CDH1, DLEC1 and SFRP5 Methylation Panel As a Prognostic Marker for Advanced Epithelial Ovarian Cancer

Figures & data

Table 1.  Characteristics of advanced-stage patients.

Clinical items	Value
Patient numbers	177
Age (years old)	55.4 + 11.4
CA125 (U/ml)	1046.5 (16.6–39,370)
Histology:
– Serous	121 (68.4%)
– Endometrioid	24 (13.5%)
– Clear cell	32 (18.1%)
Grade:
– Low	21 (11.8%)
– High	124 (70.1%)
– Not available	32 (18.1%)
Residual tumor size after debulking surgery:
– <1 cm	91 (51.4%)
– ≥1 cm	86 (48.6%)
Median follow-up time (months)	32 (1–78)
Frequency of gene methylation:
– CDH1	55 (31.1%)
– DLEC1	116 (65.5%)
– SFRP5	34 (19.2%)
– ≥2 methylated gene	54 (30.5%)

Age is presented as mean ± standard error. Follow-up time is presented as median (minimum–maximum). Other values are presented as number (percentage).

CA125: Cancer antigen 125.

Figure 1.  Capillary electrophoresis of methylation-specific PCR for DLEC1.

Gene methylation of DLEC1 was found in patients 1, 2 and 3, while patients 4 and 5 had unmethylated DLEC1.

^†Positive findings of methylation-specific PCR products.

M: Primers for the methylated gene promoter; U: Primers for the unmethylated gene promoter.

Table 2.  Correlations between gene methylation and clinical parameters.

Our gene panel	Histology				Grade^†				FIGO stage
	Serous (n = 121)	N-serous^‡ (n = 56)	p-value	B–H p-value	Low (n = 21)	High (n = 124)	p-value	B–H p-value	Early (n = 0)	Advanced (n = 177)	p-value	B–H p-value
CDH1	40 (33.1%)	15 (26.8%)	0.486	0.486	3 (14.3%)	40 (32.3%)	0.123	0.492	NA	55 (31.1%)	NA	NA
DLEC1	83 (68.6%)	33 (58.9%)	0.236	0.472	13 (61.9%)	81 (65.3%)	0.807	0.638	NA	116 (65.5%)	NA	NA
SFRP5	9 (7.4%)	25 (44.6%)	<0.001	0.004	5 (23.8%)	17 (13.7%)	0.319	1.000	NA	34 (19.2%)	NA	NA
≥2 methylated genes	34 (28.1%)	20 (35.7%)	0.380	0.486	6 (28.6%)	36 (29.0%)	1.000	1.000	NA	54 (30.5%)	NA	NA
	Recurrence				Chemoresponse				Outcome
	No (n = 50)	Yes (n = 127)	p-value	B–H p-value	Sensitive (n = 110)	Resistant (n = 67)	p-value	B–H p-value	Alive (n = 91)	Death (n = 86)	p-value	B–H p-value
CDH1	10 (20.0%)	45 (35.4%)	0.049	0.071	28 (25.5%)	27 (40.3%)	0.045	0.090	27 (29.7%)	28 (32.6%)	0.746	0.746
DLEC1	27 (54.0%)	89 (70.1%)	0.053	0.071	68 (61.8%)	48 (71.6%)	0.196	0.261	57 (62.6%)	59 (68.6%)	0.432	0.576
SFRP5	10 (20.0%)	24 (18.9%)	0.836	0.836	19 (17.3%)	15 (22.4%)	0.435	0.435	14 (15.4%)	20 (23.3%)	0.252	0.504
≥2 methylated genes	6 (12.0%)	48 (37.8%)	0.001	0.004	25 (22.7%)	29 (43.3%)	0.007	0.028	21 (23.1%)	33 (38.4%)	0.034	0.136

† The histological grade was not available in 32 patients. There were 145 patients with available histological grade who were analyzed in the group.

‡ N-serous indicated nonserous. p-value was calculated by the Fisher’s exact test; B–H p-value indicated Benjamini–Hochberg multiple testing correction p-value.

FIGO: International Federation of Gynecology and Obstetric; NA: Not available.

Figure 2.  Kaplan–Meier analysis of disease-free survival and overall survival of all ovarian cancer patients by gene methylation.

(A) Patients with CDH1 methylation had a significantly shorter disease-free survival (DFS; p = 0.021; log-rank test). (B) Patients with CDH1 methylation did not have significant different overall survival (OS; p = 0.313; log-rank test). (C) Patients with DLEC1 methylation had a significantly shorter DFS (p = 0.026; log-rank test). (D) Patients with DLEC1 methylation did not have significant different OS (p = 0.183; log-rank test). (E) Patients with SFRP5 methylation did not have significant different DFS (p = 0.524; log-rank test). (F) Patients with SFRP5 methylation had a significantly shorter OS (p = 0.026; log-rank test). (G) Patients with two or three methylated genes had a significantly shorter DFS (p < 0.001; log-rank test). (H) Patients with two or three methylated genes had a significantly shorter OS (p = 0.001; log-rank test).

Figure 3.  Kaplan–Meier analysis of disease-free survival and overall survival of serous ovarian cancer patients by gene methylation.

(A) Patients with CDH1 methylation did not have significant different disease-free survival (DFS; p = 0.145; log-rank test). (B) Patients with CDH1 methylation did not have significant different overall survival (OS; p = 0.758; log-rank test). (C) Patients with DLEC1 methylation had shorter DFS close to the significance level (p = 0.050; log-rank test). (D) Patients with DLEC1 methylation did not have significant different OS (p = 0.152; log-rank test). (E) Patients with SFRP5 methylation did not have significant different DFS (p = 0.781; log-rank test). (F) Patients with SFRP5 methylation did not have significant different OS (p = 0.514; log-rank test). (G) Patients with two or three methylated genes had a significantly shorter DFS (p = 0.004; log-rank test). (H) Patients with two or three methylated genes did not have significant different OS (p = 0.067; log-rank test).

Table 3.  Cox regression model for the risk factors for recurrence and death in all patients.

Clinical items and our gene panel	n	Recurrence						Death
		Univariate			Multivariate			Univariate			Multivariate
		HR (95% CI)	p-value	B–H	HR (95% CI)	p-value	B–H	HR (95% CI)	p-value	B–H	HR (95% CI)	p-value	B–H
Histology:
– Serous adenocarcinoma	121	1 (reference)						1 (reference)
– Endometrioid adenocarcinoma	24	0.92 (0.52–1.63)	0.770	0.770				1.21 (0.62–2.36)	0.572	0.572
– Clear cell carcinoma	32	1.38 (0.87–2.20)	0.168	0.224				1.43 (0.79–2.60)	0.241	0.386
Grade:
– Low	21	1 (reference)						1 (reference)
– High	124	1.98 (1.05–3.72)	0.035	0.070				1.23 (0.61–2.50)	0.561	0.572
Residual tumor size after debulking surgery:
– ≥1 cm	86	1 (reference)			1 (reference)			1 (reference)			1 (reference)
– <1 cm	91	0.59 (0.41–0.84)	0.003	0.012	0.60 (0.42–0.86)	0.006	0.006	0.53 (0.34–0.83)	0.005	0.020	0.55 (0.35–0.85)	0.008	0.008
CDH1:
– Unmethylated	122	1 (reference)						1 (reference)
– Methylated	55	1.50 (1.04–2.18)	0.032	0.070				1.26 (0.79–1.99)	0.335	0.447
DLEC1:
– Unmethylated	61	1 (reference)						1 (reference)
– Methylated	116	1.49 (1.01–2.20)	0.045	0.072				1.34 (0.83–2.16)	0.232	0.386
SFRP5:
– Unmethylated	143	1 (reference)						1 (reference)
– Methylated	34	1.08 (0.68–1.71)	0.754	0.770				1.73 (1.02–2.93)	0.041	0.109
CDH1, DLEC1, SFRP5:
– <2 methylated gene	123	1 (reference)			1 (reference)			1 (reference)			1 (reference)
– ≥2 methylated gene	54	1.97 (1.36–2.84)	<0.001	0.008	1.91 (1.33–2.76)	0.001	0.002	2.02 (1.30–3.15)	0.002	0.016	1.96 (1.26–3.06)	0.003	0.006

n indicated patient number; B–H indicated Benjamini–Hochberg multiple testing correction p-value.

HR: Hazard ratio.

Table 4.  Cox regression model for the risk factors for recurrence and death in serous-type patients.

Clinical items and our gene panel	n	Recurrence						Death
		Univariate			Multivariate			Univariate			Multivariate
		HR (95% CI)	p-value	B–H	HR (95% CI)	p-value	B–H	HR (95% CI)	p-value	B–H	HR (95% CI)	p-value	B–H
Grade:
– Low	16	1 (reference)			1 (reference)			1 (reference)
– High	105	2.27 (1.13–4.57)	0.022	0.044	1.94 (0.96–3.93)	0.066	0.066	1.60 (0.72–3.53)	0.253	0.379
Residual tumor size after debulking surgery:
– ≥1 cm	69	1 (reference)			1 (reference)			1 (reference)			1 (reference)
– <1 cm	52	0.57 (0.37–0.87)	0.010	0.030	0.64 (0.42–0.98)	0.045	0.066	0.37 (0.21–0.65)	0.001	0.006	0.37 (0.21–0.65)	0.001	0.001
CDH1:
– Unmethylated	81	1 (reference)						1 (reference)
– Methylated	40	1.37 (0.88–2.11)	0.160	0.192				1.09 (0.63–1.88)	0.759	0.759
DLEC1:
– Unmethylated	38	1 (reference)						1 (reference)
– Methylated	83	1.57 (0.98–2.50)	0.059	0.089				1.51 (0.85–2.65)	0.158	0.316
SFRP5:
– Unmethylated	112	1 (reference)						1 (reference)
– Methylated	9	0.90 (0.41–1.95)	0.787	0.787				1.32 (0.57–3.09)	0.518	0.622
CDH1, DLEC1, SFRP5:
– <2 methylated gene	87	1 (reference)			1 (reference)			1 (reference)
– ≥2 methylated gene	34	1.85 (1.19–2.87)	0.006	0.030	1.67 (1.06–2.60)	0.023	0.066	1.65 (0.96–2.83)	0.072	0.216

n indicated patient number; B–H indicated Benjamini–Hochberg multiple testing correction p-value.

HR: Hazard ratio.

Figure 4.  Kaplan–Meier analysis of disease-free survival and overall survival of ovarian cancer patients from The Cancer Genome Atlas database.

(A) Patients with two or three methylated genes had a significantly shorter disease-free survival (p = 0.023; log-rank test). (B) Patients with two or three methylated genes had a significantly shorter overall survival (p = 0.003; log-rank test).

TGCA: The Cancer Genome Atlas.

Table 5.  Multivariate Cox regression model for the risk factors for recurrence and death in The Cancer Genome Atlas dataset.

Clinical items and our gene panel	Recurrence		Death
	HR (95% CI)	p-value	HR (95% CI)	p-value
Age	1.00 (0.99–1.01)	0.229	1.02 (1.01–1.03)	8.14 × 10^-5
Grade:
– I	0.38 (0.05–2.89)	0.346	0.98 (0.25–3.80)	0.976
– II	0.41 (0.06–3.02)	0.383	2.27 (0.72–7.12)	0.158
– III	0.40 (0.05–3.00)	0.372	3.28 (1.02–10.54)	0.046
Stage:
– II	1.34 (0.41–4.36)	0.630	1.33 (0.32–5.55)	0.698
– III	1.78 (0.57–5.59)	0.321	1.68 (0.41–6.81)	0.467
– IV	67.10 (6.27–715.59)	0.001	3.09 (0.28–34.25)	0.358
Our panel	1.37 (0.99–1.88)	0.058	1.59 (1.15–2.18)	0.0047

HR: Hazard ratio.