The first suggestion that molecular genetic observations of HB may have prognostic significance was provided by Weber et al. (2000), who observed chromosomal gains of 8q and 20 by CGH more often in children who died of disease. The PLAG1 gene at 8q11.2–q13 is overexpressed in HB and contributes to IGF2 overexpression (Zatkova et al. 2004). CGH of a pre- and postchemotherapy HB showed 2q and 12 in a mixed tumor with fetal and embryonal epithelium that were not present in the large-cell anaplastic tumor that persisted after therapy and later metastasized. Chromosome 10 was enhanced in the recurrence. In two other tumors that had CGH before and after chemotherapy, only the posttreatment samples showed loss of 15q and gains of 18q and 22q. CGH results have been inconsistent, but gains of chromosome 1q, 2q, 8q, and 17 and losses of 4 are frequent. Since the first edition of this text, advances in molecular analysis have provided prognostic value as well as a better understanding of pathogenesis. For example, genome-wide SNP in GSD1A found simultaneous gain of 6p and loss of 6q in three of ten adenomas. Expression of IGF2R and LATS 1 candidate tumor suppressor genes on 6q was reduced over 50 % (Kishnani et al. 2009).

Children with the Beckwith–Wiedemann syndrome (BWS) have a relative risk of having an HB of 2280 (DeBaun and Tucker 1998), particularly in patients with hemihypertrophy. Eight percent of tumors in BWS are HB (Fig. 14.7a), and almost every other form of hepatic neoplasia has been observed in this syndrome (Fig. 14.23). The development of an embryonal malignancy in BWS patients is not as ominous as in other children. Vaughan et al. (1995) found 100 % disease-free survival in 13 affected children. Two of the tumors were advanced stage HBs that responded completely to chemotherapy. One patient with HB had an interstitial deletion of 11p, but 19 of 28 other BWS patients had no constitutional karyotypic abnormality. Of the nine cases with aberrations, six had triplication for 11p15. The p57KIP2 gene is located at 11p15.5 and is a negative regulator of cell proliferation. This is an imprinted gene with expression of only the maternal allele in normal cells. Mutation in the maternal allele has been observed in 4 of 49 BWS patients (Table 14.6). Many imprinted genes are dysregulated in HB, not only in 11p15; they include DLK1, PEG10, PEG3, BEX1, and RASGRF1. DLK1 is most heavily upregulated, especially in embryonal HB, reflecting its role in liver differentiation as a NOTCH ligand. Upregulation was not observed in a small undifferentiated HB (Lopez-Terrada et al. 2009), adding further evidence to the small cell not being a stem cell for HB (Badve et al. 2003).

Epigenetic modifications of the genome are of growing interest in tumors. Hypermethylation of DLK1–MEG3 is common in HB (Roy et al. 2010), and this could be particularly relevant to the striking excess of this embryonic tumor among survivors of prematurity, whose hepatocellular precursors are dividing rapidly while exposed to endogenous molecules that normally are eliminated by the placenta, as well as environmental challenges for which the immature liver is unprepared. Hypermethylation of both RASSFIA (Honda et al. 2008b) and MTIG (Sakamoto et al. 2010) has been found in HB with poor prognosis. Aberrant epigenetic silencing of SFRP1 by hypermethylation has been demonstrated in HCC, leading to Wnt activation and upregulation of c-Myc and cyclin D1 (Kaur et al. 2012).

Like Drut et al. (1992), we have observed hepatic hemangioendotheliomas in an infant with BWS (Fig. 14.7b). IGF2 is highly expressed during the proliferative phase of infantile hemangiomas and downregulated during regression, as shown with DNA microarray analysis, quantitative reverse transcription polymerase chain reaction (RT-PCR), and immunohistochemistry (Haas et al. 1986). Albrecht et al. (1994) found LOH for 11p15.5 in 6 of 18 HBs, one of which was from a BWS patient. This suggested loss of a tumor suppressor while preserving the growth stimulation activity of IGF2. Activation of the IGF axis and abnormal expression of molecules in this pathway are thought to be important in HB pathogenesis (Gray et al. 2000a) and have been proposed as HB therapeutic targets. IGF2 (insulin-like growth factor 2 or somatomedin A) is an imprinted gene located on chromosome 11p15.5 that regulates downstream phosphatidylinositol (PI) 3-kinase or mitogen-activated protein (MAP) kinase activation. Preferential induction of MAPK pathway signaling was reported in aggressive histologic epithelial HB subtypes (Adesina et al. 2009). Hartmann and colleagues (2009) identified strong p-AKT, p-GSK-3 beta, and p-mTOR expression in most HB tumors, as well as a PIK3CA gene mutation in one HB. Downregulation of mRNA for H19, a noncoding candidate for the suppressor role, was found in 10 of 12 HB by Hartmann et al. (2000). The same authors noted upregulation of p57 in 9 of 12 tumors, suggesting reactivation of the paternal allele in those tumors. Interestingly, LOH for 11p15 markers in an HB was found in a child with FAP who had lost the maternal APC gene in the tumor while retaining a mutant allele from the father. This work refines the first observation of LOH of 11p in diverse tumors of BWS patients by Koufos et al. (1985). There have been many studies of gene expression in the 11p15 region (Table 14.6). The tetraspanin gene CD81 that complexes with integrins and thereby influences cell–cell interactions and LSP1, which binds f-actin and regulates intracellular calcium, are also found at this locus. Insulin and IGF2 are stimulants of embryonal cell growth, so their upregulation or loss of imprinting in HB is suggestive of an early genetic contribution to fetal oncogenesis (Honda et al. 2008a). Elevated serum levels of hepatocyte growth factor (HGF) have been observed in HB patients at the time of diagnosis and proposed to be at least partly responsible for tumor progression. HGF/c-Met-related activation of beta-catenin was reported in a proportion of wild-type CTNNB1 tumors (Purcell et al. 2011). Hence, HGF and its receptor c-Met are candidates for targeted therapy in these tumors (Fig. 14.8).

HB occurs in children of families with heritable polyposis (FAP) at a frequency of 1,000–2,000 times that of the population at large (Fig. 14.5b). De Chadarevian et al. (2002) found a constitutional inversion of chromosome 5 in a family with FAP and two siblings with hepatocellular neoplasia. Nevertheless, no karyotypic alterations in tumors have been detected at 5q21. Oda et al. (1996) found mutations in APC in 8 of 13 sporadic HB, with no abnormality in the nontumoral liver. The kinds of germ line mutations of APC in patients with HB are not different from families without liver tumors; all the survivors of HB have later developed colonic polyposis. Other hepatobiliary tumors have been reported in FAP cases, including HCC in a 9-year-old boy and fibrolamellar carcinoma in a 15-year-old girl, adenoma in a 2-year-old child with biallelic inactivation of the APC gene in the tumor, bile duct adenoma, and gallbladder adenomatous proliferation and dysplasia. We have elsewhere depicted the simultaneous presence of adenoma and HB in an 11-year-old (Hadzic and Finegold 2011).

The APC protein regulates beta-catenin, an E-cadherin-binding protein normally involved in intercellular adhesion. When APC is mutated, beta-catenin accumulates in cells, where it stimulates expression of Wnt pathway. Wnt is important for the embryologic differentiation and zonation of the liver (Thompson and Monga 2007). Activating mutations in beta-catenin have been observed in 50–90 % of HB (Koch et al. 1999) and in 20–26 % of HCC (Lopez-Terrada et al. 2009). In the 30 % of HB found not to have chromosomal gains or losses by CGH by Buendia, all had beta-catenin mutations (Nhieu et al. 1999). Mutations of beta-catenin lead to nuclear translocation, activation of diverse genes, and increased cell proliferation. With immunohistochemistry, a shift of beta-catenin from its normal localization at the hepatocyte plasma membrane to the nucleus is found, especially at the invasion fronts, whereas well-differentiated fetal cells often preserve membranous staining (Table 14.7).

Lopez-Terrada et al. (2009) investigated the canonical Wnt signaling pathway in HBs and correlated the histologic subtype, beta-catenin immunostaining, and mutation status of the CTNNB1 gene (Fig. 14.8). All tumors with embryonal or small-cell histology showed either point mutations in exon 3, deletions within or confined to exon 3, or showed no CTNNB1 mutations. By contrast, HBs with pure fetal epithelial histology demonstrated predominantly large deletions of CTNNB1 including the entire exon 3 and most of exon 4. Immunohistochemistry demonstrated a correlation between the presence of CTNNB1 mutations confined to exon 3 and increased nuclear translocation of beta-catenin, particularly in the higher-grade tumor components, whereas HBs with large CTNNB1 deletions showed nuclear translocation of beta-catenin confined to a much smaller proportion of tumor cells or only the normal staining of cell plasma membranes. Ueda and colleagues recently reported Wnt activation in a small subset of HBs with wild-type beta-catenin, associated with high expression levels of telomerase reverse transcriptase (TERT), chemoresistance, and poor prognosis.

The NOTCH developmental pathway is involved in stem cell self-renewal and is necessary for cholangiocytic lineage differentiation. NOTCH pathway is also activated in some cancers, including HB. DLK1 (delta-like protein/preadipocyte factor 1/fetal antigen 1 or DLK/Pref1) is a NOTCH ligand which is highly expressed in bipotential transit amplifying oval cells and upregulated in hepatocellular tumors, including HCC and HB. NOTCH2 overexpression has been recently reported in HB (Litten et al. 2011).

Another pathway in development and liver regeneration is Sonic Hedgehog, which is activated in HCC (Huang et al. 2006). Increased transcription of GLII and PTCH1, HHIP gene promoter methylation, and a strong inhibitory effect on proliferation by blocking Hh signaling have been reported using HB cell lines. Increased expression of Sonic Hedgehog (SHH), Patched (PTCH), and the transcription factor GLI has also been detected by immunohistochemical analysis in HB specimens (Eichenmuller et al. 2009). However, no PTCH1 mutations were identified in a recently reported series of HBs (Chavan et al. 2012).

HBs derive from pluripotent stem cells (hepatoblasts), defined by their ability to differentiate into multiple cell lineages, self renew, and regenerate. Tumor heterogeneity characteristic of HB may be explained either by ongoing mutagenesis or by their origin from a stem cell arrested at different developmental stages and able to give rise to diverse forms of cancer, or by a combination of both (Fig. 14.9).

Expression profiling analysis has also been reported in HB by several groups. Nagata and colleagues (2003) identified 26 differentially expressed genes in HBs, when compared to the adjacent liver tissue, including 4 located on the 1q21 chromosomal region (ETV3, TPR, CD34, and NR113), and other genes encoding cell division and growth regulators (IGF2 and IGFBP4). Gene expression study of HB and the adjacent liver identified 6 genes (IGF2, Fibronectin, DLK1, TGFb1, MALAT1, and MIG6) differentially overexpressed in HB (Luo et al. 2006).

Comprehensive HB profiling studies by Cairo and colleagues (2008) described liver stem-like phenotype resulting from a combined Wnt/beta-catenin and c-Myc signaling, recapitulating a developmental stage with a profile similar to a hepatic stem/progenitor. Cairo et al. (2008) developed a 16-gene signature whose altered expression correlated with great significance to the prognosis of HB. The most dramatically expressed gene was DLK1 at 61.7× that of the normal liver, a member of the Wnt pathway, whereas ANNEXIN 10 was most significantly downregulated (tenfold). Figure 14.8 depicts the many genes and pathways in which abnormal expression has been reported.

miR profiling of HBs and HCCs demonstrated that Myc-driven reprogramming of miR expression contributes to an aggressive hepatic progenitor cell tumor phenotype and revealed a four-miR signature that may be useful to stratify HB patients (Cairo et al. 2010) (Table 14.8).

Expression of cyclins in HB is linked to the Wnt pathway. Significant downregulation of cyclin D1 was detected in four of seven tumors, whereas cyclin D2 and D3 were overexpressed versus nonneoplastic tissue using quantitative RT-PCR for mRNA and Western blotting for proteins (Iolascon et al. 1998). With immunohistochemistry, cyclin D1 was overexpressed in comparison to adjacent nontumoral liver in 13 of 17 cases, and cdk4 was increased in 15–17. In 11 patients both were overexpressed. Expression of the cdk inhibitor p27 has been lost in many aggressive neoplasms. We found a similar result in 27 HB (Brotto and Finegold 2002). The most interesting correlation was seen in the well-differentiated fetal component that has been associated with a favorable outcome when present as a pure population but not necessarily when admixed with other cell types (Haas et al. 1989). Fetal-type tumor cells that remained after chemotherapy in ten cases showed an average decline in the percentage of p27-positive nuclei from 75 to 12 % (Fig. 14.10a–d). All ten cases had decreased expression, and it was completely absent in two. Embryonal epithelium before and after therapy had 23 % positive cells. Small, undifferentiated cells in 3 cases were p27 negative in 2 and only 5 % were positive in the other. Stem cell markers were identified histochemically among fetal hepatoblasts rather than small cells by Fiegel et al. (2004).

The first application of CHIP technology to screen for mRNA expression in HB revealed a surprise, which was BRCA-2 expression in three cases (Schofield et al. 1999) (Fig. 14.5c). BRCA-2 is constitutionally mutated in familial breast and ovarian cancers and was mutated in 3 of 60 HCCs. Interestingly no APC expression was found with CHIP probes in the three tumors. BRCA-2 is a member of the Fanconi gene family and involved in breakpoint repair. Antibody to BRCA-2 stained six of six HBs but not fetal liver.

Chromosome 1q, 4q, 8p, 8q, 16p, and 17p abnormalities have been most commonly identified in HCC, relying mainly on CGH and microsatellite polymorphisms (Table 14.5). Few of the many studies include pediatric cases, most of which are not associated with chronic infection and cirrhosis, as in adults. From 58 to 72 % of HCC had increased copies of 1q. The high incidence of 8q excess may account for increased c-Myc expression, as it is located at 8q24 (Wong et al. 1999). Fluorescence in situ hybridization (FISH) showed that polysomy for chromosome 1 was the most common abnormality in HCC and trisomy 8 was next (Nasarek et al. 1995). The most frequent abnormalities found with CGH were of 4q, with decreased copies in 43–70 % and LOH in 77 % of cases. 6q, where many HCC have LOH or decreased copies, is not a site of abnormality in HB. Interestingly, 20, 8, and i(8 q10), as well as rearrangement of 1, were found in the cytogenetic analysis of a metastatic fibrolamellar carcinoma, resembling HB (Lowichik et al. 1996). CGH has been helpful in distinguishing well-differentiated HCC from adenomas, which do not show any of the several abnormalities in HCC (Wilkens et al. 2001). LOH for a DNA repair gene has led to microsatellite instability in HCC.

The rapidly expanding application of molecular genetic analyses of HCC has provided much data relevant to pathogenesis and prognosis. Table 14.6 shows the genes that have received the most attention. For most genes the proportion of carcinomas in which abnormalities of expression have been demonstrated is generally less than 50 %, and many of the defects occur only in the advanced stage of the disease, when many secondary mutations have accumulated. As in a growing number of cancers, defects in DNA repair contribute to HCC as in Fanconi anemia, BRCA-2 mutation, and Bloom syndrome (Jain et al. 2010).

Molecular profiling of HCC has resulted in the recognition of stem-cell signatures and characteristic profiles associated with response to therapy and survival. Thorgeirsson and colleagues demonstrated that HCCs with the cancer stem-cell (CSC) profile had worse prognosis, similar to what had been reported in other tumor types (Lee et al. 2006). A summary of work from many laboratories by Andrisani et al. (2011) provides the following expression data that strengthens the correlation with poor prognosis in HCC: increased expression of EpCAM, CK19, DLK1, DKK1, PROM1 (CD133), AFP, and Myc and decreased expression of UGT2B7 and CYP3A4. Just as in HB, microinhibitory RNAs (miRNA) have become increasingly relevant to HCC (Cairo et al. 2010) (Table 14.8). Detection of miR-15b and miR-130b in the serum of patients with HCC by RT-PCR was strikingly reduced after resection, particularly in patients with normal serum AFP (Liu et al. 2012).

The most consistent alteration in HCC has been codon 249 mutations of p53 (TP53 gene), which was found in 67 % of patients exposed to aflatoxin in Mexico, Mozambique, South China, and Africa. The frequency of p53 mutations elsewhere in the world is lower and involves other loci in the gene. LOH for p53 was found in 57 % of HCCs and 63 % of adjacent cirrhotic nodules, indicating premalignant loss of the tumor suppressor in some cases (Kishimoto et al. 1997). The importance of p53 is to regulate cell division via p21 WAF by sensing abnormalities in the DNA and preventing defective cells from dividing. The overexpression of p53 in HB and other liver malignancies, when detected immunohistochemically, does not signify mutations in the gene (Kennedy et al. 1994). p53 also activates expression of HIC-1, a recently discovered candidate for tumor suppression in a nearby chromosomal region (17p 13.3) (Wales et al. 1995). HIC-1 mRNA is markedly reduced in precancerous liver nodules of chronic hepatitis and cirrhosis and even more so in HCC, especially in poorly differentiated tumors. This is due to hypermethylation at the D17S5 locus on 17p. There was LOH at the locus in 54 % of carcinomas. Reduced expression of E-cadherin in HCCs also was attributable to methylation of the promoter on 16q 22.1 (Kanai et al. 1997). High levels of CpG methylation also account for loss of the tumor suppressor p16INK4 expression in half of the HCC (Hui et al. 1996). Silencing of the JAK/STAT regulator, SOCS-1, with aberrant methylation was found in 65 % of primary HCC (Yoshikawa et al. 2001). The Mdm2 proto-oncogene was overexpressed in 6 of 23 HCC. Mdm2 is known to downregulate p53 activity by competing with it, but it appears to have a p53-independent role in carcinogenesis because transgenic mice deficient in p53 also develop many sarcomas. No amplification of Mdm2 was found in 19 HB (Ohnishi et al. 1996). However Mdm4, whose product interacts with Mdm2 to inactivate p53, is overexpressed in 35 % of HB, especially in embryonal cells (Fig. 14.6c and Roy et al. 2012). The tumor suppressor, P21, is similarly inactivated (Hui et al. 1996) by excess Mdm4. When p53 mutations in other loci were detected in a group of HCC that were treated with surgical excision, the mean patient survival was reduced from 60 months to 15 months, compared to patients with wild-type p53 in their tumors (Honda et al. 1998). The presence of antibody to p53 in the serum of patients with HCC (32 % of 86 patients) was predictive of shorter survival (Shiota et al. 1997).

Independent clonal origin of multiple HCC in the same liver has been demonstrated with different patterns of LOH in the separate tumors (Tsuda et al. 1992). This observation was superbly elaborated by Gerlinger et al. (2012) in relation to the marked intratumoral heterogeneity for mutations in several tumor suppressors. As a result of the divergencies, expression signatures from one part of a tumor may suggest a poor prognosis while the opposite may be concluded from a second sample. This careful study emphasizes the potential danger of “personalized” cancer gene therapy from a single small sample (Fig. 14.11). When LOH for a particular locus is observed in noncirrhotic livers, it may have more significance. That is the case for 5q LOH in five of seven patients from England (Gray et al. 2000b). Nine cirrhotic patients with HCC failed to show this abnormality, but both groups had LOH at 17p13 (the p53 and HIC 1 loci). The 5q locus was at q35-qter, distant from the APC and MCC genes at 5q 21–22.

Telomerase activity was not detectable in a normal liver but was increased in 17 % of nontumoral tissue from patients with HCC and was present in 38 % of the tumors. It was more prevalent in moderately differentiated than well-differentiated tumors. Eighty-six percent of large regenerative nodules in patients without carcinoma had telomerase activities equal to carcinomas (Hytiroglou et al. 1998). Telomerase activity in the nontumoral tissues was associated with early recurrence (Ohta et al. 1997).

The Hippo pathway of growth control in Drosophila has received much attention recently in diverse human cancers (Liu et al. 2010). Even without amplification of the gene on 11q22, overexpression of nuclear YAP (Yes-associated protein) is observed in many HCC (Li et al. 2012) along with its targets, glypican 3, CTGF, and survivin. YAP1 phosphorylation is downregulated in 30 % of HCC, leading to a loss of targeted Mst1, a tumor suppressor (Zhou et al. 2009). This pathway also influences contact inhibition and hepatocellular growth through E-cadherin (Kim et al. 2011). And this provides for a cytoplasmic interaction with beta-catenin and the Wnt pathway. YAP also associates with beta-catenin in the nucleus to activate gene expression and excessive hepatocellular replication (Imajo et al. 2012). Thus, each new observation of genes involved in the control of the cell cycle, apoptosis, and cell–cell interaction points to nodes of interaction among the pathways and the likelihood of redundancy as well as additive effects.

The development of benign as well as malignant hepatocellular proliferation in patients with constitutional defects in DNA replication and repair (Fanconi anemia), the Wnt pathway (familial polyposis), or metabolic disease (GSD1a) has been illustrated (Fig. 14.3). Adenomas characterized by Wnt pathway disturbances, including beta-catenin activation, were particularly prone to the simultaneous or subsequent presence of HCC in the large series from France (Bioulac-Sage et al. 2009). This group delineated a second set of adenomas due to mutations of TCF1 that inactivate HNF1α and could be identified immunohistochemically and by RT-PCR to have lost FABP1 expression. A third form of adenoma was characterized by steatosis and inflammation (Fig. 14.12c), especially in obese and alcoholic adults and often hemorrhagic and telangiectatic. Immunohistochemistry revealed serum amyloid A only in this group. Restoration of G6Pase activity by adenovirus-mediated gene therapy to the liver in mice lacking the gene not only corrected their hypoglycemia and hepatic steatosis but prevented adenoma formation, providing an exciting prospect for humans with this condition (Lee et al. 2012).

Chromosomal aberrations in hepatic neoplasms of mesenchymal origin are shown in Table 14.5. It is particularly interesting that in contrast to HB, in which triploidy for chromosomes 20 and 2 are the most common findings, those chromosomes are frequently lost in the undifferentiated or embryonal sarcoma (Lauwers et al. 1997). It is also noteworthy that mesenchymal hamartomas, which some authors attributed to a vascular accident in utero, have a characteristic translocation involving t(19q 13.4), consistent with a neoplastic process rather than a post-injury reparative phenomenon (Shehata et al. 2011). This is clinically important because some publications have advocated conservative management, such as marsupialization of cysts without resection. Failure of marsupialization does not necessarily lead to sarcoma but to recurrence. At least seven cases of mesenchymal hamartoma have been reported to evolve into a sarcoma, and the translocation t(11;19)(11q,13.3–13.4) was demonstrated in one sarcoma (Rajaram et al. 2007). An association between mesenchymal hamartoma and placental mesenchymal dysplasia has been reported. Androgenic biparental mosaicism, as observed in the latter, has now been demonstrated in the liver lesion (Lin et al. 2011). This is particularly interesting because both conditions occur in the Beckwith–Wiedemann syndrome, which often involves uniparental disomy of the imprinted locus on 11p15. We have described a BWS patent with MH in infancy whose embryonal HB was overlooked and 4 years later was found to have HCC and cholangiocarcinoma in the hepatectomy specimen (Hadzic et al. 2011) (Fig. 14.23f).

A variety of molecular genetic alterations in hemangiomata associated with particular syndromes, such as von Hippel–Lindau and cerebral cavernous angiomas (KRITI/CCM1 mutation), have yet to elucidate the pathogenesis of hepatic hemangioendotheliomas. Whether the observation of VEGF receptor mutations in 2 of 15 hemangioendotheliomas of children will be applicable to liver lesions remains to be demonstrated (Walter et al. 2002). Hepatic hemangiomas were part of the maldevelopment complex in mice heterozygous for the tuberous sclerosis gene TSC 1. The tumors lost the normal allele. Hemangiomas occur in infants with BWS (Fig. 14.7b) independently or associated with mesenchymal hamartoma (Hadzic et al. 2011). One of three epithelioid hemangioendotheliomas found to have a t(1;3)(p36.3;q25) translocation with WWTR1–CAMTA1 fusion arose in the liver (Errani et al. 2011). Angiosarcomas induced by thorotrast radiation display K-ras mutations.

The genetic abnormalities of inflammatory myofibroblastic tumor include clonality by karyotyping, 9p deletion, and 2p23 abnormalities with anaplastic lymphoma kinase (ALK) rearrangements (Coffin et al. 2001). The latter occurred in slightly younger children and recurred more often. Malignant transformation was observed twice in children with ALK1 abnormalities and twice with aneuploidy but no ALK abnormality. A more aggressive variant of IMT arising in the abdomen has been designated as epithelioid inflammatory myofibroblastic sarcoma and is characterized by ALK expression on or near the nuclear membrane (Marino-Enriquez et al. 2011). Nine of the eleven patients had ALK rearrangements by FISH and three had RANBP2–ALK fusion by RT-PCR.