Currently, it is suggested that HCC relates to the following pathogenic factors:

Based on the statistics of HCC patients who received surgical resection during the past 30-year period in the EHBH, the infection rate of HBV (either serum or immunohistochemical HBsAg positive) in these HCC patients was 85.86%. Due to the large population infected with HBV, the carrying rate of hepatitis B surface antigen in people aged from 15 to 59 years old was 8.57%. And according to the data released by Chinese Society of Hepatology, Chinese Medical Association, among about 93 million people with chronic HBV infection in China, there were 20 million patients, and the number of HBV infections would continue to increase in the future. Furthermore, 15–25% of people with chronic HBV infection develop into hepatic cirrhosis or liver cancer, which is the major cause responsible for the high incidence of HCC in China currently; thus, prevention and treatment of liver cancers are long and arduous tasks.

Researchers showed that the insertion of HBV-DNA gene into adjacent regions of the cancer gene or suppressor genes in the liver cells would cis-change the function of the gene resulting in disruption of related protein-encoding sequence. The integration of HBx gene into the host genome is the most crucial event, which can be found in genes of any length, and in 96% of the HCC specimens, all X genes are found to insert into the host cell DNA in the truncated form, suggesting trans-carcinogenic effects of the truncated X protein, such as exertion of the trans-transcription activity by the interaction between protein and protein. X protein encoded by HBx gene is a multifunctional regulatory protein and plays a key regulatory role in viral infection, replication, pathogenic, and carcinogenic processes. HBV integration results in a series of molecular variations, such as insertion mutation into the host genome, upregulated transcription driven by virus promoter, human-virus transcriptional fusion, variation of DNA copy number and induction of genomic instability, including activation of oncogenes (such as C-myc, KRAS, C-fos, IGF- II, IGF-IR) via trans-activation mechanism, inactivation of tumor suppressor genes (such as p53, RB, Bcl-2, DNA mismatch repair genes), regulation of epigenetic proteins via mediation of DNA methylation, or regulation of cell apoptosis, DNA damage and repair, cell cycle, and miRNA functions through transcriptional activation of various signal transduction pathways to promote the abnormal proliferation of liver cells leading to the formation of HCC, which is HBV-DNA integrated-type HCC. Recent studies demonstrated HBx can induce epithelium-mesenchymal transformation (EMT) via PI3K/AKT/GSK3β/Snail signaling pathway, promote the invasion and metastasis of HBV-HCC via formation of HBx-YAP conjugates [6, 7], or exert target inhibition of the function of tumor suppressor genes by regulating miRNA.

HCV is a single-stranded RNA virus encoding a single polyprotein precursor of about 3000 amino acids, generating more than ten proteins, among which NS5A protein, a transcription factor for cell growth, plays a key role in the canceration of liver cells through interaction with other proteins, such as participation in HCV protein mutation and replication of RNA, regulation of the expression of multiple genes in the host cells, stimulation of cell proliferation, inhibition of apoptosis, and reducing the curative effects of interferon [8]. Wurmbach et al. (2007) studied the signal pathways involving multiple stages in the development of HCV-related HCC and found dysregulation of several pathways, including Notch- and Toll-like receptor in the precancerous stage of hepatic cirrhosis, and several components of JAK/STAT pathway in the early stage of carcinogenesis, upregulation of expression of genes participating in DNA replication, and repair and cell cycle in late stage of carcinogenesis [9]. Because of no discovery of reverse transcriptase in the liver cells, HCV cannot integrate into the host genome but produce severe cytotoxic damage on the infected liver cells and modification of the host’s immune system through indirect interaction similar to oncogene proteins. In order to evade the host immune surveillance, HCV can constantly mutate to form variant strains after infection of host cells, resulting in constant degeneration necrosis, repeated regeneration, and proliferation of the host cells and finally causing chronic hepatic injury [10].

Generally, about 20% of the chronic HCV hepatitis will develop into hepatic cirrhosis, including 1–4% of the cases evolving into HCC. Therefore, the development from infection of HBV or HCV to HCC generally includes a stage ofchronic hepatitis and cirrhosis. Compared with HBV hepatitis, the period for chronic hepatitis HCV to develop into hepatic cirrhosis may be shorter. According to statistics of 26,330 cases of surgically excised HCC diagnosed in the Department of Pathology of EHBH, patients with cirrhosis accounted for 73% of all the cases. From the temporal perspective, the proportion of HCC cases with hepatic cirrhosis reached 95–100% before 2000, while it was 50–95% for HCC cases diagnosed after 2001, suggesting a potential increase in the number of non-cirrhotic HCC, and the pathological significance of this type of HCC needs further studies. For patients of chronic hepatitis or cirrhosis with persistent positive serum HBV/HCV, regardless of clinical symptoms and signs, physical examinations should be carried out regularly, including the monitoring of HCC markers, such as serum AFP, and standardized antiviral treatment should be adopted.

According to a recent survey, children aged 7–13 years of age with an excess body mass index (BMI) have a significantly higher risk of HCC in their adulthood. An American study showed that patients with BMI ≥ 35 kg/m² have a morality rate 4.5 times higher than that of the people with a normal BMI, and the relative risks of HCC in people with overweight and obesity are 117% and 189%, respectively. The risk of HCC developing in diabetic patients increases by three times compared to their nondiabetic counterparts [12]. The incidences of nonalcoholic fatty liver disease (NAFLD) in many countries have increased significantly, and it has become the second major liver disease after viral hepatitis. The cumulative incidence rate of HCC with NAFLD-related cirrhosis is 2.6%. And in the past 10 years, at least 300 cases of NAFLD with HCC have been reported in the literature, and defined risk factors for NAFLD include obesity, diabetes, hyperlipidemia, and insulin resistance. About 20% cases of NAFLD are complicated by fatty hepatitis, the latter of which is a risk factor for the development of cirrhosis and HCC. A German study showed that fatty hepatitis is an underlying cause in 24% of HCC patients, and average 55% of HCV patients in Western countries have NAFLD. In addition, there are also cases of HCC developed from non-cirrhosis NAFLD and nonfibrous fatty hepatitis.

Mutations of cancer genes and/or inactivation of tumor suppressor genes (TSG) results in canceration of liver cells due to lack of regulation in signal transduction, cell cycle and growth, and proliferation by normal genes. The various molecular mechanisms and molecular types of HCC genomic variation are complex, and it has been found that human body has at least more than 1000 HCC-related genes with increasing discoveries of new HCC-related oncogenes, tumor suppressor genes, signal transduction pathways, and molecular targets. For example, He et al. (2011) conducted a genome-wide SNP microarray analysis on HCC tissues, screened out 1241 somatic copy number variation regions, and further identified 362 differentially expressed genes. They found that 60% of the HCC demonstrated lower expression >twofolds of TRIM35 gene which is a tumor suppressor and inhibits the proliferation of HCC cells. Higher expression (>twofolds) of HEY1 gene was found in 42.6% of the HCC, which is a cancer gene and promotes the proliferation of HCC cells [13]. Li et al. (2011) conducted a detection study on 18 thousand exons of coding genes using massively parallel sequencing technique, finding that 18.2% of HCV-HCC cases contain inactivating mutations of ARID2 gene, suggesting that it is a tumor suppressor gene for HCC [14]. A China-US joint research demonstrated a study using whole genome sequencing (WGS), and the results showed that β-catenin gene was the most common oncogene which mutate (15.9%) in HBV-HCC tissue, and TP53 gene was most susceptible to inactivation of anti-oncogenes (35.2%), while Wnt/beta-catenin pathway (62.5%) and JAK/STAT pathway (45.5%) were two signal pathways in which variations are the most frequently found for HCC [15].

Liu et al. (2014) investigated chromosomal DNA copy number changes in HCC by comparative genomic hybridization and detected the overexpression (>2-folds) of a new oncogene Maelstrom (MAEL) in 59.7% of the HCC cases. The experimental results showed that the MAEL is located on chromosome 1q24 and can activate Akt/ GSK-3β/Snail signaling pathway, inducing epithelial-mesenchymal transition (EMT) to promote the invasion and metastasis of HCC, and it also correlates with the recurrence and prognosis of these patients [16]. DLC-1 (deleted in liver cancer-1) gene is located on chromosome 8p21–22, encoding GTPase activator and can inhibit tumor metastasis. Deletion of DLC-1 gene has been found in 20% of HCC samples and 40% of HCC cell lines, and the growth of HCC cell strains transfected with DLC-1 was significantly inhibited, suggesting it is an inhibitory gene for HCC.

The biological characteristics of HCC are regulated by a complex cell signaling system, which are studied widely in aspects including the pathogenesis, growth, apoptosis, angiogenesis, invasion, metastasis, molecular therapeutic targets, and prognosis evaluation of HCC. And the representatives are tyrosine kinase pathway, Wnt/β-catenin pathway, P53 pathway, Notch pathway, NF-κB pathway, Hedgehog (Hh) pathway, Ras/Raf/MAPK pathway, VEGF pathway, JAK-STAT pathway, PI3K/Akt/mTOR pathway, HGF/c-Met pathway, and TGFβ1/Smads pathway, each of which is composed by diverse and complex key molecules. These pathways may be regulated by upstream miRNAs or target genes and exert the functions via the downstream target genes. Molecular analysis of molecules and function modes of HCC-related signal pathways is of clinical practice in aspects such as molecular typing, molecular diagnosis, and molecular-targeted therapy.

The association between alcoholic fatty liver disease (AFLD) and HCC has been recognized, and smoking can increase the risk of HCC development. And cases of HCC in patients with hereditary, congenital, allergic, or metabolic liver diseases, such as α1-antitrypsin protease deficiency, hereditary hemochromatosis, hereditary tyrosinemia, autoimmune hepatitis, primary biliary cirrhosis, have also been reported.

In a word, the genesis of HCC is a complex process involving multiple causes, mechanisms, steps, and genes. We briefly summarize the common causes of HCC, 16 key canceration mechanisms or research focus, and multistages during the pathological development (Fig. 7.3).

According to the statistics of clinical data in 28,869 cases of surgically excised HCC cases in the Department of Pathology, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, the ratio of male to female was 6.72:1, and the average age of HCC patients in each year during the 30 years was stable at around the age of 50 years old, younger than the average age of ICC patients of 54.9 years old and older than the average age of patients with benign liver tumors of 44.9 years old. And 86% of the HCC patients had a history of HBV infection, while about 10% of the patients had a history of HCV infection. The serum AFP level increased with the growth of the tumor, and the positive rates of serum AFP were 69.6, 59.1, 57.6, and 68.2% in HCC patients with tumors <1 cm, <2 cm, <3 cm, and >3 cm in diameter, respectively, with 20 μg/ml as the threshold, while the positive rates of serum AFP were 39.6, 25.7, 26.4, and 44.7% with 400 μg/ml as the threshold, showing that the serum AFP is still one of the serological markers for diagnosis of HCC, but negative serum AFP was found in more than 50% of these patients with HCC.

Most of HCC patients with tumor <3–5 cm in diameter are in early subclinical stage and may manifest no obvious clinical symptoms or signs, while the manifestations in HCC patients with tumors >3–5 cm in diameter include pain in hepatic zone, hepatosplenomegaly, general gastrointestinal symptoms (abdominal distention, diarrhea, decreased appetite), upper abdominal mass, fatigue, emaciation, persistent fever, jaundice, weight loss, abnormal liver function, and paraneoplastic syndrome.

According to clinical features, HCC can be divided into cirrhosis-type HCC characterized by hepatic cirrhosis, portal hypertension, and upper gastrointestinal hemorrhage; fever-type HCC with fever, increased white blood cells, and multiple concurrent infections, which is similar to liver abscess as the main characteristics; hepatitis-type HCC with progressive liver failure and hepatic encephalopathy (hepatic coma) in severe cases, similar to acute severe hepatitis; acute abdomen-type HCC with tumor rupture hemorrhage as the first symptom; cholestasis-type HCC manifested obstructive jaundice involving common bile duct; and metastasis-type HCC with metastasis of extrahepatic organs as the first clinical manifestation.

Type B ultrasonic (B-US) images display hypoechoic lesions in small HCC and hypoechoes or mixed echoes in large HCC, and the detection rate was 85–95% for lesions of 3–5 cm in diameter, while the sensitivity can reach 60–80% for lesions of 1 cm in diameter. Computer tomography (CT) images are more clear and stable with high resolution and show low densities for HCC lesions. Dynamic enhancement CT shows a curve of rapidly increased and then rapidly decreased densities of contrast agent in arterial phase, showing the characteristic “fast in fast out” performance. Magnetic resonance imaging (MRI) images of HCC is characterized by low signal on T₁-weighted images and high signal on T₂-weighted images, and MRI is especially sensitive for small lesions.

Current gross classification of HCC mainly includes the following three models:

According to the statistics on the pathological data of 8580 cases of HCC with surgical resection in our hospital during 2009–2011, huge, massive, nodular, and small types of HCC accounted for 15.1, 30.2, 27.8, and 26.9%, respectively. Following the late 1970s when Chinese Cooperation Group on Pathology of Liver Cancers first proposed the pathological characteristics of small HCC ≤3 cm in diameter and classified it as an independent type, we suggested that ≤3 cm small HCC was a key transition of biological characteristics between benign and malignant tumors in the 1990s (refer to the chapter Small Hepatocellular Carcinoma ). In the Practice guidelines for the pathological diagnosis of primary liver cancer: 2015 update issued by the Chinese Pathological Group of Hepatobiliary Tumor and Liver Transplantation, a single tumor ≤1 cm in diameter is defined as minute HCC, and a single tumor with a diameter from >1 cm to ≤3 cm is defined as SHCC (Fig. 7.11). All the above viewpoints represent the basic understanding of gross typing of liver cancers in China at present (Fig. 7.12). In a word, HCC is currently in a lack of a unified international standard on the gross classification, but the major trend is to combine morphological, biological, and molecular characteristics affecting the clinical prognosis, and analysis of gene spectrum may lead to the suggestion of a new model of HCC molecular typing.

Exophytic HCC may be connected to the hepatic capsule by fibrous pedicles or directly adheres to the visceral or diaphragmatic surface of the hepatic capsule, and the outward growth of the main part of the tumor into extrahepatic regions can be found due to low resistance, compressing the surrounding organs, while the liver parenchyma is less involved, similar to abdominal massed clinically. One case of HCC in the caudate lobe of the liver was reported in China, with a tumor of 21 cm in diameter protruding into the lessor omental bursa. Among the eight cases of huge exophytic HCC with surgical resection reported by Zhang HB of our hospital, the average diameter of the tumors was 18 cm, and all the patients had a history of HBV infection, of which seven cases concerned lesions on the visceral surface of the liver, seven cases with different degrees of invasion into the adjacent hepatic lobes, and patients in six cases demonstrated long-term survival.

Morphological features of HCC include the tumor size, number, and the association with the surrounding liver tissue, such as capsular integrity, focal infiltration, cancer embolus in the vessels, satellite nodules, intrahepatic metastasis, and other biological behaviors, all of which are the main basis for pathological typing of HCC. The section of a HCC mass is often solid, gray white, and soft in texture, with hemorrhage and necrosis, or dark green in cases with cholestasis (Fig. 7.13), dark brown in cases with severe hemorrhage (Fig. 7.14), and pale yellow due to severe fatty degeneration (Fig. 7.15). Cystic degeneration can be observed in cases with severe liquefaction necrosis, and fibrous scars can be found in the lesions of sclerosing HCC. Special attention should be paid to the invasion of capsule and the boundary invasion (Fig. 7.16).

In addition, cirrhosis-like HCC (CL-HCC) was also reported in the literature, characterized by diffuse micronodular cirrhosis like microcarcinoma in the background of cirrhosis [18], with mild elevation in serum AFP level and difficulty in distinguishing it from liver cirrhosis nodules. Although it is similar to diffuse HCC, most CL-HCC nodules have a fibrous capsule and a clear boundary. Histologically, it is composed by moderate, well-differentiated HCC cells, often with visible pseudoglandular tubular structures, and 80% of the cases contain visible Mallory bodies. These morphological features of CL-HCC suggest its polyclonal origin, and liver transplantation is a choice for the treatment during which cirrhosis lesions and tumor nodules in the liver can be removed.

With the deepening understanding on the biological characteristics and tumor microenvironment of HCC, and based on the clinical demand for prognosis evaluation and individualized treatment, more attention should be paid to the examination on invasion of the surrounding liver tissue (microvascular invasion and satellite lesions) and the lesions around the tumors (precancerous lesions ). Therefore, the pathological sampling should focus on comprehensive evaluation of the condition of the tumor and the adjacent liver tissues, rather than tumor itself as previous experience, and the specification of sampling will directly affect the statistical accuracy and scientific significance of pathological parameters (number and distribution of microvascular invasion and satellite foci). According to our experience in operability of practical work in the Department of Pathology, the basic method of sampling for HCC is as follows. Make vertical sections in an interval of 0.5–1 cm, select a representative one, and harvest 7-point baseline sampling protocol, which stipulates that at least four tissue specimens should be sampled at the junction of the tumor and adjacent liver tissues (1:1 ratio) at the 12, 3, 6, and 9 o’clock reference positions, making sure that every sample contains both the tumor tissue and the peritumorous liver tissue for observation of the invasion of capsule, blood vessels, and the surrounding liver tissue. For the purposes of molecular pathological examination, at least one specimen should be sampled at the intratumoral zone. In addition, harvest of liver tissue within the distance of ≤1 cm (adjacent peritumoral liver tissues) and >1 cm (distant peritumoral liver tissues) (Fig. 7.17) is for observation of satellite foci, microvascular invasion , residual cancer cells, as well as the background of the liver tissue (inflammation, fibrosis, cirrhosis). Sampling in the cutting edge should be used to determine whether a positive cutting edge can be found.

Of course, the number and sites of the samples depend on the size, shape, and number of the lesions of the tumor. For ≤3 cm small HCC, the whole tumor with peritumorous tissue should harvested, and more samples should be harvested corresponding the increased amount of peritumorous liver tissue and number of tumor nodules. The size of each sample should be 1.5–2.0 cm × 1.0 cm × 0.2 cm, and the sampling sites should be marked, with picture of the samples taken for file keeping.

The architectural patterns of HCC mainly include the following types:

Cases with diffuse pseudoglandular structures should be distinguished from intrahepatic cholangiocarcinoma and metastatic adenocarcinoma in the liver. These pseodoglandular tubes are positive for a hepatocellular marker Hep Par-1 (Fig. 7.29), and polyclonal carcinoembryonic antigen (CEA) and CD10 staining shows a characteristic canalicular staining pattern on the membrane of the pseudoglandular cells (Fig. 7.30), which suggest these are liver cells rather than real glandular epithelium. CK19 staining demonstrates generally negative results, and its positive results may suggest bile duct epithelial differentiation of the HCC cells (Fig. 7.31), and it belongs to a new subtype of HCC, so we named dual-phenotype HCC (see below). Pseudoglands do not contain myxoid components, and AB/PAS mucus staining is negative, and MUC-1 staining shows bile-like secretion (Fig. 7.32), also suggesting the lumen derives from specialized transformation of capillary bile ducts.

Commonly used markers for liver cells are Hep Par-1, CD10 (Fig. 7.98), pCEA (Fig. 7.99), GPC-3, CD34 (Figs. 7.21 and 7.24), AFP, etc. The former three markers cannot be used to distinguish the nature of liver cells, while the latter three markers are characteristically expressed in HCC tissues. And in differentiation from non-hepatocellular tumors, a combination of liver cell markers is very effective, and positive HBsAg staining is of certain reference value in the diagnosis of HCC (Fig. 7.100). The American Association for the Study of Liver Diseases (AASLD), the European Association of Liver Diseases, and the Panel of International Consensus Group all make the recommendation of diagnostic marker combination for HCC, “GPC-3+HSP70+GS,” which has a sensitivity and a specificity of 72% and 100%, respectively [23]. In addition, Yong et al. (2013) recently found high expression of the carcinoembryonic gene SALL4 in 55.6% of HCC tissues, but no expression was detected in pericarcinoma liver tissues and was associated with a poor prognosis. It can be used as a potential diagnostic marker in immunohistochemistry for HCC [24], and the evaluation of the background (inflammation, fibrosis) in the pericarcinoma liver tissue can rely on Masson staining.

The acinar (pseudoglandular)-type HCC should be differentiated from intrahepatic cholangiocarcinoma (ICC ) and hepatic metastatic adenocarcinoma (HMA), and the main differential points of the three are shown in Table 7.1. The immunohistochemical features of HCC are shown in Chap. 6 Immunohistochemistry and Special Staining for Liver Tissue.

Many factors have an impact on the prognosis of HCC. And to accurately evaluate the risk of postsurgical recurrence, survival, and prognosis in HCC patients, approximately 20 clinical pathological staging schemes for HCC have been proposed in the current literature, including the following HCC staging systems with great influence, such as the seventh edition of the American Joint Committee on Cancer (AJCC)/The Union for International Cancer Control (UICC) tumor-node-metastasis (TNM) staging system (Table 7.2), Okuda staging, the Cancer of the Liver Italian Program (CLIP) index, the Barcelona Clinic Liver Cancer (BCLC) score, the French staging, the Chinese University Prognostic Index (CUPI), Japan Integrated Staging (JIS), the Tokyo score, etc. The index system is usually composed of serological indexes and pathological parameters (such as tumor size, tumor number, MVI , satellite nodules, differentiation, grading, etc.), while the currently available staging systems in the literature have not received unanimous verdict in the assessment of HCC biological characteristics and prognosis of patients with HCC, each of which has advantages and disadvantages. Kee et al. (2013) identified that the seventh edition of the TNM staging was of better predictive value for the prognosis of HCC than its sixth edition [25]. In view of the high heterogeneity and malignancy of HCC, it is not sufficient to predict its complex biological behaviors based on the limited clinical and pathological indexes. Therefore, several indexes have been included into the staging of HCC, such as serum albumin, bilirubin, vascular endothelial growth (VEGF), and insulin-like growth factor-1 (IGF-1) [26]. It can be expected that, with the development of understanding on the biological characteristics of HCC, there will be consistent improvement and perfection in the prognostic evaluation system of HCC, including the international TNM staging.

In addition, Shirabe et al. (2014) proposed that in cases of HCC with the tumor diameter of 3.6 cm, the maximum standard uptake value (SUVmax) of 18F- fluorodeoxyglucose (FDG) in positron emission tomography (PET) was 4.2, and the abnormal prothrombin in serum reached 10 mAU/ml, both of which have the highest sensitivity and specificity in prediction of MVI [27]. Tsujita et al. (2012) proposed the indexes affecting the prognosis of patients with recurrent HCC: ①indocyanine green retention rate at 15 minutes, ②the disease-free interval, ③tumor size, ④portal vein invasion at resection of the primary HCC, ⑤ gender, and ⑥blood loss [28]. Kadalayil et al. (2013) proposed the prognosis indexes for TACE in HCC, including albumin <36 g/dl, bilirubin >17 μmol/L, and AFP >400 ng/ml or the diameter of the primary tumor >7 cm, each of which was recorded as 1 point. The median survival time for patients scoring 0, 1, 2, and >2 points was 27.6 months, 18.5 months, 9 months, and 3.6 months [29], respectively. Furthermore, the molecular typing of HCC will be an important trend for HCC research in the future.

As stated, factors related to the therapeutic effect and survival of patients with HCC include growth pattern, proliferation activity, invasion and metastasis potential, postoperative recurrence risk, and variation of genes and proteins associated with biological characteristics, the main carrier for the above important information is the tumor tissue specimens. Obviously, the traditional pathology focuses on the diagnosis model mainly based on the morphological features of HCC tissues and cells; however, it does not fit the concern about the pathobiological features of HCC in modern hepatic surgery, and new adjustments and supplements in the diagnostic concept, modes, and contents should be made to establish a comprehensive mode of pathological diagnosis based on both pathology and biology, further providing practical pathological information for the improvement of clinical curative effects. This is an important trend for future pathology of hepatic tumors.

Therefore, pathological diagnosis report for HCC should meet the clinical demands and concerns for the assessment of tumor growth pattern, prediction of metastasis and recurrence risk, formulation of individualized treatment strategy and evaluation of prognosis. No uniform standards on the content or format of the pathological diagnosis report for HCC have been determined, and it mainly includes the following aspects depending on the specific circumstances:

Early discovery, early treatment, and early cure and the smaller the tumor, the better the therapeutic efficacy have always been the basic principles and the ideal goals of modern surgical oncology. Many studies have suggested that the size of solid tumors can be used as an important reference index to assess the clinical prognosis of the patients. Despite many clinical and pathological factors that affect the malignancy and prognosis of the tumor, tumor size remains the most intuitive and simple index for presurgical assessment on tumorous progression stages. A typical example is the concepts of early gastric carcinoma, small gastric carcinoma, and micro-gastric carcinoma which were proposed 30 years ago and has greatly improved the level of clinical diagnosis and treatment of gastric cancer. In the late 1970s, Chinese scholars represented by academician Prof. Zhao-You Tang and Prof. Meng-Chao Wu first put forward the important concept of SHCC , being the milestone in development of clinical research of HCC into a new era of SHCC . In those days, the understanding of SHCC features in the field of hepatic surgery mainly include the following: 70% of the HCC patients with no obvious clinical symptoms had tumors ≤5 cm in diameter, and more than 70% of the HCC patients with obvious clinical symptoms had tumors >5 cm in diameter. Therefore, the tumor volume of ≤5 cm in diameter is widely accepted as the diagnostic standard for SHCC both at home and abroad. Under the condition of limited diagnostic and treatment techniques of HCC in that time, low rates of diagnosis and surgical resection of SHCC ≤5 cm were observed, and there were even reports in which surgically excised HCC <4.5 cm was called microcarcinoma [30]. And a diameter of 5 cm was still adopted as the pathological staging criteria in the seventh edition of TNM staging of HCC issued by AJCC and UICC in 2009.

On the basis of the study of 500 autopsy cases of HCC pathological specimens, the pathological classification of the “Five Major Types and Six Sub-Types” of HCC was put forward by the Chinese Cooperative Pathology Group of Liver Cancer in 1982, and three nodules ≤3 cm in diameter were called SHCC , which was especially proposed for the first time and was one of the most prominent contributions in the field of pathology of HCC in China. Since then, we have conducted systematic studies on the relationship between HCC size and its pathological biological behaviors based on the rat carcinogenesis model and surgically excised human HCC of different tumor sizes, demonstrating that SHCC ≤3 cm is often DNA diploid dominant, with relatively benign biological behaviors of HCC in the early stage, good prognosis, and low recurrence rate, while LHCC >3 cm is often DNA ploidy dominant, with obvious malignant biological behaviors, high postoperative recurrence rate, and low long-term survival rate. Thus, it has been proposed that HCC which is approximate 3 cm in diameter is in a key period in which its biological characteristics transform from relatively benign nature to marked malignancy, and the early diagnosis and treatment of SHCC can lead to relatively good therapeutic effects based on its pathobiological feature of radical resectability. Right now, BCLC staging system-defined HCC in the early stage may include patients with single or up to three tumor nodules, each ≤3 cm. However, based on the clonal origin theory of multinodular HCC, we think there is a great possibility of intrahepatic metastasis for three tumor nodules ≤3 cm. Therefore, in the Practice guidelines for the pathological diagnosis of primary liver cancer: 2015 update issued by the Chinese Pathological Group of Hepatobiliary Tumor and Liver Transplantation, a single tumor with a diameter from >1 cm to ≤3 cm is defined as SHCC .

Based on the statistics of data from the Department of Pathology in our hospital, the proportion of SHCC ≤3 cm treated by surgical resection has continuously increased (Fig. 7.101), and the postoperative survival rate at 5 years was 67.8%. Interestingly, Moribe et al. (2009) detected the methylation of 12 genes in 25 cases of HCC using quantitative methylation-specific PCR (MSP), and the results showed that all the SHCC ≤3 cm contained three gene methylation of RASSF1A, SPINT2, and CCND2, and its specificity, sensitivity, and accuracy were 100%, ≥75%, and 95%, respectively [31]. Llovet et al. (2006) found a successively increasing expression of three-gene spectrum consisting of GPC-3, survivin, and LYVE1 spectrum in dysplastic nodules , SHCC ( diameter (2 ± 0.6) cm, ranged 0.9–3 cm) and LHCC, and the diagnostic accuracy of the gene spectrum in the three kinds of lesions reached 94% [32]. These two studies further suggest that there are corresponding molecular variations in the transformation from SHCC (≤3 cm) to LHCC (>3 cm) which is worth further study.

However, no uniform criterion for the tumor volume of SHCC has been accepted at home and abroad, and 5 cm of SHCC in diameter is still in use by many scholars, which is not correspondent to the biological characteristics of early HCC or current clinical techniques in early detection and early diagnosis of HCC. The present international staging systems, such as BCLC and AASLD, defined the early HCC as tumors ≤3 cm and very early HCC as tumors ≤2 cm. Nevertheless, up to now, most studies on SHCC ≤2 cm are based on multicenter joint studies with long-term data collection, and among the cases of surgically resected HCC in centers for hepatic diseases all around the world, reports of SHCC ≤2 cm are still very rare with only a few studies on their biological characteristics (Table 7.3). For instance, Minagawa et al. (2007) collected 2767 cases of ≤2 cm SHCC from 829 units in Japan during a 30-year period. This is a summary of a huge sample, but the actual number of cases in each year for each unit is minimal. Farinati et al. (2009) analyzed the data of 1834 HCC cases (partly confirmed by pathology) during 10 years accumulated by the Cancer of the Liver Italian Program (ITA.​LI.​CA) and found that cases of ≤2 cm SHCC accounted for only 3%, which could not be analyzed for internal validation due to the insufficient number of cases. Thus, the authors suggested that the tumor volume of ≤2 cm as a criterion for the diagnosis of SHCC is of less staging significance in clinical practice because these cases are few in number.

Many studies have been reported at home and abroad; the long-term survival rate of patients with SHCC is significantly higher than that of LHCC patients, and postoperative recurrence rate was significantly lower than that of LHCC patients. We (2011) conducted a control study of 618 cases of HCC patients, who were divided into five groups according to the diameter of the tumors, ≤1 cm, 1.1–2 cm, 2.1–3 cm, 3.1–5 cm, and >5 cm, and the results showed that no significant difference in pathologic parameters and operative prognosis is found between 1–3 cm and >5 cm groups except the group of patients with tumors ≤1 cm in diameter (Fig. 7.102). However, if the patients were divided into two groups with the tumor diameter of 3 cm as a threshold, there were significant difference in pathologic parameters and operative prognosis between the two groups (Fig. 7.103) [33].

Based on our observations, the main biological characteristics of SHCC include DNA diploid dominant, relatively slow growth with single and localized nodule, high degrees of differentiation, thin-trabecular type in histology, with or without a fibrous capsule, clear boundaries, rare MVI or satellite focus which are usually found in liver tissues 0.5–1 cm from the surgical margin, high postsurgical survival rate, and low recurrence rate. Tumors larger than 3 cm in diameter have pathobiological indexes which indicate transformation into malignant LHCC in progression, such as significantly increased rates of capsular and microvascular invasion , occurrence of satellite focus, and postsurgical recurrence. Combining our researching results and understanding, the growth and progression of HCC can be generally divided into four stages correspondent to four patterns of tumor growth, including microcarcinoma (≤1 cm, non-capsule stage) with transition between the tumor margin and pericarcinoma liver tissues → SHCC (1–3 cm, capsule formation stage) with dominantly solitary nodular growth, a clear border and an intact capsule in most of the cases, or with capsular invasion in a few cases → medium-sized HCC (3–5 cm, accelerated growth stage) with more extracapsular sub-carcinoma foci and increased microvascular invasion → large HCC (>5 cm, malignant advanced stage) with multifocal infiltration in most cases, capsular invasion, MVI , and commonly seen satellite nodules (Fig. 7.104) [33, 34].

Early HCC (EHCC) should be referred to a small and well-differentiated HCC in the early stage of development, with relatively benign biological behaviors, no vascular invasion, and intrahepatic metastasis. However, the current conception and criterion of EHCC and SHCC is not consistent in the world. In BCLC staging classification, three nodules ≤3 cm in diameter were included in the early stage (stage A). However, from the point of view of pathology, three HCC nodules have a great possibility of intrahepatic metastasis, suggesting not in early stage in a biological sense. Nathan et al. (2013) defined EHCC as HCC ≤5 cm in diameter, without metastatic foci, lymph node metastasis, extrahepatic expansion, or invasion of major blood vessels. Sakamoto et al. (1998) defined EHCC as well-differentiated (Edmondson pathological grade I or grade I with grade II in a few region) HCC with negative tumor staining in angiography regardless of the size of the tumor. EHCC was also adopted in the WHO’s Pathological Classification of Hepatobiliary Tumors (2010), which was defined as well-differentiated SHCC with a diameter of <2 cm. In general, SHCC refers to a HCC in small size, which is a morphological concept, while EHCC refers to HCC in early stages of tumorigenesis and progression with relatively benign biological behaviors, which is a biological concept. Therefore, it is inappropriate to simply equate SHCC and EHCC, since a few cases of SHCC have malignant biological behaviors including DNA ploidy, capsular invasion, and MVI formation in earlier stages. Table 7.4 shows the differences in criteria of staging systems on SHCC and EHCC in the literature, each of which has a certain influence.

When evaluating the biological characteristics according to the tumor size of HCC, most SHCC ≤3 cm presents relatively benign performances of EHCC in early stage of tumorigenesis and development, with long-term survival rate and low recurrence rate after radical resection, indicating SHCC ≤3 cm has the pathological basis of radical resectability. However, we have found that about 26% of the SHCC contain DNA ploidy, presenting significantly malignant biological behaviors, such as poor differentiation, capsular invasion, and formation of microvascular thrombus, with postsurgical early recurrence; even a microcarcinoma of 0.6 cm in diameter may contain MVI , suggesting that these SHCC have an increased growth activity, and has an accelerated progress of malignancy, which does not belong to the biological category of EHCC. It is certain that part of the LHCC may have the morphological representation of SHCC and the biological characteristics of EHCC, with long-term survival rate and low recurrence rate after radical resection. Thus, SHCC is not completely equal to EHCC in the strict sense. In clinical surgery, the individualized therapeutic strategy for SHCC should be formulated according to the pathological type and growth pattern with a certain extent of surgical resection. Generally, for SHCC ≤1–3 cm in diameter, a retaining resection extension of 0.5–1 cm should be considered, and for HCC larger than 3 cm, it should be >1–2 cm to completely remove possible residual satellite foci or MVI , which also fits in radiofrequency ablation in order to improve the long-term treatment efficacy.

We have noticed in pathological practice that a typical HCC in morphology can express markers of both the liver cell line (e.g., Hep Par-1, CK18, pCEA, etc.) and bile duct cell line (e.g., CK7, CK19, MUC-1, etc.), different from the cHCC-CC containing both HCC and CC components, and we named it as dual-phenotype HCC (DPHCC) [54]. Clearly, morphopathologically, DPHCC is not completely the same as cHCC-CC .

Based on the analysis on the characteristics of immunohistochemical phenotype, DPHCC accounts for about 5% of HCC. In clinical practice, DPHCC’s performance is similar to that of HCC, including a history of HBV or HCV infection, as well as chronic hepatitis and cirrhosis background, and elevation of both serum AFP and CA19-9 can be found in some cases. Histologically, DPHCC represents typical HCC features, such as polygon-shaped cancer cells, trabecular arrangement, sinuses, and pseudoglandular structure. In immunohistochemistry, the carcinoma cells, which express markers of liver cells with canalicular staining pattern for CD10 and pCEA, show the phenotypic characteristics of immunohistochemical proteins in CC (Fig. 7.136). Interestingly, the MVI found in DPHCC tissue maintained the dual phenotype, suggesting it is composed by cell populations with high proliferation and invasion activities (Fig. 7.137). According to our diagnostic criteria, the number of dual-phenotype positive cancer cells should be more than 15%, and they often show high expression of stem cell markers, such as EpCAM. DPHCC showed stronger invasiveness in biological behavior, and the incidence of satellite nodules and MVI is higher than that of common HCC. In the aspect of prognosis, the total survival rate and recurrence rate in patients with DPHCC are significantly poorer than those of the common HCC [54]. And our experimental studies showed that inhibition or upregulation of the expression of CK19 mRNA in HCC cells can markedly affect the proliferation and invasion of cancer cells. We hypothesized that DPHCC is a unique molecular subtype of HCC, originating from hepatic progenitor cells in the liver and maintaining a bidirectional differentiation potential, and it has a higher malignancy due to its dual biological behavior of HCC and ICC (Fig. 7.138). Govaere et al. (2013) also reported a lower invasive activity in the CK19 knockout HCC cell strain, as well as decreased drug resistance to 5-FU and sorafenib, suggesting the multiple drug resistance of DPHCC in clinical treatment [55]. Therefore, the routine pathological diagnosis should contain careful classification of special subtypes of HCC including DPHCC, to provide elaborate pathological information for clinical development of individualized diagnosis and treatment strategies.