DNA Methylation Profiles Distinguish Different Subtypes of Gastroenteropancreatic Neuroendocrine Tumors

Figures & data

Figure 1. Hierarchical clustering of tumoral and peritumoral samples according to DNA methylation patterns.

(A) Dendrogram of all tumoral and all peritumoral samples. (B) Dendrogram of all tumoral samples. Values above each cluster correspond to the approximately unbiased value obtained after 10,000 multiscale bootstrap resampling tests. The numbers under the SIETs in the dendrogram presented in the (B) panel correspond to the two groups of SIETs identified according to the cluster analysis presented in the (A) panel (1 or 2 for the SIETs clustering respectively with the SIET peritumoral samples or the other tumors).

Pan.: Pancreatic; Duo.: Duodenal; NF-PET: Non-functioning pancreatic endocrine tumor; SIET: Small intestine endocrine tumor.

Figure 2. Main diseases, disorders, molecular and cellular functions significantly associated with each tumor type.

(A) Diseases and disorders. (B) Molecular and cellular functions. The threshold represents a p-value of 0.05.

DG: Duodenal gastrinoma; G1S: Group 1 small intestine endocrine tumors; G2S: Group 2 small intestine endocrine tumors; Ins: Insulinomas; NFP: Non-functioning pancreatic endocrine tumor; PG: Pancreatic gastrinoma; SIET: Small intestine endocrine tumor.

Figure 3. Methylation levels of four candidate genes classified according to the WHO criteria for all pancreatic endocrine tumors.

(A) Methylation level of EGF, FGF1, KLK11 and ZIM3 according to GoldenGate. (B) Methylation level of EGF, FGF1, KLK11 and ZIM3 according to pyrosequencing. Statistical significance of DNA methylation differences between the three histological groups using the GoldenGate and pyrosequencing was assessed by the nonparametric Kruskal–Wallis test.

B: Benign; UB: Uncertain behavior; WDEC: Well-differentiated endocrine carcinoma.

Figure 4. Methylation and expression patterns of four selected genes in gastroenteropancreatic neuroendocrine tumors.

(A) The DNA methylation level obtained by the BeadArray analysis in function of the tumor types. (B) Relative expression level according to tumor type.

Gast.: Gastrinomas; Ins.: Insulinomas; NF-PET: Non-functioning pancreatic endocrine tumor, Panc: Pancreatic normal/peritumoral tissue; SIET: Small intestine endocrine tumor.

Table 1. Clinical characteristics of the patient cohort.

Clinical characteristics	Insulinomas	Gastrinomas		NF-PETs	SIETs	Total
		Pancreatic	Duodenal
Number of patients	12	5	7	20	18	62
Age
– Min	20	25	22	18	44	18
– Max	76	68	63	75	80	80
– Average	48	45	51	50	62	52
Sex
– Male	5	4	6	11	12	38
– Female	7	1	1	9	6	24
Tumors	11	5	7	19	18	60
Peritumoral samples	5	3	4	7	11	30
Number of patients with matched tumor and peritumoral samples available	4	2	4	6	11	28
Metastases
– Positive	0	4	6	14	18	42
– Negative	11	1	1	5	0	18
WHO classification
– B	5	0	0	0	–	5
– UB	6	1	1	5	–	13
– WDEC	0	4	6	14	–	24

B: Benign; NF-PET: Non-functioning pancreatic endocrine tumor; SIET: Small intestine endocrine tumor; UB: Uncertain behavior; WDEC: Well-differentiated endocrine carcinoma.

Table 2. List of genes presenting methylation variation according to the WHO criteria.

Target ID	Description	B (%)	UB (%)	WDEC (%)
CDH17^†	Cadherin 17, LI cadherin	78.39	66.67	46.04
CEACAM^†	Carcinoembryonic antigen-related cell adhesion molecule 1	51.37	27.78	16.93
EGF^†	Epidermal growth factor	69.96	56.00	23.69
FAS^†	Fas (tnf receptor superfamily, member 6)	40.25	31.76	15.98
FGF^†	Fibroblast growth factor 1	82.66	63.07	25.42
KLK11^†	Kallikrein-related peptidase 11	80.48	66.43	48.15
MKRN3^‡	Makorin ring finger protein 3	93.55	87.62	52.06
S100A2^‡	S100 calcium binding protein a2	70.50	61.08	59.95
S100A4^‡	S100 calcium binding protein a4	63.01	41.44	16.69
SNRPN^†	Small nuclear ribonucleoprotein polypeptide n	66.77	47.58	34.88
THBS2^†	Thrombospondin 2	81.75	70.07	27.20
ZIM3^†	Zinc finger, imprinted 3	76.35	73.52	39.61
PDGFRA^‡	Platelet-derived growth factor receptor, alpha polypeptide	24.04	41.04	70.80
PRKCDBP^‡	PKC, delta binding protein	11.68	26.97	62.07
TRIM29^†	Tripartite motif-containing 29	35.62	48.58	80.43

^†Promoter region analyzed.

^‡Exonic region analyzed.

B: Benign; UB: Uncertain behavior; WDEC: Well-differentiated endocrine carcinoma.