Supplementary Appendix Supplementary tables: 7 Supplementary figures: 2 Supplementary Table S1

Supplementary Appendix
Supplementary tables: 7

Supplementary figures: 2

Supplementary Table S1: Clinicopathological Characteristics of 24 Nasopharyngeal Carcinoma (NPC) Tissues and 24 Non-cancer Nasopharyngitis Biopsy Tissues (NCNBT)

Variables	NPC N (%)	NCNBT N (%)
Age (Mean ± SD)	42.8± 10.5	43.1±10.2
Gender
Male	15 (63)	15(63)
Female	9 (37)	9(37)
T stage
T1	0
T2	4(17)
T3	15(63)
T4	5(20)
N stage
N0	3(13)
N1	12(50)
N2	9(37)
N3	0
TNM stage
I-II	2(8)
III-IV	22(92)
WHO pathologic type
Undifferentiated non-keratinizing	24(100)

TNM: tumor-node-metastasis.

Supplementary Table S2: Clinical Characteristics of Nasopharyngeal Carcinoma Patients According to Gene Methylation Panel in Training, Validation, and Independent Cohorts.

	Training Cohort (n=151)		P-value^*	Validation Cohort (n=150)		P-value^*	Independent Cohort (n=153)		P-value^*
Characteristic	Low Methylation Group (%)	High Methylation Group (%)		Low Methylation Group (%)	High Methylation Group (%)		Low Methylation Group (%)	High Methylation Group (%)
	n=75	n=76		n=79	n=71		n=69	n=84
Age (mean ± SD)
<45	35 (46.7)	36 (47.4)	0.931	39 (49.4)	32 (45.1)	0.599	22 (31.9)	28 (33.3)	0.849
≥45	40 (53.3)	40 (52.6)		40 (50.6)	39 (54.9)		47 (68.1)	56 (66.7)
Sex
Male	56 (74.7)	58 (76.3)	0.814	58 (73.4)	54 (76.1)	0.711	46 (66.7)	63 (75.0)	0.257
Female	19 (25.3)	18 (23.7)		21 (26.6)	17 (23.9)		23 (33.3)	21 (25.0)
WHO pathologic type
Undifferentiated non-keratinizing	74 (98.7)	73 (96.1)	0.317	78 (98.7)	70 (98.6)	0.939	61 (88.4)	78 (92.9)	0.342
Differentiated non-keratinizing	1 (1.3)	3 (3.9)		1 (1.3)	1 (1.4)		8 (11.6)	6 (7.1)
T Stage
T1	11 (14.7)	5 (6.6)	0.277	11 (13.9)	8 (11.3)	0.505	14 (20.3)	15 (17.9)	0.775
T2	20 (26.7)	20 (26.3)		21 (26.6)	20 (28.2)		35 (50.7)	38 (45.2)
T3	28 (37.3)	27 (35.5)		29 (36.7)	20 (28.2)		13 (18.8)	21 (25.0)
T4	16 (21.3)	24 (31.6)		18 (22.8)	23 (32.4)		7 (10.1)	10 (11.9)
N Stage
N0	14 (18.7)	10 (13.2)	0.629	10 (12.7)	16 (22.5)	0.193	9 (13.0)	19 (22.6)	0.372
N1	36 (48.0)	35 (46.1)		37 (46.8)	37 (52.1)		22 (31.9)	26 (31.0)
N2	15 (20.0)	20 (26.3)		22 (27.8)	14 (19.7)		32 (46.4)	30 (35.7)
N3	10 (13.3)	11 (14.5)		10 (12.7)	4 (5.6)		6 (8.7)	9 (10.7)
TNM Stage
I	1 (1.3)	2 (2.6)	0.576	2 (2.5)	3 (4.2)	0.815	1 (1.4)	1 (1.2)	0·589
II	18 (24.0)	12 (15.8)		20 (25.3)	19 (26.8)		17 (24.6)	21 (25.0)
III	30 (40.0)	31 (40.8)		32 (40.5)	24 (33.8)		39 (56.5)	40 (47.6)
IV	26 (34.7)	31 (40.8)		25 (31.6)	25 (35.2)		12 (17.4)	22 (26.2)
Concurrent chemotherapy
Yes	42 (56.0)	44 (55.3)	0.927	42 (53.2)	45 (63.4)	0.206	45 (65.2)	57 (67.9)	0.595
No	33 (44.0)	34 (44.7)		37 (46.8)	26 (36.6)		24 (34.8)	27 (32.1)

*χ² test or Fisher’s exact test. TNM tumor-node-metastasis, RT radiotherapy

Supplementary Table S3: Primer Sequences of 28 Genes and GAPDH

Gene	Forward primer (5'-3')	Reverse primer (5'-3')	Tm (°C)	Product (bp)
ALDH1A3	CTCTGCTGTGGGAACACCAT	TTTCCAACCTCTGTGGAGCC	60	206
CCNA1	GCCTGGCAAACTATACTGTGA	AAGCCTTGTACTTCTCCCTAAT	60	165
CD38	GAGGCCTGGGTGATACATGG	AAAACAACCACAGCGACTGG	60	210
CD44	CCCCAGCAACCCTACTGATG	TTGCCTCTTGGTTGCTGTCT	60	211
CDKN1B	GCCTCAGAAGACGTCAAACG	TCCAACGCTTTTAGAGGCAGA	60	224
DKK1	GAGTACTGCGCTAGTCCCAC	TGGAATACCCATCCAAGGTGC	60	226
DPP4	GCAGAATGTCCAGATGCCCT	GTGCTTGCAAGGTAAGTGGC	60	198
F2R	GTCAGGAGAGAGGGTGAAGC	CGGGGATCTAAGGTGGCATT	60	198
GAPDH (1)	AAGGCTGAGAACGGGAAGC	GAGGGATCTCGCTCCTGGA	60	68
G0S2	AAAGATATAAGCGGCCCCCG	GGAGGCGGGAATGACCTTAG	60	186
GREM1	AAGTTGGCAGCAGTAATCT	CCCTCTTTGGCTAGTGATA	56	155
HCK	CAGGATGGGGTGCATGAAGT	CCTCCCTGATTCCTGGTGTG	60	167
HIC1	TAAATCGGGAGAGTGTGCTGG	TCCAGGTTGAGCAGGTTGTC	60	225
IGFBP3	GTGCGGCATCTACACCGA	TCTTCCTCCGACTCACTAGCA	60	189
MT3	CCCCTGCCCTTCTGGTGG	CGCCTTTGCACACACAGTCCT	60	143
NT5E	TGATGAACGCAACAATGGAATC	ACCACGTTGATATCTTGGTCAC	60	240
PER1	AGGCTGCGTGGACTCGACAG	GCCCGAGGGTTCTGGTGGT	61	143
PRKCDBP	AGGAAGATCCCGGGAGACC	CGCTCCTTATTAGGGCGTGA	60	154
RASSF1	TGAAGGAAAATGACTCTGGGGA	GCGGCAATAGGAGTACTTCTGC	60	134
RPRM	GGGGTCTCACAAATCCGTGT	CTGTAACTCCTCAGGCAGGC	60	196
SFRP1	AGTTCTTCGGCTTCTACTGGC	CAGGGAGGACACACCGTTG	60	133
SOCS1	CACTTCCGCACATTCCGTTC	AGGCCATCTTCACGCTAAGG	60	202
SULF1	GGAGCTGTGTGGTCTTCTCC	TCTGCCTTGAAGAACCTGCAT	60	158
TNFRSF10C	GTTAGGGAACTCTGGGGACA	TGGTGGCAGAGTAAGCTAGG	60	173
TP73	TGGAGACGAGGACACGTACTACC	CTGCCGATAGGAGTCCACCA	60	127
TWIST1	GGACAGTGATTCCCAGACGG	CCTTTCAGTGGCTGATTGGC	60	173
UCHL1	AACGTGGATGGCCACCTCTA	AAGTCCCTCCCACAGAGCAT	60	193
VIM	AGGCGAGGAGAGCAGGATTT	CTGCACTGAGTGTGTGCAATTT	60	209
WIF1	ACTGCTCAACCACCTGCTTT	GACAGGGTTGTGGGCATTTG	60	114

Supplementary Table S4: Primer Used for Pyrosequencing Methylation Analysis of Candidate Genes

Gene	Primer		Primer sequence		No. of CpGs		Annealing temperature (°C)
WIF1	Forward primer		5'- biotin-GAGGGATTTTAGAGGTTGTATTTATAGT-3'		6		54
		Reverse primer		5'-AACCTTATCTACTCTCCCCATTTC-3'
		Sequencing primer		5'- AAAATCTCTAAATACCCTTCT-3'
UCHL1	Forward primer		5'-GGTTTTGTTTTTGTTTTTTTTGTATAGG-3'		3		54
		Reverse primer		5'-biotin-AATCTCCATCCACTTAAACTACATCTTC-3'
		Sequencing primer		5'-TTGTATAGGTTTTATAGTG-3'
RASSF1	Forward primer		5'- biotin-CTCAAAAATTCCAAATAAAAAATAACA-3'		3		54
		Reverse primer		5 '-CTCTCCCTAAAACTTCCCTTCAA-3'
		Sequencing primer		5'-TCAATCTCCRAAACATT-3'
CCNA1	Forward primer		5'-GTTGTTAGAGGTTGTTGGGAGAA-3'		4		54
		Reverse primer		5'-biotin-TCTACACACCTCCCCTCTAATCC-3'
		Sequencing primer		5'-GGAAATAGTTTTTTTTAAAGT-3'
TP73	Forward primer		5'-GTTTAGTATGTAGGGTTTTTTAGTTAGGG-3'		3		54
		Reverse primer		5'-biotin- CCCCTACTCCTCCTAAACCAT-3'
		Sequencing primer		5'-TTTAGTTAGGGTTTGGTGTA-3'
SFRP1	Forward primer		5'-biotin-GTTAAAATTAAGGGTTTTTATTAGGGTAGA-3'		3		56
(For fresh tissues)	Reverse primer		5'-TCACTCCCAACTCTCCAAAACT-3'
	Sequencing primer		5'-TAACAAAAAAACTTCTATTCC-3'
SFRP1	Forward primer		5'-GTTAAAATTAAGGGTTTTTATTAGGGTAGA-3'		1		56
(For FFPE specimens)	Reverse primer		5'- biotin-CTCAAAAATTCCAAATAAAAAATAACA-3'
	Sequencing primer		5'-TTAAGAGGAAGGTATTAGTATAA-3'

FFPE:  Formalin-Fixed Paraffin-Embedded

Supplementary Table S5: Differentially Methylated CpG Sites (2173 Sites) in 24 Nasopharyngeal Carcinoma (NPC) Tissues and 24 Non-cancer Nasopharyngitis Biopsy Tissues (NCNBT)

(See supplementary table S5 in a PDF file)
Supplementary Table S6: Data Analysis with PubMeth and NCBI PubMed

	Gene in PubMeth	Cancer with Methylation-associated Prognosis Published in NCBI PubMed
1	ALDH1A3	Bladder cance^(2); glioblastoma ^(3); melanoma ^(4);
2	CCNA1	Acute myeloid leukaemia^(5); prostate cancer ^(6); head and neck squamous cell carcinoma ^(7)；
3	CD38	Chronic lymphocytic leukemia ^(8);
4	CD44	Breast Cancer ^(9); oral cavity squamous cell carcinoma^(10);
5	CDKN1B	Breast cancer ^(11); Lung cancer^(12); prostate cancer^(13) ;
6	DKK1	Intrahepatic cholangiocarcinoma ^(14); esophageal squamous cell carcinoma^(15); hepatocellular carcinomas ^(16)；
7	DPP4	Colon cancer ^(17); renal cancer ^(18);
8	F2R	Prostate cancer ^(19)
9	G0S2	Hepatocellular Carcinoma^(20); squamous lung cancer ^(21);
10	GREM1	Renal cell carcinoma ^(22); pancreatic neuroendocrine tumors ^(23)；
11	HCK	Pancreatic adenocarcinoma ^(24); acute lymphocytic leukemia ^(25); renal clear cell carcinoma ^(26);
12	HIC1	Prostate cancer ^(27); hepatocellular carcinoma ^(28); colon cancer ^(29);
13	IGFBP3	Liver cancer ^(30); glioblastoma ^(31); ovarian endometrioid carcinoma ^(32)；
14	MT3	Esophageal adenocarcinomas^(33); breast cancer ^(34); prostate cancer ^(35);
15	MX1	None
16	NT5E	Breast cancer ^(36); melanoma ^(37);
17	PER1	Buccal squamous cell carcinoma ^(38); colorectal cancer^(39); prostate cancer ^(40)；
18	PLEKHG5	None
19	POU3F1	None
20	PRKCDBP	Colorectal cancer ^(41); neuroblastoma ^(42); breast cancer ^(43);
21	PRTFDC1	None
22	RASSF1	Lung cancer ^(44); oral squamous cell carcinoma ^(45); melanoma^(46), Breast Cancer^(47);
23	RPRM	Prostate cancer ^(48); pancreatic ductal adenocarcinoma ^(49)；
24	SFRP1	Renal cell carcinoma ^(50); breast cancer ^(51); colorectal cancer ^(52); gastric cancer ^(53);
25	SLC1A2	None
26	SOCS1	Multiple myeloma ^(54);colorectal cancer ^(55); melanoma ^(56)；
27	SULF1	Gastric cancer^(57); ovarian cancer ^(58);
28	TNFRSF10C	Colorectal carcinomas ^(59)
29	TP73	Pancreatic cancer ^(60); colon cancer^(61); glioma ^(62)
30	TWIST1	Breast cancer ^(63); colorectal carcinomas ^(64)；
31	UCHL1	Renal cell cancer ^(65); esophageal squamous cell carcinoma ^(66);
32	VIM	Bladder cancer ^(67) ; colorectal cancer^(68);breast cancer ^(69);
33	WIF1	Melanoma^(56); acute lymphoblastic leukemia ^(70); renal cell carcinoma ^(50)

Supplementary Table S7: Correlation Analysis between Methylation of Six Genes in the Training Cohort

	UCHL1	RASSF1	CCNA1	TP73	SFRP1
WIF1	R=0.406 P<0.001	R=0.436 P<0.001	R=0.389 P<0.001	R=0.535 P<0.001	R=0.486 P<0.001
UCHL1		R=0.465 P<0.001	R=0.317 P<0.001	R=0.524 P<0.001	R=0.269 P<0.001
RASSF1			R=0.379 P<0.001	R=0.605 P<0.001	R=0.394 P<0.001
CCNA1				R=0.433 P<0.001	R=0.222 P<0.009
TP73					R=0.464 P<0.001

Supplementary Figure Legends

Supplementary Figure S1: Representative Pyrograms of Methylation Analysis by Bisulfite Pyrosequencing. Percentages of methylation represent the ratio between signal intensities of C and T in each C of a CpG site.

Supplementary Figure S2. Kaplan-Meier curves estimation of disease-free survival (DFS) and overall survival (OS) for patients with high or low methylation levels stratified by tumor stage or the receipt of concurrent chemotherapy.

(A, B) Stage I-II patient DFS and OS. (C, D) Stage III-IV patients with high methylation DFS and OS. HR and P values were calculated with adjusted multivariate Cox proportional hazards models. The following parameters were included in the model as the covariates for each analysis: methylation gene panel (high methylation vs. low methylation), sex, age (<45 years vs. ≥45 years), World Health Organization (WHO) pathology type (undifferentiated non-keratinizing vs. differentiated non-keratinizing) and concurrent chemotherapy (yes vs. no). HR: hazard ratio, CI: confidence interval. CT: chemotherapy.

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