Methylation changes at the GNAS imprinted locus in pancreatic cystic neoplasms are important for the diagnosis of malignant cysts

Table 1 Demographics and clinical characteristics of the study population

Characteristics	Value
Female gender, n (%) (n = 52)	35 (67.3)
Mean age at EUS-FNA, y, mean ± SD (interval)	59.1 ± 14.8 (29-91)
Cyst location, n (%) (n = 52)
Head	22 (42.3)
Body	20 (38.5)
Tail	9 (17.3)
Multiple cyst locations	1 (1.9)
Cyst size, cm, mean ± SD (interval)	3.9 ± 2.3 (1-10)
Cyst size > 3 cm, n (%)	29 (55.8)
Cyst with nodule/mass, n (%)	18 (34.6)
EUS imaging, n (%) (n = 52)1
No high risk features	13 (25)
1 high risk feature	29 (55.8)
≥ 2 risk features	10 (19.2)
PCF CEA, n (%) (n = 52)
CEA < 192 ng/mL	31 (59.6)
CEA ≥ 192 ng/mL	17 (32.7)
No result available	4 (7.7)
PCF cytology, n (%) (n = 52)
Non-diagnostic	27 (51.9)
Negative for malignancy	14 (26.9)
Suspicious/malignant	10 (19.2)
NET	1 (2)
Treatment decision, n (%) (n = 52)
Follow up	34 (65.4)
Surgery	11 (21.2)
Endoscopic drainage	1 (1.9)
Palliation (symptomatic or chemotherapy)	6 (11.5)

¹High-risk features: cyst size ≥ 3 and solid component or thick wall or dilated Wirsung (> 10 mm). CEA: Carcinoembryonic antigen; EUS-FNA: Endoscopic ultrasound with fine needle aspiration; NET: Neuroendocrine tumor; PCF: Pancreatic cyst fluid; SD: standard deviation.

Table 2 Frequency of GNAS locus methylation changes in malignant, mucinous, and benign cysts, n (%)

Informative cyst fluid methylation analysis, 38 samples	Malignant, n = 8	Mucinous pre-malignant, n = 10	Benign, n = 20	P value
NESP55 hypermethylation	3 (37.5)	0 (0.0 )	1 (5.0)	0.053
GNASAS hypermethylation	3 (37.5)	1 (10.0)	3 (15.0)	0.065
GNASXL hypermethylation	4 (50)	0 (0.0)	2 (10.0)	0.004
GNAS1A hypermethylation	1 (12.5)	0 (0.0)	0 (0)	0.0355
GNAS locus methylation changes	6 (75.0)	0 (0.0)	2 (6.7)	0.000

GNAS locus methylation changes: DMR hypermethylation or GNAS intragenic hypomethylation. GNAS: Guanine nucleotide-binding protein, alpha stimulating.

Table 3 Correlation between methylation status and malignancy with partial correlation controlling for patients’ gender and age

Rank correlation	Kendall		Partial
Possible confounders			Gender	Age
Malignant cysts	Correlation	P value	Correlation	Correlation
NESP55 hypermethylation	0.539	0.003	0.545	0.519
GNASAS hypermethylation	0.431	0.015	0.459	0.584
GNASXL hypermethylation	0.434	0.011	0.461	0.356
GNAS1A hypermethylation	0.160	0.361	0.191	0.147
GNAS locus methylation changes	0.837	<0.001	0.870	0.825

GNAS: Guanine nucleotide-binding protein, alpha stimulating.

Table 4 Frequencies of distinct clinical features and pancreatic cystic fluid analysis in the two groups, with or without GNAS locus methylation changes

Cyst fluid samples	GNAS locus methylation changes	No GNAS locus methylation changes	P value
Female	63%	75%	0.486
Age > 65 yr	50%	40%	0.216
Symptoms	63%	17%	0.008
CEA > 192 ng/mL	63%	25%	0.133
KRAS/GNAS mutation	63%	11%	0.008
Cytology, malignant/atypical	63%	7%	0.003

GNAS locus methylation changes: DMR hypermethylation or GNAS intragenic hypomethylation. CEA: Carcinoembryonic antigen; GNAS: Guanine nucleotide-binding protein, alpha stimulating.

Table 5 Area under the curve for diagnosis of mucinous and malignant cysts

Variables	Mucinous cysts				Malignant cysts
	AUC	P value	Confidence interval		AUC	P value	Confidence interval
			Lower limit	Upper limit			Lower limit	Upper limit
CEA in mg/dL	0.889	0.002	0.720	1.000	0.812	0.038	0.579	1.000
Cytology	0.598	0.443	0.349	0.847	0.771	0.072	0.571	0.970
Mutation (KRAS/GNAS)	0.833	0.009	0.634	1.000	0.841	0.023	0.615	1.000
Met_NESP55	0.620	0.35	0.370	0.869	0.759	0.085	0.481	1.000
Met_AS	0.590	0.483	0.339	0.841	0.741	0.108	0.461	1.000
Met_XL	0.474	0.841	0.228	0.721	0.629	0.389	0.357	0.902
Met_1A	0.513	0.92	0.262	0.764	0.565	0.667	0.261	0.868
GNAS locus methylation changes	0.645	0.256	0.400	0.891	0.971	0.002	0.901	1.000

AUC: Area under the curve; CEA: Carcinoembryonic antigen; GNAS: Guanine nucleotide-binding protein, alpha stimulating; Met: Methylation changes.