Methylome sequencing for fibrolamellar hepatocellular carcinoma depicts distinctive features

Figures & data

Table 1. List of cases assessed by DREAM for DNA methylation

Patient No	Histology	Sex	Age	size (cm)	TNM	Total number of tags	Median coverage of CG sites (minimum coverage of 3 tags/site)	Median coverage of CG sites (minimum coverage of 10 tags/site)
2*	p-FLC	M	21	22	N+	28,019,595	233	647
13	m-FLC	F	58	7	No	28,012,433	68	474
29	HCC	M	56	20	No	29,444,400	33	108
32	HCC	M	51	14	No	29,886,194	29	87
107	p-FLC	F	9	7	No	31,229,859	44	125
110*	p-FLC	F	13	11	N+	22,160,609	120	515
133	p-FLC	F	21	10	No	32,684,467	81	204
111	N	F	13			24,805,598	198	638
35	N	M	21			23,857,540	123	435

* The 2 p-FLC with asterisks are those which showed LINE-1 demethylation as compared to p-FLC 107 and p-FLC 133.

Figure 1. (A) Distribution of LINE-1 methylation levels in pure fibrolamellar carcinoma (p-FLC) and normal adjacent liver. Each dot represents the methylation level of individual samples. Horizontal lines represent the mean methylation levels for each group. (B) Kaplan-Meier curves for recurrence-free survival in low LINE-1 methylation group (n = 6) versus intermediate and high LINE-1 methylation group (n = 14). (C) Kaplan-Meier curves for overall survival in low LINE-1 methylation group (n = 6) vs. intermediate and high LINE-1 methylation group (n = 14).

Figure 2. (A) Correlation of DNA methylation % within CG sites detected by DREAM in 2 normal liver samples. Minimum coverage 10+ reads. Spearman r = 0.95, p < 0.0001. (B) Bar graphs of the number of genes gaining DNA methylation in each pure fibrolamellar carcinoma (p-FLC) and non-cirrhotic hepatocellular carcinoma (HCC) sample. (C) Bar graphs of the number of genes losing DNA methylation in each p-FLC and HCC sample. (D) Unsupervised clustering analysis using differentially methylated CG sites located in CG islands (CGI). (E) Unsupervised clustering analysis using differentially methylated CG sites located outside CGI.

Figure 3. (A) Box-plots of percentage of CG sites covered by DREAM method and gaining DNA methylation, whether located in promoters or outside promoters according to their methylation levels in normal livers. Green is related to CG sites with methylation level < 20% in normal liver. Red is related to CG sites with methylation level ≥ 20% in normal liver. (B) Box-plots of percentage of CG sites covered by DREAM method and losing DNA methylation, whether located in promoters or outside promoters according to their methylation levels in normal livers. Green is related to CG sites with methylation level < 70% in normal liver. Red is related to CG sites with methylation level ≥ 70 % in normal liver. (C) Box-plots of percentage of CG sites gaining DNA methylation, according to different genomic regions. (D) Box-plots of percentage of CG sites losing DNA methylation, according to different genomic regions. Abbreviations: PMD: partially methylated domains; LAD: lamina associated domains. For statistical analysis: ns: non significant; *P-value < 0.05; **P-value < 0.001; ***P-value < 0.0001.

Figure 4. (A–B) Box-plots for expression levels of SMARCA4 and RXRA in pure fibrolamellar carcinoma and normal adjacent livers using our previously published microarray data related to 17 p-FLC and 10 normal livers cases. (C–D) Network of genes coordinated by SMARCA4 and RXRA according to Ingenuity Pathway Analysis. Red: upregulated; green: down-regulated.

Figure 5. Unsupervised clustering for DNA methylation using 4 sets of differentially methylated genes. Note that all but one pure fibrolamellar hepatocellular carcinoma (FLC) belong to cluster C1, while mixed fibrolamellar carcinoma and hepatocellular carcinoma in non-cirrhotic liver belong to cluster C2. For the only pure FLC which clustered within C2 cases, no RNA was available to check for the presence of fusion transcript.

Figure 6. (A) Distribution of DLEU7 methylation levels in the validation set of pure fibrolamellar carcinoma (p-FLC), mixed FLC (m-FLC), non-cirrhotic hepatocellular carcinoma (nc-HCC) and normal adjacent liver. Each dot represents the methylation level of individual samples. Horizontal lines represent the mean methylation levels for each group. (B) Distribution of ZNF709 methylation levels in p-FLC, m-FLC, nc-HCC and normal adjacent liver. (C) Distribution of DUSP4 methylation levels in p-FLC, m-FLC, nc-HCC and normal adjacent liver. (D) Distribution of IGF2 methylation levels in p-FLC, m-FLC, nc-HCC and normal adjacent liver.