Epigenetic analysis of sporadic and Lynch-associated ovarian cancers reveals histology-specific patterns of DNA methylation

Figures & data

Figure 1. Flowchart of this investigation.

Figure 2. Average Dm values from MS-MLPA analyses on non-serous and serous ovarian carcinomas and the corresponding normal tissue references. Asterisks denote significantly elevated methylation in tumor vs. normal tissue by t-test for independent samples. The average Dm values of tumor DNAs may in fact be somewhat higher than those shown if possible “contamination” with normal cells is taken into account (see Materials and Methods).

Table 1. Clinicopathological data of sporadic and Lynch-associated ovarian carcinoma

	Sporadic				Lynch-associated^c
	Clear cell	Endometrioid^a	Serous	Total	Clear cell	Endometrioid	Total
No. of cases	36	28	20	84	7	12	19
Grade
G1	NA	11	0	11	NA	6	6
G2 and G3	NA	17	20	37	NA	5	5
Stage
I and II	22	14	3	39	6	9	15
III and IV	14	14	17	45	1	3	4
Overall MMR status
MMR deficient	6 (17%)	4 (14%)	1 (5%)	11 (13%)	7 (100%)	11 (92%)	18 (95%)
MMR proficient	30 (83%)	24 (86%)	19 (95%)	73 (87%)	0 (0%)	1 (8%)	1 (5%)
Average no. of TSGs methylated out of 24^b	2.5	3.0	0.6		5.0	3.8
Average no. of TSGs methylated out of 13^c	6.7	6.4	2.8		8.3	6.4

^aOne of the Lynch endometrioid tumors is borderline tumor, which is not graded.

^bNiskakoski et al.¹⁸

^cThe predisposing mutation affected MLH1 in 15 and MSH2 in 4 cases (see Niskakoski et al.¹⁸ for details).

Table 2. Hypermethylation frequencies in different patient groups

		Percentage of hypermethylated tumors^a
Tumor category	Number of tumors	RSK4	SPARC	PROM1	WT1	CABLES1	HOXA10	HOXA9	WT1-AS	SFRP2	OPCML	SFRP5	MIR34B	let-7a-3
(a) Lynch clear cell	7	100	86	71	0	14	71	100	100	43	100	14	71	57
(b) Lynch endometrioid	12	83	75	33	8	0	67	92	92	25	83	8	42	33
(c) Sporadic clear cell	36	58	86	28	8	8	78	94	94	33	86	31	22	39
(d) Sporadic endometrioid	28	64	64	54	21	4	64	79	64	39	71	25	36	57
(e) Sporadic serous	20	65	15	20	0	0	30	35	30	5	30	5	5	35
(f) Lynch non-serous ( = a + b)	19	89	79	47	5	5	68	95	95	32	89	11	53	42
(g) Sporadic non-serous ( = c + d)	64	61	77	39	14	6	72	56	81	36	80	28	28	47
P value ^b
I Sporadic versus hereditary
(a) vs. (c)		ns	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns
(b) versus (d)		ns	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns
(f) vs. (g)		ns	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns
II Sporadic non-serous versus serous
(g) vs. (e)		ns	< 0.001	ns	ns	ns	<0.05	<0.001	<0.001	ns	<0.001	ns	ns	ns
III Sporadic and Lynch non-serous versus sporadic serous
(f + g) vs. (e)		ns	< 0.001	ns	ns	ns	<0.05	<0.001	<0.001	ns	<0.001	ns	ns	ns

^a Using cutoffs determined by methylation in the respective normal tissues (Suppl. Table 4.)

^b Determined by Fisher exact test and corrected for multiple testing

ns = Nonsignificant

Figure 3. Distribution of methylation dosage ratios (Dm values) for RSK4, SPARC, and HOXA9 in endometrioid ovarian carcinomas (sporadic and Lynch-associated combined) stratified by grade (low refers to grade 1 and high to grades 2 and 3). The horizontal line denotes the median and each triangle represents the Dm value of individual data point. Significance values by t-test for independent samples are shown.

Figure 4. Correlation analysis of expression (Y-axis, RMA normalized values for protein coding genes and quantile normalized values for MIR34B from arrays) and methylation (X-axis, Dm values from MS-MLPA). The analysis includes cancer cell lines and normal tissue references for which high molecular weight DNA and RNA were available. Data points for normal tissues predominantly clustered in the left top quadrants, compatible with low methylation and high expression. Cancer cells with high degree of methylation often showed low expression (hence, were located in the right bottom quadrants), whereas cancer cells with low methylation showed high or low expression depending on the intrinsic properties of the genes and tissue types in question. While methylation for all these genes significantly correlated with transcriptional repression overall, subsets of specimens occasionally showed transcriptional regulation apparently unrelated to methylation (see, e.g., MIR34B).