CpG island methylator phenotype in adenocarcinomas from the digestive tract: Methods, conclusions, and controversies

Table 1 Overview of previous studies of CpG island methylator phenotype in tumors from the gastrointestinal track

Year	Event	Ref.
1999	CIMP is first reported in a set of CRC patients	[5]
2004	Nature Reviews paper discussing CIMP in a variety of tumors besides CRC	[23]
2006	Refined molecular subtyping includes CIMP-low and CIMP-0 categories in CRC, with associations to KRAS mutations	[47]
	New insights are gained about the interplay between BRAF V600E mutations, MSI status, MLH1 promoter methylation and CIMP in CRC	[14]
2006-2012	High throughput DNA methylation arrays become widely available, enabling the use of larger gene panels for CIMP characterization	[45,46]
2014	TCGA marker paper on gastric cancer highlights the biological relevance of CIMP for molecular subtyping, exploring associations with EBV infection	[64]
	A better mechanistic understanding of CIMP in CRC is gained through elucidation of the role of MAFG in the context of MLH1 silencing and BRAF V600E mutations	[76]
2015	Pan-cancer stratification of solid tumors reveals similarities in CIMP across a wide variety of cancer types	[51]

CIMP: CpG island methylator phenotype; CRC: Colorectal cancer; MSI: Microsatellite instability; TCGA: The Cancer Genome Atlas.

Table 2 Gastrointestinal adenocarcinoma types, sample sizes, probe set sizes, and CpG island methylator phenotype status

Cancer type	Differentially methylated probes	Control samples	Tumor samples	CIMP-	CIMPi	CIMP+
EAC	6717	11	87	26	31	30
STAD	1110	2	260	109	95	56
COAD	2656	38	274	96	92	86
READ	1255	7	96	31	39	26

CIMP: CpG island methylator phenotype; CIMPi: CIMP intermediate; COAD: Colon adenocarcinoma; EAC: Esophageal adenocarcinoma; READ: Rectal adenocarcinoma; STAD: Stomach adenocarcinoma.

Table 3 Comparison between our CpG island methylator phenotype classification of stomach adenocarcinomas and the four subtypes defined by The Cancer Genome Atlas Research Network¹

	CIN	EBV+	GS	MSI	Total
CIMP+	212 (26.6)	313 (5.4)	22 (11.3)	327 (10.7)	-54
CIMPi	43 (45.75)	12 (9.4)	20 (19.5)	18 (18.4)	93
CIMP-	367 (49.68)	20 (10.2)	330 (21.2)	24 (20.0)	101
Total	122	25	52	49	248

¹Numbers outside parentheses correspond to actual sample counts, whereas numbers in parentheses show expected counts under the null model of independent classification;

²Indicates under-represented counts;

³Indicates cells with significantly over-represented counts (P < 0.05, based on Fisher’s exact test). CIMP: CpG island methylator phenotype; CIMPi: CIMP intermediate; CIN: Chromosomal instability; EBV+: Epstein-Barr virus positive; GS: Genomically stable; MSI: Microsatellite instability.

Table 4 Somatic mutations found in the tumor suppressor gene MLH1 in gastrointestinal adenocarcinoma samples

Sample	Cancer type	CIMP class	Mutation	Mutation type	AA pos.	Aff. AAs	VEST score1
TCGA-A6-6780-01	COAD	CIMP+	chr3:37038192.G>A	Missense substitution	67	1	0.994
TCGA-CA-6719-01	COAD	CIMP+	chr3:37067243.G>A	Missense substitution	385	1	0.701
TCGA-CM-6171-01	COAD	CIMP+	chr3:37070349.C>-	Frameshift deletion	495	262	-
TCGA-EI-6917-01	READ	CIMP+	chr3:37058999.C>T	Missense substitution	265	1	0.981
TCGA-BR-6452-01	STAD	CIMP+	chr3:37107356.A>G	3' UTR	-	-	-
TCGA-FP-A4BE-01	STAD	CIMP+	chr3:37090086.C>T	Nonsense substitution	659	98	-
TCGA-A6-6138-01	COAD	CIMPi	chr3:37035084.G>A	Missense substitution	16	1	0.901
TCGA-AD-6889-01	COAD	CIMPi	chr3:37053348.->A	Frameshift insertion	195	562	-
TCGA-AZ-6601-01	COAD	CIMPi	chr3:37067242.C>T	Missense substitution	385	1	0.952
TCGA-CM-4746-01	COAD	CIMPi	chr3:37059062.A>-	Frameshift deletion	286	471	-
TCGA-EI-6884-01	READ	CIMPi	chr3:37058995.A>G	Acceptor splice site	264	493	-
TCGA-BR-6802-01	STAD	CIMPi	chr3:37053348.A>-	Frameshift deletion	195	562	-
TCGA-F1-6874-01	STAD	CIMPi	chr3:37050312.ACCTTTTTTACAACATAGCC>-	Frameshift deletion	154	603	-
TCGA-A6-6781-01	COAD	CIMP-	chr3:37053348.A>-	Frameshift deletion	195	562	-
TCGA-CM-6674-01	COAD	CIMP-	chr3:37058999.C>-	Frameshift deletion	265	492	-
TCGA-F4-6856-01	COAD	CIMP-	chr3:37089123.GAA>-	In-frame deletion	615	1	-
TCGA-R6-A6KZ-01	EAC	CIMP-	chr3:37034874.T>C	5’ UTR	-	-	-
TCGA-CG-5723-01	STAD	CIMP-	chr3:37053550.G>-	Frameshift deletion	213	544	-

¹Computed using the VEST tool[100], which evaluates only the effect of missense substitutions. AA pos.: Amino acid position; Aff. AAs: Number of affected amino acids (the MLH1 protein contains 756 residues); CIMP: CpG island methylator phenotype; CIMPi: CIMP intermediate; COAD: Colon adenocarcinoma; EAC: Esophageal adenocarcinoma; READ: Rectal adenocarcinoma; STAD: Stomach adenocarcinoma; VEST: Variant effect scoring tool.

Table 5 Association between methylation and gene expression in tumor suppressor genes with significantly hypermethylated promoters in CpG island methylator phenotype + samples across four gastrointestinal adenocarcinoma types¹

		Differential methylation				Correlation with expression
Gene symbol	Promoter probes	Significant probes per cancer type				EAC		STAD		COAD		READ
		COAD	EAC	READ	STAD	cor	p-val	cor	p-val	cor	p-val	cor	p-val
TP73	24	18	3	2	23	-0.34	4.E-01	-0.24	1.E-01	-0.10	1.E+00	-0.20	1.E+00
MAL	8	6	5	2	7	-0.37	3.E-01	-0.46	2.E-07	-0.47	7.E-09	-0.45	3.E-02
C2orf40	8	5	3	1	7	-0.51	2.E-03	-0.57	5.E-13	-0.39	2.E-05	-0.24	6.E-01
TMEFF2	7	7	7	7	7	-0.54	2.E-03	-0.49	2.E-08	-0.41	2.E-03	-0.32	5.E-01
ERBB4	6	7	7	2	7	-0.26	5.E-01	-0.15	5.E-01	NA	1.E+00	-0.30	6.E-01
TWIST2	5	5	4	1	4	-0.26	3.E-01	-0.33	3.E-04	-0.38	2.E-06	-0.37	3.E-02
LRRC3B	13	9	7	1	12	-0.36	5.E-01	-0.38	2.E-04	-0.41	4.E-03	-0.11	1.E+00
HTRA3	10	6	3	1	6	0.02	1.E+00	-0.03	1.E+00	-0.10	1.E+00	-0.07	1.E+00
UNC5C	13	13	13	8	13	-0.38	7.E-02	-0.34	5.E-04	-0.43	4.E-08	-0.40	3.E-02
FAT4	13	13	9	2	13	-0.33	2.E-01	-0.44	4.E-07	-0.34	4.E-05	-0.28	4.E-01
IRX1	5	3	4	3	4	-0.37	2.E-01	-0.37	3.E-04	NA	1.E+00	NA	1.E+00
SCGB3A1	9	9	4	2	9	-0.27	4.E-01	-0.37	3.E-05	-0.22	3.E-01	-0.12	1.E+00
AKAP12	10	9	5	1	10	-0.19	1.E+00	-0.42	1.E-06	-0.42	7.E-08	-0.27	6.E-01
DFNA5	12	10	8	1	9	-0.75	0.E+00	-0.56	1.E-12	-0.36	1.E-05	-0.34	1.E-01
TFPI2	15	22	14	19	22	-0.49	3.E-03	-0.54	2.E-11	-0.38	4.E-06	-0.22	1.E+00
NRCAM	7	6	1	1	6	-0.52	3.E-04	-0.47	2.E-08	-0.19	8.E-02	-0.17	1.E+00
CNTNAP2	14	14	11	1	14	-0.14	1.E+00	-0.27	2.E-02	-0.14	1.E+00	-0.12	1.E+00
PAX6	12	12	5	3	11	-0.22	1.E+00	-0.18	6.E-01	-0.04	1.E+00	-0.32	3.E-01
WT1	12	12	12	3	12	-0.25	1.E+00	-0.04	1.E+00	-0.26	8.E-03	-0.23	1.E+00
PHOX2A	11	11	6	5	11	-0.26	1.E+00	-0.13	1.E+00	-0.38	3.E-03	-0.26	1.E+00
WIF1	8	5	5	3	7	-0.57	2.E-03	-0.32	5.E-03	-0.44	9.E-07	-0.56	2.E-04
SLC5A8	9	11	10	4	12	-0.26	1.E+00	-0.28	2.E-01	-0.17	1.E+00	-0.33	9.E-01
TBX5	17	11	7	1	16	-0.32	5.E-01	-0.09	1.E+00	0.03	1.E+00	-0.16	1.E+00
ATP8A2	8	5	4	2	5	-0.28	1.E-01	-0.37	2.E-05	-0.24	5.E-03	-0.24	3.E-01
ADAMTS18	8	7	5	3	7	-0.27	3.E-01	-0.36	6.E-05	-0.34	5.E-05	-0.30	2.E-01
GALR1	29	27	5	8	27	-0.17	1.E+00	-0.47	2.E-04	0.00	1.E+00	-0.14	1.E+00
RASSF2	5	6	4	3	6	-0.52	5.E-04	-0.31	1.E-03	-0.41	1.E-07	-0.40	2.E-02
CDH4	3	2	2	2	4	-0.17	6.E-01	-0.09	8.E-01	-0.16	3.E-01	-0.32	1.E-01

¹Promoter regions were designated as 2-kb regions encompassing 1.5 kb upstream and 0.5 kb downstream of the transcription start site. For each cancer type, probes were considered significant if the P-value after a one-sided Mann-Whitney U test with Bonferroni correction for multiple testing was < 0.05. The total number of probes considered across all four cancer types was 395814. COAD: Colon adenocarcinoma; EAC: Esophageal adenocarcinoma; READ: Rectal adenocarcinoma; STAD: Stomach adenocarcinoma.

Table 6 Genes differentially mutated between CpG island methylator phenotype+ and CpG island methylator phenotype- gastrointestinal adenocarcinoma samples¹

Gene	Count CIMP+	% CIMP+	Count CIMP-	% CIMP-	P% Diff	P-value	FDR	Pathway
KMT2D	35	20.30%	10	4.30%	16.00%	6.22E-07	2.24E-05	Chromatin
ARID1A	60	34.90%	32	13.90%	21.00%	1.15E-06	2.24E-05	Chromatin
RNF43	42	24.40%	17	7.40%	17.10%	3.04E-06	3.79E-05	Wnt
CSF3R	19	11.00%	2	0.90%	10.20%	4.19E-06	3.79E-05	ERK
SOX7	14	8.10%	0	0.00%	8.10%	4.86E-06	3.79E-05	ERK
PIK3CA	48	27.90%	26	11.30%	16.70%	2.62E-05	1.70E-04	PI3K/RAS
PAX6	17	9.90%	2	0.90%	9.00%	3.96E-05	2.21E-04	Differentiation
ATM	37	21.50%	17	7.40%	14.20%	5.05E-05	2.46E-04	DNA damage
KRAS	52	30.20%	32	13.90%	16.40%	1.04E-04	4.53E-04	PI3K/RAS
EGR1	15	8.70%	2	0.90%	7.90%	1.63E-04	6.37E-04	Differentiation
GATA3	19	11.00%	5	2.20%	8.90%	2.22E-04	7.87E-04	NF-KB
KMT2C	38	22.10%	22	9.50%	12.60%	6.15E-04	2.00E-03	Chromatin
ALDH2	10	5.80%	1	0.40%	5.40%	1.18E-03	3.30E-03	Metabolic
CDK12	18	10.50%	6	2.60%	7.90%	1.18E-03	3.30E-03	PI3K/RAS
SAFB	15	8.70%	4	1.70%	7.00%	1.44E-03	3.73E-03	Chromatin
BCOR	19	11.00%	7	3.00%	8.00%	1.68E-03	4.09E-03	Chromatin
PTEN	24	14.00%	11	4.80%	9.20%	1.97E-03	4.32E-03	PI3K/RAS
AXIN2	21	12.20%	9	3.90%	8.30%	2.00E-03	4.32E-03	Wnt
CTCF	14	8.10%	4	1.70%	6.40%	2.73E-03	5.41E-03	Chromatin
PALB2	11	6.40%	2	0.90%	5.50%	2.77E-03	5.41E-03	DNA repair
ERBB3	18	10.50%	7	3.00%	7.40%	2.96E-03	5.49E-03	PI3K/RAS
ERBB4	29	16.90%	17	7.40%	9.50%	4.05E-03	6.97E-03	PI3K/RAS
FBXW7	32	18.60%	20	8.70%	9.90%	4.11E-03	6.97E-03	Notch
CIC	23	13.40%	12	5.20%	8.20%	6.55E-03	1.06E-02	Proliferation
HLA.A	17	9.90%	8	3.50%	6.40%	1.13E-02	1.71E-02	Immune
MSH6	19	11.00%	10	4.30%	6.70%	1.14E-02	1.71E-02	MMR
ERBB2	15	8.70%	8	3.50%	5.30%	2.98E-02	4.21E-02	PI3K/RAS
CASP8	13	7.60%	6	2.60%	5.00%	3.02E-02	4.21E-02	Apoptosis
SMAD4	27	15.70%	20	8.70%	7.00%	4.05E-02	5.45E-02	Wnt
TFE3	6	3.50%	1	0.40%	3.10%	4.53E-02	5.90E-02	Wnt
APC	82	47.70%	87	37.70%	10.00%	5.24E-02	6.60E-02	Wnt
NRAS	10	5.80%	5	2.20%	3.60%	6.55E-02	7.74E-02	PI3K/RAS
SMARCB1	10	5.80%	5	2.20%	3.60%	6.55E-02	7.74E-02	Chromatin
IGFBP7	3	1.70%	0	0.00%	1.70%	7.70E-02	8.65E-02	DNA Damage
TBL1XR1	6	3.50%	2	0.90%	2.60%	7.76E-02	8.65E-02	Wnt

¹Results are based on a combined set of 179 CIMP- and 154 CIMP+ gastrointestinal adenocarcinoma samples. P-values were computed using a two-tailed Fishers’ exact test. Only genes with FDR < 10% are shown. CIMP: CpG island methylator phenotype.