Von Hippel-Lindau status influences phenotype of liver cancers arising from PTEN loss

Figures & data

Figure 1 Liver and tumor tissue from mice with different genotypes show differing phenotypes on histological examination.

Notes: Livers from Vhl^-/- mice had no evidence of tumor or fatty liver formation, but did show a mildly expanded vasculature, magnified in the inset (Aand B). Tumors were found in Pten^-/-;Vhl^-/- and Pten^-/- mouse livers and had different phenotypes and varying levels of fatty liver. Pten^-/-;Vhl^-/- tumors had a poorly demarcated, trabecular morphology and only occasional lipid vesicles (yellow arrow) (C–E), while Pten^-/- tumors had mixed glandular and solid morphologies (black arrow) and widespread lipid deposition (yellow arrow) (F–H). Large pictures are 10× magnification, inset pictures, 20×. Sizing measurements are indicated in each panel.

Figure 2 Genes related to hypoxia and fatty liver are differentially expressed between mouse genotypes.

Notes: (A) Selected HIF target genes are upregulated in Pten^-/-;Vhl^-/- mouse tumors compared to Pten^-/- mouse tumors. Significantly upregulated genes include: VEGFA, SLC2A1, PGK1, and HK1. Vhl is downregulated in Pten^-/-;Vhl^-/- due to the induced deletion. (B) Genes known to be downregulated in human fatty liver disease show reduced or unchanged expression in the Pten^-/- mouse tumors compared to the Pten^-/-;Vhl^-/- tumors. (C) Genes known to be upregulated in human fatty liver show increased or unchanged expression in the Pten^-/- mouse tumors compared to the Pten-/-; Vhl-/- tumors. *P<0.05; **P<0.01. Error bars indicate standard deviation.

Figure 3 Human HCC and CC gene expression datasets show different regulation of genes related to fatty liver.

Notes: An assembled fatty liver gene set was used to cluster the human HCC/CC expression data. (A) The resulting heat map shows that tumors cluster by histologic type using a fatty liver profile. (B) Genes known to be downregulated in fatty liver show reduced expression in the human HCC dataset compared to the human CC dataset. (C) Genes known to be upregulated in fatty liver show increased expression in the human HCC dataset compared to human CC. **P<0.01. Error bars represent standard deviation.
Abbreviations: HCC, hepatocellular carcinoma; CC, cholangiocarcinoma.

Figure 4 Genes specific to the hypoxia response show variable expression between human HCC and CC.

Notes: (A) Expression of two genes specific to the HIF pathway (HK and SLC2A1) were significantly elevated in CC relative to HCC. (B) CC HIF pathway expression was identified as either CC HIF UP or CC HIF DOWN following median centering. The CC tumors were then clustered by HIF target gene expression. (C) Using the CC HIF UP or CC HIF DOWN designation, genes involved in fatty liver disease were able to cluster CC cases using expression datasets. **P<0.01.
Abbreviations: HCC, hepatocellular carcinoma; CC, cholangiocarcinoma.

Table 1 Clinicopathologic characteristics of an independent cohort of human cholangiocarcinoma samples

Characteristic	Frequency Number	%
Age, years
Median	64
Range	32–80
Sex
Female	13	48
Male	14	52
Primary tumor site
Intrahepatic	19	70
Extrahepatic	8	30
Distant metastases	11	41
Status of primary tumor
Resected with no residual	8	30
Resected with known residual or positive margins	11	40
Unresectable	8	30
Received adjuvant radiation ± chemotherapy	10	37

Table 2 Clinicopathologic characteristics of human cholangio-carcinoma samples based on HIF-1α expression

Characteristic	HIF-1α expression
	Positive (%)	Negative (%)
Number of patients	15 (56)	12 (44)
Median age, years	63	59
Sex
Female	7 (47)	6 (50)
Primary tumor site
Intrahepatic cholangiocarcinoma	9 (60)	10 (83)
Extrahepatic cholangiocarcinoma	6 (40)	2 (17)
Distant metastases	4 (27)	7 (58)
Status of primary tumor
Resected with no residual	4 (27)	4 (33)
Resected with known residual or positive margins	9 (60)	2 (17)
Unresectable	2 (13)	6 (50)

Figure 5 HIF-1α is variably expressed in a UNC cohort of human CC and may influence disease severity.

Notes: (A and B) human CC tumors were stained for HIF-1α expression, demonstrating an example of high expression (A) and low expression (B). Human tumors were also stained for phosphor-S6, with all tumors displaying positive expression of this marker, example shown in (C). (D) Kaplan–Meier curve demonstrates survival data from a cohort of 27 human CC patients based on HIF-1α expression.
Abbreviations: UNC, University of North Carolina; CC, cholangiocarcinoma.

Figure S1 Keratin 18 staining in normal liver identifies sites of probable tumor origin.

Notes: Because induced mutations were restricted to cells expressing Keratin 18, immunohistochemistry was used to identify the expression of this gene. Livers were mostly negative for Keratin 18, except for bile ducts, where positive staining revealed Keratin 18 expression (A and B).