The process of tumor initiation and progression in cirrhosis is well established. Progressive accumulation of genetic and epigenetic abnormalities marks the temporal steps of hepatocarcinogenesis [1]. In the early stages of hepatocarcinogenesis the epigenetic mechanisms of gene expression upregulation prevail, involving particularly the transforming growth factor-α[alpha] (TGF α[alpha]) and the insulin-like growth factor-2 (IGF-2). Chronic hepatitis (both viral and nonviral) and cirrhosis are considered predisposing situations, since the combined action of inflammatory cytokines, viral transactivation, and regenerative response can determine changes in telomerase length, gene methylation, microsatellite instability, and fraction of alleles that were observed both in hepatocytes during chronic inflammation and cirrhosis, and in dysplastic nodules as well as HCC [21]. In these inflammatory situations there is an increased expression of DNA methyltransferases (DNMTs), that catalyze the methylation and demethylation of CpG groups, and both DNMT1 and DNMT3a are upregulated in HCC [21].

Loss of heterozygosity (LOH) and microsatellite instability (MSI) are detectable in preneoplastic liver lesions, with the most frequent deletion occurring at 8p, both in HGDNs and HCC. Similarly, RNs, dysplastic nodules, and adjacent HCC may share an MSI phenotype at identical loci and similar allelic deletions or gene mutations. Oncogene activation (v-akt homolog 2), loss of tumor suppressor genes (WT1, RB1), and DNA repair genes are deregulated in HGDN. Similarly, the expression at the protein level of growth factors (EGFR, IGFBP3) cytokines, adhesion proteins, signal transduction proteins, transcription factors, and housekeeping genes is altered in precursor and neoplastic hepatocellular lesions [1, 21–23]. The AKT-mTORC1 signaling pathway is activated in cirrhotic liver with and without HCC. As a result, increased expression of phospho-S6 (an AKT effector) was observed, while PTEN staining was negative in 24 % of HCC cases [23].

Allelic deletions are rare in chronic hepatitis and cirrhosis, but their number steadily increases in dysplastic nodules and in HCC and that suggests that HCCs often arise as clonal outgrowths of cirrhotic (dysplastic) nodules. Although several of the these structural alterations in preneoplastic cell populations differ from those found in adjacent HCCs, suggesting that most of the cells harboring the early genomic aberrations do not evolve into the malignant phenotype [21].

Hepatocyte dysplasia, especially the “large-cell” HGDN, is often accompanied by alterations that can also be found in HCC, such as telomerase shortening, microsatellite instability, inactivation of cell cycle checkpoints CDKN2A and CDKN1A, resulting in increased proliferation index [22]. However, the most common genetic alterations in HCC (i.e., β[beta]-catenin or TP53 mutations) have never been described in preneoplastic liver lesions [24].

The macroscopic features of HCC largely depend on the status of the surrounding liver. In cirrhotic livers, HCC is seen as a single lesion or as multiple lesions, and is encapsulated, resembling larger cirrhotic nodules (Fig. 24.3). The neoplastic nodules are soft, with a grayish-white to greenish color, according to the amount of bile content. The macroscopic diagnosis of HCC in a cirrhotic background is not always easy, especially in eHCC < 2 cm in size. HCC in noncirrhotic livers is usually a single large mass, with irregular, poorly defined borders, sometimes with satellite lesions and grossly visible vascular invasion. Vascular invasion is a common feature of any HCC at the time of diagnosis, involving the portal and the hepatic veins, or the vena cava [15].

Small nodules (≤2 cm) within a cirrhotic background with the architectural and cytologic atypical of a HGDN, but affecting 100 % of the cells, is classified as (eHCC) [19]. eHCC is by definition grade 1 according to the Edmondson and Steiner grading system (see below). At a histological level, hypercellularity, loss of reticulin staining, and the presence of hepatocellular trabeculae more than two-cell thick are helpful diagnostic criteria if present throughout. In addition, no portal tracts are visible within the lesion. Nearly half of eHCCs show fatty changes (macrovescicular steatosis) [24]. An important diagnostic feature is the occurrence of stromal invasion, defined as the presence of HCC cells in the stroma of the surrounding portal tracts (or in fibrous septa) [25]. Although stromal invasion is the most specific histological feature for the diagnosis of eHCC, it is not always recognizable on needle biopsies, and even when present, it is difficult to distinguish from non-neoplastic hepatocytes entrapped within the fibrosis.

Unlike many other solid tumors the neoplastic cells in HCC are quite similar to the non-neoplastic hepatocytes, i.e., they are polygonal cells with a variable amount of eosinophilic granular cytoplasm and large polymorphic vesicular nuclei, with open chromatin and one or more prominent nucleoli. Many cell features seen in not-neoplastic hepatocytes are common also in HCC, such as Mallory bodies, hyaline bodies, glycogen inclusions, fatty changes, bile production, and nuclear inclusions [15].

HCCs are routinely graded according to the original Edmondson and Steiner system of 1954 [26]. Like the grading systems of other cancers, Edmondson and Steiner’s is based on the dedifferentiation of the neoplastic hepatocytes, i.e., their progressive loss of resemblance with the normal hepatocyte (Fig. 24.4). More practically, the grade of HCC is based on the nuclear-to-cytoplasmic ration, nuclear hyperchromasia, and cell polymorphism. Grade 1 HCCs are characterized by neoplastic hepatocytes with preserved eosinophilic cytoplasms, mild (or no) atypia, while in grade 2 and 3 HCCs a progression of the above-mentioned features is observed. Grade 4 HCCs include anaplastic, small-cell and giant-cell tumors.

The architectural appearance of HCC is variable, with different, often intermingled patterns. The trabecular, acinar, and solid growth patterns are recognized. The most common pattern, particularly in well-to-moderately differentiated HCC, is the trabecular pattern, characterized by cords of neoplastic cells surrounded by vascular sinusoids [15]. These cords resemble the normal hepatic trabeculae, but are generally more than two neoplastic cell thick, with a high N/C ratio and nuclear overcrowding. With tumor progression, the neoplastic trabeculae can become distorted, creating a pseudoglandular pattern. In the acinar growth pattern, the neoplastic hepatocytes form luminal structures resembling bile canaliculi. Interestingly, bile production is detectable in nearly half of the cases simply by light microscopy [1]. These acini are not true glands, hence the term pseudoglandular, and may contain bile. Their biliary derivation can be demonstrated by electron microscopy or by the immunoreaction for CEA. The acini may contain PAS-positive proteinaceous fluid as well. The solid pattern is the least frequent, and occurs in poorly differentiated HCC, generally in association with at least one of the other architectural patterns. In the solid pattern the tumor cells are densely aggregated, without visible trabeculae or sinusoids. Immunostaining for CD34, however, often reveals compressed sinusoids within the lesion [22]. Other histological variants of HCC include: fibrolamellar HCC, scirrhous HCC, sarcomatoid HCC, clear-cell HCC, giant-cell HCC, HCC with a predominant inflammatory infiltrate, HCC with predominant hematopoiesis [1].

Due to the lack of strong diagnostic criteria, the differential diagnosis between HGDN and eHCC is challenging, and often requires immunohistochemistry (IHC). The American Association for the Study of Liver Diseases (AASLD) and the European Association for the Study of the Liver/European Organization for Research and Treatment of Cancer EASL/EORTC guidelines recommend the use of the three markers: Glutamine Synthase (GS), Glypican-3 and heat shock protein-70 (HSP70) [17, 18]. The combination of these three markers is considered sensitive and specific for the diagnosis of HCC in small liver lesions, while their diagnostic value in biopsy samples is less valuable (Fig. 24.5a–l). Although additional markers, such as EZH2 (Fig. 24.5m–o) [27] and Agrin [28] have been proposed, the differential diagnosis between a HGDN and eHCC in needle biopsies still represents a major challenge for the pathologist. A new promising class of biomarkers is represented by the cleavage products of the endoplasmatic reticulum protein, in particular calreticulin, PDIA3, PDI, and GRP78 [23]. A role as potential biomarker for HCC was postulated for the ubiquitin-conjugating enzyme E2C (UBE2C) due to its overexpression in human HCC at significantly higher levels compared to normal hepatocytes. In addition, high-UBE2C levels were associated with significantly lower disease-free survival rates [23].