total	Sens*	Spec*
NDRG2	127	98	225	88.9	93.9
GREM1	127	99	226	69.3	91.9
TAC1	126	101	227	72.2	90.1
HOXD1	120	94	214	64.2	95.7
NDRG2 and GREM1	130	103	233	91.5	86.4
NDRG2 and TAC1	131	105	236	93.9	84.8
NDRG2 and HOXD1	129	98	227	94.6	89.8
GREM1 and TAC1	129	103	232	82.2	86.4
GREM1 and HOXD1	128	99	227	75.8	87.9
TAC1 and HOXD1	128	101	229	79.7	86.1
NDRG2 and GREM1 and HOXD1	130	103	233	94.6	82.5
NDRG2 and TAC1 and HOXD1	131	105	236	94.7	80.9
(B) MDx Health (DCIS/stage 0)
NDRG2 and HOXD1	21	100	121	85.7	89.9
(C) MDx Health (stages 0 to 1)
NDRG2 and HOXD1	40	100	140	90.0	89.9
(D) MDx Health (stages 0 to 2)
NDRG2 and HOXD1	70	100	170	92.9	89.9

* Maximum sensitivity/specificity trade offs were calculated using the fisher exact test

Table 3. Statistical association between clinocopathological characteristics and methylation frequencies (%) of genes in Hopkins extended training cohort

Hopkins extended training cohort (n = 97)
Stage*		Stage 0,1 and 2	Stage 3 and 4	p value
	CDO1	50.00	100	< 0.001
	KL	5.00	23	0.018
	SFRP1	24.00	54	0.026
	TAC1	48.00	100	< 0.001
	ZNF702	12.00	38	0.015
Grade*		Grade 1 and 2	Grade 3	p value
	CKM	17.00	36	0.038
	GREM1	29.00	57	0.010
Lymphovascular or Perinodal Invasion*		Absent	Present	p value
	TAC1	49.00	80	0.011
	TF	32.00	64	0.009
ER/PR receptor*		Positive	Negative	p value
	CRIP1	7.00	29	0.007
	TNFRSF11B	9.00	26	0.048
Ki67 Proliferation Index*		Low (< 30%)	High (≥ 30%)	p value
	ZNF702	9.00	28	0.042

* p values were computed using pearson chi 2 test

Figure 2. Single gene and gene combinations for prognostication of mortality risk in BC patients. (A) Forest plots depicting univariate and multivariate HRs and corresponding 95% CIs for overall mortality risk associated with DNA methylation of single or combinations of genes in Hopkins extended training cohort (n = 97) and (B) Hopkins validation cohort (n = 89). Vertical line at 1 indicates HR = 1. Single gene marker and selected statistically significant gene combinations are shown. (C) Univariate and multivariate Cox proportional hazards regression based survival curves for prognostic gene marker combination TAC1 and CKM in Hopkins extended training cohort and Hopkins validation cohort.

Figure 3. Distinct methylation frequency pattern for BC detection vs. BC prognostication marker. Methylation frequency of single marker genes (GREM1, HOXD1, NDRG2, TAC1, CDO1, CKM, CRIP1 and KL) was plotted against cancer stage as obtained in Hopkins training cohort (n = 30). Two groups, BC detection marker and BC prognostication marker, of genes showing distinct methylation frequency pattern were identified. Upper plot shows BC detection markers (GREM1, HOXD1, TAC1) that are characterized by methylation frequency ≥ 40% in early stage BC’s (DCIS and stage 1). Lower plot shows BC prognostication markers (CDO1, CKM, KL and TAC1) that are characterized by stage increasing methylation frequency. Note NDRG2, an early detection marker, did not reach the 40% methylation frequency cut off in early stage BCs by highly stringent MSP assay and CRIP1, a prognostic marker, did not follow the stage increasing methylation frequency pattern as seen with the other prognostic markers.