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Additional Supporting Information may be found in the online version of this article.

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HEP_23488_sm_SupFig1.tif2677KSupporting Fig. S1. Schematic representation of the experimental protocol. (A) Male F344 rats were injected intraperitoneally with diethylnitrosamine (DENA, Sigma) at a dose of 150 mg/kg body weight. Following a 2-week recovery, rats were fed a diet containing 0.02% 2-acetylaminofluorene (2-AAF, Sigma) for 1 week followed by a two-thirds partial hepatectomy (PHx), and an additional week of 2-AAF diet. The animals were then returned to a basal diet, and euthanized at 10 weeks, 9, and 14 months in groups of three.
HEP_23488_sm_SupFig2.tif3058KSupporting Fig. S2. Morphology of the representative hepatic lesions in rats subjected to RH protocol. (A) Adenoma at 9 months. (B) Early HCC at 9 months. (C) HCC at 14 months after DENA initiation. Inset shows multiple-cell thick plates of hepatocytes with atypical nuclei. H&E staining. Original magnification: X100 (A-C), insert, X200.
HEP_23488_sm_SupFig3.tif10613KSupporting Fig. S3. Expression of GSTP and CK19 in focal lesions. Rats were treated with DENA and 2-AAF, and sacrificed 10 weeks after initiation. Parallel liver sections were immunostained for GSTP (A, C, E) and CK19 (B, D, F). GSTP+ nodules displayed different degrees of CK19 expression. Original magnification, X40.
HEP_23488_sm_SupFig4.tif7949KSupporting Fig. S4. Supervised analysis of functional gene connectivity. Ingenuity Pathway Analysis (IPA) tools were used to analyze genes unique to each cluster. Genes upregulated in the CK19+ focal lesions as compared to normal liver are shown in red and downregulated genes are shown in green. Dashed lines represent indirect connectivity. The top 5 networks are involved in cancer, metabolism, and immune response (P<10−22 to 10−35). The significance is estimated for each network in IPA by determining the number of the differentially expressed genes within the network and the strength of each connection.
HEP_23488_sm_SupTab1.doc50KSupporting Table S1. Clinicopathological data
HEP_23488_sm_SupTab2.doc37KSupporting Table S2. Gene set overlaps with the Molecular Signatures Database (MSigDB)
HEP_23488_sm_SupTab3.doc182KSupporting Table S3. List of the 110 classifier genes
HEP_23488_sm_SupTab4A.doc49KSupporting Table S4A. Performance of the 110 gene classifier
HEP_23488_sm_SupTab4B.doc25KSupporting Table S4B. Performance of classifier during cross-validation
HEP_23488_sm_SupDocument.doc30KSupporting Methods

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