Quantitative assessment of aberrant P16^INK4a methylation in ovarian cancer: a meta-analysis based on literature and TCGA datasets

Figures & data

Figure 1 Flow diagram of study selection.

Table 1 Characteristics of studies included for the association between P16^INK4a methylation and ovarian cancer risk

Authors	Year	Country	Geographical location	Sample sizea	Case number				Age (years)d	Sample type	Method	Methylation site	NOS score
					C (M/n)	LMP (M/n)	B (M/n)	NT (M/n)
Moselhy et al^Citation17	2015	Saudi Arabia	Asia	Small	14/18	NA	12/32	NA	52.3±12.1	FFPET	MSP	Promoter	7
Bhagat et al^Citation20	2014	India	Asia	Large	58/134	5/23	11/26	0/15	49.55±9.72	FFT	qMSP	Promoter	7
Ozdemir et al^Citation24	2012	Turkeyb	Asia	Large	1/75	NA	NA	0/75	NA	Tissue	MS-MLPA	Promoter	7
Ho et al^Citation37	2012	Taiwan	Asia	Small	1/47	NA	6/29	NA	50(32–66)	FFT	MS-MLPA	Promoter	7
Cuľbová et al^Citation38	2011	Slovakia	Europe	Small	5/13	0/2	5/19	NA	54.8 (34–74)	FFT	MSP	Promoter	6
Abou-Zeid et al^Citation22	2011	Egyptc	Africa	Large	21/52	NA	9/43	4/40	60(49–74)	FFT	qMSP	Promoter	7
Gu et al^Citation39	2009	China	Asia	Large	8/87	NA	13/42	NA	51(21–69)	Tissue	MethyLight	Promoter	7
Shen et al^Citation25	2008	China	Asia	Large	13/63	NA	NA	0/30	52.8 (33–76)	FFT	MSP	Promoter	6
Wu et al^Citation40	2007	Norway	Europe	Large	0/52	0/2	0/2	NA	NA	FFT	MSP	Promoter	6
Tam et al^Citation26	2007	HongKong	Asia	Large	17/89	1/16	1/19	4/16	53.1±1.4	FFT	MSP	Promoter	7
Wiley et al^Citation42	2006	Italy	Europe	Large	89/215	4/19	NA	NA	57.7±11.4	FFT	MSP	Promoter	7
Li et al^Citation27	2006	China	Asia	Small	6/18	NA	NA	0/10	NA	Tissue	MSP	Promoter	5
Makarla et al^Citation29	2005	USA	America	Small	7/23	5/23	3/23	0/16	51.5 (20–86)	FFT	MSP	Promoter	7
Liu et al^Citation28	2005	USA	America	Large	13/52	NA	NA	15/40	61.5±9.4	FFT	MSP	Promoter	5
Dhillon et al^Citation23	2004	India	Asia	Small	10/25	NA	NA	1/75	NA	Tissue	MSP	Promoter	7
Rathi et al^Citation30	2002	USA	America	Small	5/49	NA	NA	0/16	56(40–79)	FFT	MSP	Promoter	7
Strathdee	2001	UK	Europe	Large	0/93	NA	NA	0/18	NA	FFT	MSP	Promoter	6
et al^Citation46
Brown et al^Citation45	2001	UK	Europe	Small	0/30	0/13	0/14	NA	NA	FFT	MSP	Promoter	5
McCluskey et al^Citation47	1999	USA	America	Small	21/37	11/15	14/20	NA	NA	FFT	MSP	Promoter	6
Shih et al^Citation49	1997	Australia	Oceania	Small	0/45	0/3	0/2	NA	NA	Tissue	Southern analysis	Promoter	5

Notes:

a We defined n<50 as small size and ≥50 as large size.

b Turkey is a transcontinental Eurasian country and is usually assigned to Asia internationally.

c Egypt is a transcontinental country spanning the northeast corner of Africa and southwest corner of Asia, usually assigned to Africa internationally.

d Age data are presented as mean ± SD or median (IQR).

Abbreviations: B, benign tissues; BL, borderline; C, cancer tissues; FFPET, formalin fixed and paraffin embedded tissues; FFT, fast frozen tissues; LMP, low malignant potential or borderline tumor tissues; M, methylated; MS-MLPA, methylation-specific multiplex ligation-dependent probe amplification; MSP, methylation-specific PCR; n, number of patients in the group; NA, not available; NT, normal tissues; NOS, Newcastle Ottawa Scale; qMSP, real-time quantitative MSP.

Table 2 Characteristics of studies included for the association between P16^INK4a methylation and clinicopathological features of ovarian cancer

Authors	Year	Country	Geographical location	Number of patients	Age (years)a	Tumor stage		Tumor grade		Histological subtype
						I–II (M/n)	III–IV (M/n)	1–2 (M/n)	3 (M/n)	Serous (M/n)	Non-serous (M/n)
Bhagat et al^Citation20	2014	India	Asia	134	49.55±9.72	19/41	39/93	18/45	40/89	32/76	26/58
Cuľbová et al^Citation38	2011	Slovakia	Europe	13	54.8 (34–74)	NA	NA	NA	NA	2/6	3/7
Shen et al^Citation25	2008	China	Asia	63	52.8 (33–76)	4/22	9/41	6/36	7/27	7/34	6/29
Yang et al^Citation41	2006	Hong Kong	Asia	49	48.8 (26–79)	4/24	5/25	6/22	3/25	3/17	6/32
Makarla et al^Citation29	2005	USA	America	23	51.5 (20–86)	NA	NA	NA	NA	2/9	5/14
Liu et al^Citation28	2005	USA	America	52	61.5±9.4	NA	NA	10/41	3/11	NA	NA
Katsaros et al^Citation43	2004	Italy	Europe	249	57(19–82)	22/68	68/152	26/75	64/141	40/86	50/141
Hashiguchi et al^Citation44	2001	Japan	Asia	46	NA	4/21	2/20	7/33	0/13	2/14	5/32
McCluskey et al^Citation47	1999	USA	America	29	NA	NA	NA	NA	NA	1/14	1/15
Milde-Langosch et al^Citation48	1998	Germany	Europe	44	NA	NA	NA	NA	NA	3/11	13/33

Note:

a Age data are presented as mean ± SD or median (IQR).

Abbreviations: M, methylated; n, number of patients in the group; NA, not available.

Figure 2 Forest plots for the association between P16^INK4a methylation and ovarian cancer risk.

Notes: (A) Cancer tissues vs normal tissues; (B) cancer tissues vs benign tissues; (C) cancer tissues vs LMP tissues.

Abbreviations: LMP, low malignant potential or borderline tumor tissues; M–H, Mantel–Haenszel.

Figure 3 Forest plots for the association between P16^INK4a methylation and ovarian diseases.

Notes: (A) Benign tissues vs normal tissues; (B) LMP tissues vs normal tissues.

Abbreviations: LMP, low malignant potential or borderline tumor tissues; M–H, Mantel–Haenszel.

Table 3 Meta-regression and subgroup analyses of P16^INK4a methylation in comparison of cancer tissues vs normal tissues

Stratified analysis	Number of studies	Pooled OR (95% CI)		Meta-regression	Heterogeneity
		Random	Fixed	P-value	I^2 (%)	P-value
Publication year				0.376
≤2005	4	5.69 (0.42–76.14)	2.17 (1.17–4.05)		84	0.0003
>2005	6	4.71 (1.30–17.07)	4.65 (2.32–9.30)		56	0.05
Geographical location				0.161
Asia	6	7.85 (1.33–46.32)	5.87 (2.70–12.78)		70	0.005
America	3	2.31 (0.24–22.01)	1.15 (0.56–2.37)		68	0.05
Africa	1	6.10 (1.89–19.69)	6.10 (1.89–19.69)		–	–
Method				0.651
MSP	7	4.49 (0.97–20.64)	2.33 (1.38–3.92)		77	0.0003
Others	3	6.79 (2.43–18.94)	8.11 (2.93–22.40)		0	0.57
Case sample size				0.041
<50	4	17.21 (4.54–65.28)	15.75 (4.05–61.34)		0	0.58
≥50	6	2.21 (1.33–3.67)	2.74 (0.71–10.53)		75	0.001

Abbreviations: MSP, methylation-specific PCR; OR, odds ratio.

Figure 4 Forest plots for the association between P16^INK4a methylation and clinicopathological features in ovarian cancer.

Notes: (A) Age; (B) clinical stage; (C) tumor grade; (D) histological subtype.

Abbreviation: M–H, Mantel–Haenszel.

Figure 5 Forest plots for P16^INK4a methylation on survival analysis in univariate and multivariate Cox regression model.

Notes: (A) PFS in univariate Cox regression model; (B) PFS in multivariate Cox regression model; (C) OS in univariate Cox regression model; (D) OS in multivariate Cox regression model.

Abbreviations: OS, overall survival; PFS, progression-free survival; SE, standard error.

Figure 6 Sensitivity analyses and Begg’s test for publication bias of P16^INK4a methylation during the carcinogenesis of ovarian cancer.

Notes: (A and D) Cancer tissues vs normal tissues; (B and E) sensitivity analysis for the comparison of cancer tissues vs benign tissues; (C and F) sensitivity analysis for the comparison of cancer tissues vs LMP tissues.

Abbreviations: LMP, low malignant potential or borderline tumor tissues; SE, standard error.

Table 4 Methylation of P16^INK4a CpG sites on Illumina HumanMethylation 27 BeadChip from TCGA datasets

Probe (Illumina HumanMethylation 27)	CpG island location (chromosome: DNA range)	Normal tissue beta value (mean, n=12)	Tumor tissue beta value (mean, n=582)	Adjusted P-valuea	Pearson correlation coefficient	Adjusted P-valueb
cg00718440	9: 21983444–21986348	0.016	0.016	0.960249	0.194104	0.001719
cg03079681	9: 21983444–21986348	0.015	0.026	<0.000001	0.012972	1.0
cg07752420	9: 21958106–21958899	0.149	0.653	<0.000001	0.569887	<0.000001
cg09099744	9: 21958106–21958899	0.099	0.642	<0.000001	0.630768	<0.000001
cg10895543	9: 21958106–21958899	0.120	0.651	<0.000001	0.624147	<0.000001
cg11653709	9: 21958106–21958899	0.144	0.610	<0.000001	0.555400	<0.000001
cg12840719	9: 21958106–21958899	0.092	0.594	<0.000001	0.627484	<0.000001
cg13479669	9: 21983444–21986348	0.045	0.027	0.004226	–0.150891	0.0333435
cg26673943	9: 21983444–21986348	0.047	0.056	0.042428	–0.269361	<0.000001

Notes:

a P-value of t-test of the difference between normal tissue beta value and tumor tissue beta value.

b P-value of Pearson’s correlation between the tumor tissues beta value and CDKN2A expression (n=368).

Abbreviation: TCGA, The Cancer Genome Atlas.

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