To the authors' knowledge, this is the first report of the detection of HBV 1762T/1764A double mutation in the urine of patients with HCC and infected with HBV. This finding was achieved by developing a PCR assay that targets a template size of 40 bp optimized for use with transrenal DNA and is the first step toward developing a urine test for screening for HCC in individuals infected with HBV. The source of the HBV 1762T/1764A double mutation DNA detected in the urine of patients with HCC is unclear. It is possible that the HBV 1762T/1764A double mutation DNA is derived from the breakdown of DNA from the HBV infectious particles in circulation, from the closed circular form of the HBV genome, or from the integrated HBV genome released from infected hepatocytes. Because the DNA substrate is the low molecular weight urine DNA fraction (less than 1 Kb), the authors do not think that the HBV 1762T/1764A double mutation DNA detected was derived from infectious HBV virions in urine or in the circulation. Previous studies by the authors have shown that low molecular weight urine DNA co-migrated with DNA of 1 to 2 nucleosomes isolated in serum, which suggests that the source of this low molecular weight urine DNA is mostly from apoptotic cells (Su, Wang et al. 2004). It has been suggested that the turnover of hepatocytes in liver infected with HBV is increased, mostly by apoptosis. The authors thus speculate that the HBV 1762T/1764A double mutation detected in the urine of patients with HCC who were infected with HBV might originate from apoptotic infected hepatocytes.

In this small pilot study, the association of the detection of HBV double mutation sequences with the size of the tumor (Tables 1 and 2) was not found, perhaps because the sample size was too small. In this cohort, only 1 (20K) of 11 HCCs from patients infected with HBV was a stage I tumor. Interestingly, HBV double mutation was detected in this stage I tumor (20K, tumor size 2 cm), but not in 3 higher stage tumors [10K (stage II, tumor size 2 cm), 19K (stage III, tumor size 6 cm), and 25K (stage III, tumor size 5.5 cm)]. A larger number of samples are needed for this comparison. As discussed earlier, the amount of tumor DNA in urine could be due to the amount of apoptosis of the tumor. With heterogeneity of HCC, the rate of apoptosis might vary from tumor to tumor. Other physiological conditions such as hydration of body could also contribute to the concentration of circulation-derived DNA in urine. Nevertheless, it is encouraging that 9 of 11 urine samples from patients with HCC who were infected with HBV were positive for HBV double mutation by the HBVDM\_40 assay, suggesting that this assay could be included in a urine test for HCC screening for patients infected with HBV. The assay developed in this study is not robust enough to be used in clinical settings for routine screenings. Nevertheless, the HBV 1762T/1764A double

Individuals infected with HBV are a high-risk group for developing HCC. They need to be screened regularly for HCC. However, the latent period between contracting HBV infection and developing HCC ranges from a few to 30 years (Kim, Lee et al. 2000). Currently, the alpha-fetoprotein blood test and ultrasonography are the only screening methods available. Given the cost and the technical expertise required for blood collection and ultrasonography, a noninvasive, urine-based screening method is highly preferable and likely to have higher compliance than serum assays or imaging. It has been suggested that the HBV 1762T/1764A double mutation is a potential predictive marker for HCC because of its association with 50% to 85% of HBV-associated HCC and its detection in the circulation of patients infected with HBV who were either diagnosed with HCC at the time the blood was collected or who developed HCC a few years later (Kuang, Lekawanvijit et al. 2005).

To the authors' knowledge, this is the first report of the detection of HBV 1762T/1764A double mutation in the urine of patients with HCC and infected with HBV. This finding was achieved by developing a PCR assay that targets a template size of 40 bp optimized for use with transrenal DNA and is the first step toward developing a urine test for screening for HCC in individuals infected with HBV. The source of the HBV 1762T/1764A double mutation DNA detected in the urine of patients with HCC is unclear. It is possible that the HBV 1762T/1764A double mutation DNA is derived from the breakdown of DNA from the HBV infectious particles in circulation, from the closed circular form of the HBV genome, or from the integrated HBV genome released from infected hepatocytes. Because the DNA substrate is the low molecular weight urine DNA fraction (less than 1 Kb), the authors do not think that the HBV 1762T/1764A double mutation DNA detected was derived from infectious HBV virions in urine or in the circulation. Previous studies by the authors have shown that low molecular weight urine DNA co-migrated with DNA of 1 to 2 nucleosomes isolated in serum, which suggests that the source of this low molecular weight urine DNA is mostly from apoptotic cells (Su, Wang et al. 2004). It has been suggested that the turnover of hepatocytes in liver infected with HBV is increased, mostly by apoptosis. The authors thus speculate that the HBV 1762T/1764A double mutation detected in the urine of patients with HCC who were infected with HBV might originate from apoptotic infected hepatocytes.

In this small pilot study, the association of the detection of HBV double mutation sequences with the size of the tumor (Tables 1 and 2) was not found, perhaps because the sample size was too small. In this cohort, only 1 (20K) of 11 HCCs from patients infected with HBV was a stage I tumor. Interestingly, HBV double mutation was detected in this stage I tumor (20K, tumor size 2 cm), but not in 3 higher stage tumors [10K (stage II, tumor size 2 cm), 19K (stage III, tumor size 6 cm), and 25K (stage III, tumor size 5.5 cm)]. A larger number of samples are needed for this comparison. As discussed earlier, the amount of tumor DNA in urine could be due to the amount of apoptosis of the tumor. With heterogeneity of HCC, the rate of apoptosis might vary from tumor to tumor. Other physiological conditions such as hydration of body could also contribute to the concentration of circulation-derived DNA in urine. Nevertheless, it is encouraging that 9 of 11 urine samples from patients with HCC who were infected with HBV were positive for HBV double mutation by the HBVDM\_40 assay, suggesting that this assay could be included in a urine test for HCC screening for patients infected with HBV. The assay developed in this study is not robust enough to be used in clinical settings for routine screenings. Nevertheless, the HBV 1762T/1764A double

**4. Discussion**

mutation was detected in the urine by this two-step PCR assay; this study showed that it is necessary to minimize the targeting template size to have sufficient sensitivity to detect the circulating DNA marker in urine. The development of a high-throughput quantitative assay for this marker to translate this finding for clinical use is in progress.

It would be of great interest to compare the detectability of the HBV double mutation DNA in urine with that of the current screening methods, the serum level of alpha-fetoprotein and ultrasound. Although the sample size is small (n=15), 3 (7K, 18K, 20K) of 6 urine samples from AFP-negative (< 20 ng/mL) patients with HCC who were infected with HBV were positive for the HBV double mutation by HBVDM\_40 assay. The HBV 1762T/1764A double mutation would need to be evaluated in a large-scale study to determine how early in the disease process of HCC this marker appears in the urine by screening the urine from patients with hepatitis, cirrhosis, or HCC who were infected with HBV. Thus, the specificity and sensitivity of the marker alone for HCC screening can be determined.

The use of urine for cancer detection shows promise for potential clinical applications in HCC screening. Current literature has implicated other HBV mutations as potential HCC screening markers. A mutation in the precore region is of interest. Not only has this mutation been shown to be genotype specific but also to play a role in HBeAg secretion by forming a premature stop codon. Precore variants can be first detected around the time of HBeAg seroconversion and potentially prevent HBeAg synthesis without prohibiting production of infectious HBV virions (Kao 2003; Yang, Yeh et al. 2008). A particular precore region mutation involves position 1896 a G to A substitution (G1896A), however it depends on the nucleotide present at position 1858 as it forms a stem structure. As a result, this G1896A mutation rarely occurs in HBV genotype A due to pairing with dominate C1858 not T1858 as seen in other genotypes. The impact of this mutation is still in question. Studies have shown this mutation to be associated with a more aggressive disease such as reactivation of chronic HBV while others have shown the opposite where there is a better prognosis with the occurrence of this mutation (Kao 2003; Yang, Yeh et al. 2008).

Other mutations at position C1653T in the enhancer II region and position 1753 T to C/A/G in the core promoter region have also been found. However, the mutation at 1753 was not found to be significantly associated with HCC development, but the core promoter mutation at 1753 were found to correlate with HCC development. The C to T mutation at 1653 alters the X protein possibly contributing to hepatocarcinogenesis and shows increased frequency with progression from chronic hepatitis to cirrhosis (Yuen et al., 2008). Although the link between the core promoter mutations and development of HCC is still unclear, it is thought that these mutations play a role in altering pregenomic secondary structure that enhances viral replication and this has been demonstrated by increased HBV DNA levels in patients with core promoter mutations compared to those with no core promoter mutations.

Lastly, mutations in the PreS/S have been of interest. S mutations involving glycine to arginine substitution at position 145 (G145R) have been shown to play a role in immune escape by altering HBV envelope proteins. PreS deletions play a role in forming the hepatocyte binding site, transportation out of the hepatocyte and are essential for virion assembly (Llovet and Lok 2008; Nguyen, Law et al. 2009). These mutations are thought to occur during pathogenesis of HBV infection and transmitted vertically. Thus there is a potential for these mutations to serve as HCC screening markers.

Strategic Assay Developments for Detection of HBV 1762T/1764A Double

overall survival.

**7. References**

**6. Acknowledgment**

Coulter Foundation (YHS).

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Mutation in Urine of Patients with HBV-Associated Hepatocellular Carcinomas 151

invasive and cost-effective as possible. The urine based tests can meet both of these demands and are also suitable for developing nations with fewer resources. The next step should be to test-drive the performance of the assays for HBV mutations in a larger study population. This will allow evaluation of these mutations as diagnostic biomarkers and investigate their association with HCC development, prognosis, treatment efficacy and

This work is supported by NIH R01 CA125642 (YHS), The Prevent Cancer Foundation (Postdoctoral fellowship to SJ), and a translational research grant award from the Wallace

Aravalli, R. N., C. J. Steer, et al. (2008). "Molecular mechanisms of hepatocellular carcinoma."

Arbuthnot, P. and M. Kew (2001). "Hepatitis B virus and hepatocellular carcinoma."

Baptista, M., A. Kramvis, et al. (1999). "High prevalence of 1762T 1764A mutations in the

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Botezatu, I., O. Serdyuk, et al. (2000). "Genetic analysis of DNA excreted in urine: a new

Boyault, S., D. S. Rickman, et al. (2007). "Transcriptome classification of HCC is related to gene alterations and to new therapeutic targets." Hepatology 45 42-45. Bressac, B., M. Kew, et al. (1991). "Selective G to T mutations of p53 gene in hepatocellular

Buamah, P. K., I. Gibb, et al. (1984). "Serum alpha fetoprotein heterogeneity as a means of

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In HCC screening, it is important to take into consideration the HBV genotypes as they have a role in the clinical outcome, treatment efficacy, prevalence patterns of HBV mutants and the geographic spread of HCC. There are known genotypes A through H with specific geographic distribution. HBV genotypes B and C are predominant in Southeast Asia. Genotype C has been found to be associated with more severe liver disease with higher frequency of HBeAg, higher serum HBV DNA levels, delayed HBeAg seroconversion, higher frequency of basal core promoter mutation, and lower response rate to interferon therapy compared to genotype B. However genotype B is associated with development of HCC in younger populations. Genotype A which is frequent in North America Europe, India, and parts of Africa holds a predominant nucleotide at position 1858 and as a result incur less frequent precore mutations. While genotypes B, C, and D which hold a predominant nucleotide T at position 1858 is more frequent in the Mediterranean and Asia have a much higher frequency of precore mutations. Genotypes F is found in South America while genotypes E (Africans), F (aboriginal populations of South America), H (Amerindian populations of Central America), and G (HBV carriers in France, Georgia, USA, and Germany) are less well studied (Kao 2003; Yuen, Tanaka et al. 2008). Furthermore, in Caucasian and Indian populations genotype D is associated with greater risk of HCC development compared to genotype A indicating host factors in combination with genotype can play a role in HCC risk.

Moreover, HCC, like other cancers, is extremely heterogeneous and has a complex etiology, even within the HBV-infected group (Buamah, Gibb et al. 1984; Bressac, Kew et al. 1991; Arbuthnot and Kew 2001; Block, Mehta et al. 2003; Boyault, Rickman et al. 2007; Aravalli, Steer et al. 2008; Hoshida, Nijman et al. 2009). Any single DNA marker is unlikely to have sufficient sensitivity and specificity to detect HCC such that it can be used as a stand-alone diagnostic test. Thus, a panel of multiple genetic and epigenetic markers, such as HCCassociated HBV mutations, as discussed above, mutations in p53 and CTNNB1 genes (Bressac, Kew et al. 1991; de La Coste, Romagnolo et al. 1998; Miyoshi, Iwao et al. 1998; Huang, Fujii et al. 1999; Legoix, Bluteau et al. 1999; Terris, Pineau et al. 1999; Wong, Fan et al. 2001; Taniguchi, Roberts et al. 2002; Edamoto, Hara et al. 2003; Kirk, Lesi et al. 2005; Zhang, Rossner et al. 2006; Boyault, Rickman et al. 2007; Hussain, Schwank et al. 2007; Cieply, Zeng et al. 2009; Jain, Singhal et al. 2010) and aberrant methylation of p16, APC, and GSTP-1 genes (Lee, Lee et al. 2003; Jicai, Zongtao et al. 2006; Katoh, Shibata et al. 2006; Su, Lee et al. 2007; Chang, Yi et al. 2008; Gao, Kondo et al. 2008; Harder, Opitz et al. 2008; Su, Zhao et al. 2008; Feng, Stern et al. 2010; Hernandez-Vargas, Lambert et al. 2010; Jain, Singhal et al. 2010) could be assembled that cumulatively result in a level of sensitivity and specificity for HCC screening that is superior or complimentary to alpha fetoprotein and its L3 glycoform or to ultrasound imaging, the methods currently used for HCC screening.

### **5. Conclusion**

With the current technology in assay development, it is feasible to design assays targeting clinically relevant mutations in HBV for HCC screening. Given the heterogeneity of HCC and the genotypes of HBV, the challenge lies in developing a panel of assays which can then give a better performance than current screening tests. Also, given the long window period between HBV infection and HCC development, it is important that the test be as noninvasive and cost-effective as possible. The urine based tests can meet both of these demands and are also suitable for developing nations with fewer resources. The next step should be to test-drive the performance of the assays for HBV mutations in a larger study population. This will allow evaluation of these mutations as diagnostic biomarkers and investigate their association with HCC development, prognosis, treatment efficacy and overall survival.

### **6. Acknowledgment**

This work is supported by NIH R01 CA125642 (YHS), The Prevent Cancer Foundation (Postdoctoral fellowship to SJ), and a translational research grant award from the Wallace Coulter Foundation (YHS).

### **7. References**

150 Hepatocellular Carcinoma – Clinical Research

In HCC screening, it is important to take into consideration the HBV genotypes as they have a role in the clinical outcome, treatment efficacy, prevalence patterns of HBV mutants and the geographic spread of HCC. There are known genotypes A through H with specific geographic distribution. HBV genotypes B and C are predominant in Southeast Asia. Genotype C has been found to be associated with more severe liver disease with higher frequency of HBeAg, higher serum HBV DNA levels, delayed HBeAg seroconversion, higher frequency of basal core promoter mutation, and lower response rate to interferon therapy compared to genotype B. However genotype B is associated with development of HCC in younger populations. Genotype A which is frequent in North America Europe, India, and parts of Africa holds a predominant nucleotide at position 1858 and as a result incur less frequent precore mutations. While genotypes B, C, and D which hold a predominant nucleotide T at position 1858 is more frequent in the Mediterranean and Asia have a much higher frequency of precore mutations. Genotypes F is found in South America while genotypes E (Africans), F (aboriginal populations of South America), H (Amerindian populations of Central America), and G (HBV carriers in France, Georgia, USA, and Germany) are less well studied (Kao 2003; Yuen, Tanaka et al. 2008). Furthermore, in Caucasian and Indian populations genotype D is associated with greater risk of HCC development compared to genotype A indicating host factors in combination with genotype

Moreover, HCC, like other cancers, is extremely heterogeneous and has a complex etiology, even within the HBV-infected group (Buamah, Gibb et al. 1984; Bressac, Kew et al. 1991; Arbuthnot and Kew 2001; Block, Mehta et al. 2003; Boyault, Rickman et al. 2007; Aravalli, Steer et al. 2008; Hoshida, Nijman et al. 2009). Any single DNA marker is unlikely to have sufficient sensitivity and specificity to detect HCC such that it can be used as a stand-alone diagnostic test. Thus, a panel of multiple genetic and epigenetic markers, such as HCCassociated HBV mutations, as discussed above, mutations in p53 and CTNNB1 genes (Bressac, Kew et al. 1991; de La Coste, Romagnolo et al. 1998; Miyoshi, Iwao et al. 1998; Huang, Fujii et al. 1999; Legoix, Bluteau et al. 1999; Terris, Pineau et al. 1999; Wong, Fan et al. 2001; Taniguchi, Roberts et al. 2002; Edamoto, Hara et al. 2003; Kirk, Lesi et al. 2005; Zhang, Rossner et al. 2006; Boyault, Rickman et al. 2007; Hussain, Schwank et al. 2007; Cieply, Zeng et al. 2009; Jain, Singhal et al. 2010) and aberrant methylation of p16, APC, and GSTP-1 genes (Lee, Lee et al. 2003; Jicai, Zongtao et al. 2006; Katoh, Shibata et al. 2006; Su, Lee et al. 2007; Chang, Yi et al. 2008; Gao, Kondo et al. 2008; Harder, Opitz et al. 2008; Su, Zhao et al. 2008; Feng, Stern et al. 2010; Hernandez-Vargas, Lambert et al. 2010; Jain, Singhal et al. 2010) could be assembled that cumulatively result in a level of sensitivity and specificity for HCC screening that is superior or complimentary to alpha fetoprotein and its L3 glycoform or to ultrasound imaging, the methods currently used for HCC screening.

With the current technology in assay development, it is feasible to design assays targeting clinically relevant mutations in HBV for HCC screening. Given the heterogeneity of HCC and the genotypes of HBV, the challenge lies in developing a panel of assays which can then give a better performance than current screening tests. Also, given the long window period between HBV infection and HCC development, it is important that the test be as non-

can play a role in HCC risk.

**5. Conclusion**

Strategic Assay Developments for Detection of HBV 1762T/1764A Double

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**8**

*Japan*

**Usual and Unusual Gross Appearance of**

Kohji Miyazaki2, Hirokazu Noshiro2 and Osamu Tokunaga1

Hepatocellular carcinoma (HCC) usually shows a typical gross appearance and the gross classification and gross diagnosis of resected specimens is performed as routine clinical work. In the authors' institution, many surgical cases of HCC are encountered, including rare and unusual cases. This chapter documents the gross classification, features and diagnosis of usual and unusual HCCs with representative gross photographs and a review of the literature.

The gross appearances of HCCs are affected by various factors such as tumor size, tumor thrombus in the portal and hepatic veins, intrahepatic metastasis, necrosis and hemorrhage. Well-differentiated HCC often produces bilirubin and the tumor color will be greenish after formalin fixation. On the other hand, poorly differentiated HCC usually shows a whitish coloration on gross appearance. If the tumor is accompanied by steatosis, the tumor will be yellowish in color. HCC frequently presents with a nodular appearance showing fibrous capsules and septa. The occurrence of capsule and septum formation is closely related to tumor size. Both capsule and septum are unusual in small HCCs less than 2.0 cm in diameter. In the majority of HCCs over 2.0 cm in diameter, the tumor grows expansively

Eggel's classification [Eggel, 1901] was proposed more than 100 years ago, and has been widely used for the classification of autopsy cases, rather than surgical cases. This system classifies HCC into three major types according to gross appearance: nodular; massive; and diffuse. Nodular type consists of a single or multiple discrete nodular tumors with clear demarcation. Massive type is a large mass replacing almost the entire right or left lobe. Diffuse type is characterized by diffuse proliferation of numerous minute cirrhosis-like

**1. Introduction**

**2. Gross findings**

and a capsule and septum are formed.

**3.1 Eggel's classification**

**3. Gross classification of usual types of HCC**

tumor nodules throughout the entire lobe or liver.

**Hepatocellular Carcinomas**

*1Department of Pathology & Microbiology*

*Saga University Faculty of Medicine*

*2Department of Surgery*

Keita Kai1, Atsushi Miyoshi2, Kenji Kitahara2,

### **Usual and Unusual Gross Appearance of Hepatocellular Carcinomas**

Keita Kai1, Atsushi Miyoshi2, Kenji Kitahara2,

Kohji Miyazaki2, Hirokazu Noshiro2 and Osamu Tokunaga1 *1Department of Pathology & Microbiology 2Department of Surgery Saga University Faculty of Medicine Japan*

### **1. Introduction**

154 Hepatocellular Carcinoma – Clinical Research

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Hepatocellular carcinoma (HCC) usually shows a typical gross appearance and the gross classification and gross diagnosis of resected specimens is performed as routine clinical work. In the authors' institution, many surgical cases of HCC are encountered, including rare and unusual cases. This chapter documents the gross classification, features and diagnosis of usual and unusual HCCs with representative gross photographs and a review of the literature.

### **2. Gross findings**

The gross appearances of HCCs are affected by various factors such as tumor size, tumor thrombus in the portal and hepatic veins, intrahepatic metastasis, necrosis and hemorrhage. Well-differentiated HCC often produces bilirubin and the tumor color will be greenish after formalin fixation. On the other hand, poorly differentiated HCC usually shows a whitish coloration on gross appearance. If the tumor is accompanied by steatosis, the tumor will be yellowish in color. HCC frequently presents with a nodular appearance showing fibrous capsules and septa. The occurrence of capsule and septum formation is closely related to tumor size. Both capsule and septum are unusual in small HCCs less than 2.0 cm in diameter. In the majority of HCCs over 2.0 cm in diameter, the tumor grows expansively and a capsule and septum are formed.

### **3. Gross classification of usual types of HCC**

### **3.1 Eggel's classification**

Eggel's classification [Eggel, 1901] was proposed more than 100 years ago, and has been widely used for the classification of autopsy cases, rather than surgical cases. This system classifies HCC into three major types according to gross appearance: nodular; massive; and diffuse. Nodular type consists of a single or multiple discrete nodular tumors with clear demarcation. Massive type is a large mass replacing almost the entire right or left lobe. Diffuse type is characterized by diffuse proliferation of numerous minute cirrhosis-like tumor nodules throughout the entire lobe or liver.

Usual and Unusual Gross Appearance of Hepatocellular Carcinomas 157

with indistinct margins, distinguishing an obscure tumor nodule from surrounding cirrhotic liver is sometimes difficult. Histologically, most tumors of small nodular type with indistinct margins consist of uniformly distributed well-differentiated cancerous tissue showing a characteristic replacing growth at the tumor boundary. Although portal tracts are retained within the nodule, the number is markedly reduced [Nakashima et al., 1999]. Many of the retained portal tracts are deformed due to varying degrees of tumor cell invasion, designated as "stromal invasion", and this stromal invasion is regarded as the most helpful clue to distinguish small well-differentiated HCC from high-grade dysplastic nodules [Kondo et al., 1994, Tomizawa et al., 1995, Nakano et al, 1997]. Vascular invasion and intrahepatic micrometastasis are not usually observed in small nodular-type HCC with indistinct margins. Well-differentiated HCC of the small nodular type with indistinct margins has thus been designated as "early hepatocellular carcinoma", to separate this

particular type of HCC from the classical form of HCC [Kojiro, 2006, 2007].

a

b

Fig. 3. a) Gross appearance of small nodular type with indistinct margins. b) Characteristic

replacing growth at the tumor boundary (hematoxylin and eosin (HE), 20).

Fig. 1. Gross appearance of massive-type HCC in Eggel's classification.

### **3.2 Subclassification of nodular HCC**

Eggel's classification is not applicable to many surgically resected HCCs. In order to cope with surgically resected HCCs, the Liver Cancer Study Group of Japan (LCSGJ) divided nodular HCC into three subtypes: simple nodular type; simple nodular type with extranodular growth; and confluent multinodular type [Kanai et al., 1987; LCSGJ, 1987]. The category of small nodular type with indistinct margin, as the typical gross appearance of early HCC, has recently been added to this subclassification [LCSGJ, 2000]. Several investigators have reported the predictive value of this subclassification, with favorable outcomes for simple nodular type compared to the other two subtypes in terms of recurrence and survival after hepatectomy [Hui et al., 2000; Shimada et al., 2001; Inayoshi et al., 2003].

Fig. 2. Schema for subclassification of nodular HCC on gross appearance

### **3.2.1 Small nodular type with indistinct margins**

Grossly, small HCC less than about 2 cm in diameter can be classified into two major types: a small nodular type with indistinct margins (vaguely nodular type); and a distinctly nodular type [Kojiro & Nakashima, 1999]. The distinctly nodular type can be classified into one of the three subtypes: simple nodular type; simple nodular type with extranodular growth; and confluent multinodular type. On gross observation of the small nodular type

Eggel's classification is not applicable to many surgically resected HCCs. In order to cope with surgically resected HCCs, the Liver Cancer Study Group of Japan (LCSGJ) divided nodular HCC into three subtypes: simple nodular type; simple nodular type with extranodular growth; and confluent multinodular type [Kanai et al., 1987; LCSGJ, 1987]. The category of small nodular type with indistinct margin, as the typical gross appearance of early HCC, has recently been added to this subclassification [LCSGJ, 2000]. Several investigators have reported the predictive value of this subclassification, with favorable outcomes for simple nodular type compared to the other two subtypes in terms of recurrence and survival after hepatectomy

Fig. 1. Gross appearance of massive-type HCC in Eggel's classification.

Fig. 2. Schema for subclassification of nodular HCC on gross appearance

Grossly, small HCC less than about 2 cm in diameter can be classified into two major types: a small nodular type with indistinct margins (vaguely nodular type); and a distinctly nodular type [Kojiro & Nakashima, 1999]. The distinctly nodular type can be classified into one of the three subtypes: simple nodular type; simple nodular type with extranodular growth; and confluent multinodular type. On gross observation of the small nodular type

[Hui et al., 2000; Shimada et al., 2001; Inayoshi et al., 2003].

**3.2.1 Small nodular type with indistinct margins**

**3.2 Subclassification of nodular HCC**

with indistinct margins, distinguishing an obscure tumor nodule from surrounding cirrhotic liver is sometimes difficult. Histologically, most tumors of small nodular type with indistinct margins consist of uniformly distributed well-differentiated cancerous tissue showing a characteristic replacing growth at the tumor boundary. Although portal tracts are retained within the nodule, the number is markedly reduced [Nakashima et al., 1999]. Many of the retained portal tracts are deformed due to varying degrees of tumor cell invasion, designated as "stromal invasion", and this stromal invasion is regarded as the most helpful clue to distinguish small well-differentiated HCC from high-grade dysplastic nodules [Kondo et al., 1994, Tomizawa et al., 1995, Nakano et al, 1997]. Vascular invasion and intrahepatic micrometastasis are not usually observed in small nodular-type HCC with indistinct margins. Well-differentiated HCC of the small nodular type with indistinct margins has thus been designated as "early hepatocellular carcinoma", to separate this particular type of HCC from the classical form of HCC [Kojiro, 2006, 2007].

Fig. 3. a) Gross appearance of small nodular type with indistinct margins. b) Characteristic replacing growth at the tumor boundary (hematoxylin and eosin (HE), 20).

Usual and Unusual Gross Appearance of Hepatocellular Carcinomas 159

Fig. 5. Gross appearance of simple nodular type with extranodular growth. a) Small tumor of simple nodular type with extranodular growth. b) Large tumor of simple nodular type with extranodular growth. This type of tumor sometimes needs discrimination from

This is a tumor resulting from the joining together of a few to numerous small tumor nodules. No fibrous capsule is seen covering the entire tumor in this type and portal tracts are frequently entrapped between agglomerated small tumor nodules. The incidence of vascular invasion and/or intrahepatic metastasis is higher than in simple nodular types. Large confluent multinodular-type tumors sometimes need discrimination from massive-

massive-type and/or confluent multinodular-type HCC.

**3.2.4 Confluent multinodular type**

a

b

type HCC.

### **3.2.2 Simple nodular type**

This is a well-demarcated nodule, frequently surrounded by a fibrous capsule. Grossly, extranodular tumor growth, intrahepatic metastasis, and/or vascular invasion are not observed. The inside of the tumor often shows a mosaic appearance due to fibrous septa. Small HCC of the distinctly nodular type is also included in this category.

Fig. 4. Gross appearance of simple nodular type. A fibrous capsule and mosaic appearance due to fibrous septa are apparent.

### **3.2.3 Simple nodular type with extranodular growth**

When a tumor of simple nodular type shows varying degrees of extranodular growth beyond the capsule, vascular invasion, and/or small metastases near the nodule, the tumor should be categorized as simple nodular type with extranodular growth. This criterion is not concerned with tumor size. Small tumors showing simple nodular type with extranodular growth sometimes need discrimination from confluent multinodular type and large tumors showing simple nodular type with extranodular growth sometimes need discrimination from massive-type HCC.

This is a well-demarcated nodule, frequently surrounded by a fibrous capsule. Grossly, extranodular tumor growth, intrahepatic metastasis, and/or vascular invasion are not observed. The inside of the tumor often shows a mosaic appearance due to fibrous septa.

Fig. 4. Gross appearance of simple nodular type. A fibrous capsule and mosaic appearance

When a tumor of simple nodular type shows varying degrees of extranodular growth beyond the capsule, vascular invasion, and/or small metastases near the nodule, the tumor should be categorized as simple nodular type with extranodular growth. This criterion is not concerned with tumor size. Small tumors showing simple nodular type with extranodular growth sometimes need discrimination from confluent multinodular type and large tumors showing simple nodular type with extranodular growth sometimes need discrimination

Small HCC of the distinctly nodular type is also included in this category.

**3.2.2 Simple nodular type**

due to fibrous septa are apparent.

from massive-type HCC.

**3.2.3 Simple nodular type with extranodular growth**

Fig. 5. Gross appearance of simple nodular type with extranodular growth. a) Small tumor of simple nodular type with extranodular growth. b) Large tumor of simple nodular type with extranodular growth. This type of tumor sometimes needs discrimination from massive-type and/or confluent multinodular-type HCC.

### **3.2.4 Confluent multinodular type**

This is a tumor resulting from the joining together of a few to numerous small tumor nodules. No fibrous capsule is seen covering the entire tumor in this type and portal tracts are frequently entrapped between agglomerated small tumor nodules. The incidence of vascular invasion and/or intrahepatic metastasis is higher than in simple nodular types. Large confluent multinodular-type tumors sometimes need discrimination from massivetype HCC.

Usual and Unusual Gross Appearance of Hepatocellular Carcinomas 161

combined hepatocellular and cholangiocarcinoma, and fibrolamellar carcinoma. Scirrhous HCC is histologically distinguishable from fibrolamellar carcinoma, because the cancer cells do not differ from those of ordinary HCC. Scirrhous HCC should also not be confused with diffuse fibrosis (scirrhous change) in HCC, a well-known feature of cases treated by various anticancer therapies such as chemotherapy, irradiation and chemoembolization. Preoperatively, scirrhous HCC is often misdiagnosed as intrahepatic cholangiocarcinoma or metastatic carcinoma on diagnostic imaging, because of atypical imaging findings due to the

Fig. 7. a) Gross appearance of scirrhous HCC. Stellate-shaped fibrosis is present. b) Histology of the scirrhous HCC. Marked fibrosis (blue) is apparent along the sinusoid-like blood spaces. Cancer cells are no different from ordinary HCC (Azan staining, 100).

diffuse fibrosis [Kojiro, 2006; Bosman et al., 2010].

a

b

Fig. 6. Gross appearance of confluent multinodular-type HCC. a) Small tumor of confluent multinodular type. b) Large tumor of simple confluent multinodular type. No fibrous capsule covering the entire tumor is present.

### **4. Unusual gross types of HCC**

### **4.1 Scirrhous HCC**

About 5% of HCCs show a scirrhous growth pattern characterized by marked fibrosis along the sinusoid-like blood spaces with varying degrees of atrophy of the tumor trabeculae [Kurogi et al., 2006]. These tumors are subclassified as scirrhous HCC according to the WHO classification [Bosman et al., 2010]. Macroscopically, scirrhous HCCs are greyish-white, solid, lobulated, and unencapsulated, but well demarcated, stellate-shaped fibrosis is common. The gross appearance of scirrhous HCC mimics intrahepatic cholangiocarcinoma,

Fig. 6. Gross appearance of confluent multinodular-type HCC. a) Small tumor of confluent multinodular type. b) Large tumor of simple confluent multinodular type. No fibrous

About 5% of HCCs show a scirrhous growth pattern characterized by marked fibrosis along the sinusoid-like blood spaces with varying degrees of atrophy of the tumor trabeculae [Kurogi et al., 2006]. These tumors are subclassified as scirrhous HCC according to the WHO classification [Bosman et al., 2010]. Macroscopically, scirrhous HCCs are greyish-white, solid, lobulated, and unencapsulated, but well demarcated, stellate-shaped fibrosis is common. The gross appearance of scirrhous HCC mimics intrahepatic cholangiocarcinoma,

capsule covering the entire tumor is present.

**4. Unusual gross types of HCC**

a

b

**4.1 Scirrhous HCC**

combined hepatocellular and cholangiocarcinoma, and fibrolamellar carcinoma. Scirrhous HCC is histologically distinguishable from fibrolamellar carcinoma, because the cancer cells do not differ from those of ordinary HCC. Scirrhous HCC should also not be confused with diffuse fibrosis (scirrhous change) in HCC, a well-known feature of cases treated by various anticancer therapies such as chemotherapy, irradiation and chemoembolization. Preoperatively, scirrhous HCC is often misdiagnosed as intrahepatic cholangiocarcinoma or metastatic carcinoma on diagnostic imaging, because of atypical imaging findings due to the diffuse fibrosis [Kojiro, 2006; Bosman et al., 2010].

Fig. 7. a) Gross appearance of scirrhous HCC. Stellate-shaped fibrosis is present. b) Histology of the scirrhous HCC. Marked fibrosis (blue) is apparent along the sinusoid-like blood spaces. Cancer cells are no different from ordinary HCC (Azan staining, 100).

Usual and Unusual Gross Appearance of Hepatocellular Carcinomas 163

favorable than that of classical HCC. Grossly, the tumor is well demarcated but nonencapsulated and is light brown, tan or gray. The tumor is occasionally partially greenish due to bile production. Glistening white fibrous septa, sometimes forming a central scar, give the tumor a lobulated appearance. This gross appearance mimics focal nodular hyperplasia. Histologically, this particular type of HCC consists of tumor cells growing in sheets or small trabeculae, which are separated by fibrous collagen with a characteristic lamellar pattern of variable thickness and frequent hyalinization. Tumor cells display abundant polygonal and deeply eosinophilic cytoplasm and a distinct nucleolus. Intracytoplasmic inclusions, such as "pale bodies" that show immunohistochemically positive results for antifibrinogen and PAS-positive globules, are

Nodular-type HCC accompanied by prominent bile production shows a yellowish coloration on a fresh specimen and deep greenish coloration after formalin fixation. Such

Peliosis hepatis-like blood-filled cavities are frequently observed in HCC. HCC with this finding is generally called "HCC with peliotic change" or "pelioid-type HCC" [Ishak et al., 1999]. Another characteristic of HCC with peliotic change is that all tumors are completely or incompletely encapsulated. Several cases of extensive pelioid-type HCC have been reported [Kim, 2007, Hoshimoto, 2009, Kai, 2011]. Grossly, extensive pelioid-type HCC mostly consists of well-defined black-nodular components. Histologically, peliotic change is distinguished from hemorrhage by the presence or absence of degenerative changes and/or necrosis. In general, degenerative changes and/or necrosis are observed around

frequently observed [Ishak et al., 1999].

tumors are generally called "green HCC".

Fig. 9. Gross appearance of green HCC.

**4.4 Pelioid type of HCC**

**4.3 Green HCC**

### **4.2 Fibrolamellar HCC**

The clinical and pathological features of fibrolamellar HCC (FLC) were first described by Edmondson [Edmondson, 1958]. The term "fibrolamellar carcinoma" was proposed by Craig et al. [Craig et al., 1980], and has been adopted worldwide. FLC shows marked geographic variations in incidence, and is extremely rare in Asian countries, including Japan [Magata et al., 2001]. Soreide et al. [Soreide et al., 1986] reviewed 80 cases of FLC (including their own 9 cases) reported between 1980 and 1986. This entity most frequently occurs in noncirrhotic livers of adolescents or young adults with a mean age of 23 years; only 6% are over 50 years old. Two thirds of the cases of FLC involve the left lobe and prognosis is more

Fig. 8. a) Gross appearance of fibrolamellar hepatocellular carcinoma. White fibrous septa forming a central scar, lobulated appearance, and greenish coloration due to bile production can be observed. b) Tumor cells display abundant polygonal and deeply eosinophilic cytoplasm and characteristic lamellar fibrosis is observed (HE, 200).

favorable than that of classical HCC. Grossly, the tumor is well demarcated but nonencapsulated and is light brown, tan or gray. The tumor is occasionally partially greenish due to bile production. Glistening white fibrous septa, sometimes forming a central scar, give the tumor a lobulated appearance. This gross appearance mimics focal nodular hyperplasia. Histologically, this particular type of HCC consists of tumor cells growing in sheets or small trabeculae, which are separated by fibrous collagen with a characteristic lamellar pattern of variable thickness and frequent hyalinization. Tumor cells display abundant polygonal and deeply eosinophilic cytoplasm and a distinct nucleolus. Intracytoplasmic inclusions, such as "pale bodies" that show immunohistochemically positive results for antifibrinogen and PAS-positive globules, are frequently observed [Ishak et al., 1999].

### **4.3 Green HCC**

162 Hepatocellular Carcinoma – Clinical Research

The clinical and pathological features of fibrolamellar HCC (FLC) were first described by Edmondson [Edmondson, 1958]. The term "fibrolamellar carcinoma" was proposed by Craig et al. [Craig et al., 1980], and has been adopted worldwide. FLC shows marked geographic variations in incidence, and is extremely rare in Asian countries, including Japan [Magata et al., 2001]. Soreide et al. [Soreide et al., 1986] reviewed 80 cases of FLC (including their own 9 cases) reported between 1980 and 1986. This entity most frequently occurs in noncirrhotic livers of adolescents or young adults with a mean age of 23 years; only 6% are over 50 years old. Two thirds of the cases of FLC involve the left lobe and prognosis is more

Fig. 8. a) Gross appearance of fibrolamellar hepatocellular carcinoma. White fibrous septa forming a central scar, lobulated appearance, and greenish coloration due to bile production can be observed. b) Tumor cells display abundant polygonal and deeply eosinophilic

cytoplasm and characteristic lamellar fibrosis is observed (HE, 200).

**4.2 Fibrolamellar HCC**

a

b

Nodular-type HCC accompanied by prominent bile production shows a yellowish coloration on a fresh specimen and deep greenish coloration after formalin fixation. Such tumors are generally called "green HCC".

Fig. 9. Gross appearance of green HCC.

### **4.4 Pelioid type of HCC**

Peliosis hepatis-like blood-filled cavities are frequently observed in HCC. HCC with this finding is generally called "HCC with peliotic change" or "pelioid-type HCC" [Ishak et al., 1999]. Another characteristic of HCC with peliotic change is that all tumors are completely or incompletely encapsulated. Several cases of extensive pelioid-type HCC have been reported [Kim, 2007, Hoshimoto, 2009, Kai, 2011]. Grossly, extensive pelioid-type HCC mostly consists of well-defined black-nodular components. Histologically, peliotic change is distinguished from hemorrhage by the presence or absence of degenerative changes and/or necrosis. In general, degenerative changes and/or necrosis are observed around

Usual and Unusual Gross Appearance of Hepatocellular Carcinomas 165

[Kojiro et al., 1982; Huang et al., 1998]. Although intraductal tumor growth of HCC was once considered rare, advances in diagnostic imaging have revealed many cases of HCC presenting with intra-bile-duct growth [Kirk et al., 1994; Soyer et al., 1995; Wang et al., 2002]. Patients with the intraductal growth type of HCC rarely show no obvious intrahepatic tumor. Postoperative pathological examinations confirm the bile duct nodules causing obstructive jaundice as HCC. This type of HCC is difficult to diagnose, and only a few cases have been reported [Long et al., 2010]. In some cases, massive hemorrhage in the bile duct (hemobilia) forms due to tumor growth and can cause rapid death on rare occasions [Kojiro, 2006]. Huang et al. [Huang et al., 1998] stated that the prognosis might be improved if surgical resection of the tumor cast is performed at an earlier stage. In fact, many cases have been reported with good prognosis

after tumor cast resection [Chen et al., 1994; Lau et al., 1997; Hu et al., 1999].

a

b

Fig. 11. a) Gross appearance for the intraductal growth type of HCC. This case shows no obvious intrahepatic mass lesion. b) Tumor cells with blood clot in dilated bile ducts (HE, 20).

hemorrhage in HCC tumors. On the other hand, the tumorous tissue around peliotic change is not at all degenerative or necrotic, and blood is localized within the spaces with clear margins. However, when peliotic change becomes extensive, rupture may occur and hemorrhage may develop [Kojiro, 2006].

Fig. 10. a) The gross appearance of pelioid-type HCC. The tumor comprises well-defined black nodular components and white nodular components. b) Histologically, extensive pelioid change is apparent in the tumor tissue (HE, 25).

### **4.5 Intraductal growth type of HCC**

Rarely, HCC shows growth into the hepatic bile duct and/or common bile duct, causing progressive obstructive jaundice. In 1972, Lin [Lin, 1972] designated such cases as "icteric HCC". The reported incidence of icteric HCC varies from 0.2% to 5.4% in patients with HCC

hemorrhage in HCC tumors. On the other hand, the tumorous tissue around peliotic change is not at all degenerative or necrotic, and blood is localized within the spaces with clear margins. However, when peliotic change becomes extensive, rupture may occur and

Fig. 10. a) The gross appearance of pelioid-type HCC. The tumor comprises well-defined black nodular components and white nodular components. b) Histologically, extensive

Rarely, HCC shows growth into the hepatic bile duct and/or common bile duct, causing progressive obstructive jaundice. In 1972, Lin [Lin, 1972] designated such cases as "icteric HCC". The reported incidence of icteric HCC varies from 0.2% to 5.4% in patients with HCC

pelioid change is apparent in the tumor tissue (HE, 25).

**4.5 Intraductal growth type of HCC**

hemorrhage may develop [Kojiro, 2006].

a

b

[Kojiro et al., 1982; Huang et al., 1998]. Although intraductal tumor growth of HCC was once considered rare, advances in diagnostic imaging have revealed many cases of HCC presenting with intra-bile-duct growth [Kirk et al., 1994; Soyer et al., 1995; Wang et al., 2002]. Patients with the intraductal growth type of HCC rarely show no obvious intrahepatic tumor. Postoperative pathological examinations confirm the bile duct nodules causing obstructive jaundice as HCC. This type of HCC is difficult to diagnose, and only a few cases have been reported [Long et al., 2010]. In some cases, massive hemorrhage in the bile duct (hemobilia) forms due to tumor growth and can cause rapid death on rare occasions [Kojiro, 2006]. Huang et al. [Huang et al., 1998] stated that the prognosis might be improved if surgical resection of the tumor cast is performed at an earlier stage. In fact, many cases have been reported with good prognosis after tumor cast resection [Chen et al., 1994; Lau et al., 1997; Hu et al., 1999].

Fig. 11. a) Gross appearance for the intraductal growth type of HCC. This case shows no obvious intrahepatic mass lesion. b) Tumor cells with blood clot in dilated bile ducts (HE, 20).

Usual and Unusual Gross Appearance of Hepatocellular Carcinomas 167

have been speculated to play a role, such as alcohol withdrawal, androgen withdrawal, and intake of herbal medicines [Gottfried et al., 1982; Lam et al., 1982; McCaughan et al., 1985]. Secondary stimulation of cytokine production by tumor necrosis or bacterial infection has also been suggested to contribute to spontaneous regression [Watanabe et al., 1994; Markovic et al., 1996]. Another important factor might be insufficient blood supply to the tumor, possibly due to rapid natural tumor growth, spontaneous arterial thrombosis, thickened fibrous capsule or gastrointestinal bleeding [Suzuki et al., 1989; Gaffey et al., 1990; Tocci et al., 1990; Imaoka et al., 1994; Nakajima et al., 2004]. A few cases of spontaneous regression of HCC after angiography have been reported [Takayasu et al., 1986; Kai et al., 2010]. The characteristic histology in these cases is coagulative necrosis of tumor cells and inflammatory granulation tissue surrounding the coagulative necrosis. Grossly, yellowwhite nodular lesions reflecting coagulative necrosis and brownish lesions reflecting

Various tumors and tumor-like lesions can arise in the liver. Some of these tumors show a similar gross appearance to HCC, but some are easily discriminated from HCC on gross

Intrahepatic cholangiocellular carcinoma (ICC) is also grossly classified into three types: mass-forming type; periductal infiltrating type; and intraductal growth type. Within these three types, the mass-forming type is grossly nominated as a differential diagnosis of classical HCC. This differential diagnosis is usually easily achieved based on the characteristic gross appearance of mass-forming ICC, such as a lobulated, firm and solid

inflammatory granulation tissue coexist.

appearance.

**5. Differential diagnosis by gross appearance**

**5.1 Intrahepatic cholangiocellular carcinoma**

Fig. 13. Cut surface of the mass-forming type of ICC.

### **4.6 Spontaneous regression of HCC**

Spontaneous massive necrosis or regression of HCC is a very rare event. The precise mechanisms underlying spontaneous regression of HCC remain unclear, but various factors

Fig. 12. a) Cut surface of the resected liver in a case of spontaneous regression. Yellow-white nodular lesions and brownish lesions coexist. The HCC measuring 11 cm in diameter showed a significant reduction to 3.7 cm after angiography. b) Coagulative necrosis and inflammatory granulation tissue are apparent in the nodule and small foci of viable HCC cells are observed in another specimen (HE, 40).

Spontaneous massive necrosis or regression of HCC is a very rare event. The precise mechanisms underlying spontaneous regression of HCC remain unclear, but various factors

Fig. 12. a) Cut surface of the resected liver in a case of spontaneous regression. Yellow-white nodular lesions and brownish lesions coexist. The HCC measuring 11 cm in diameter showed a significant reduction to 3.7 cm after angiography. b) Coagulative necrosis and inflammatory granulation tissue are apparent in the nodule and small foci of viable HCC

cells are observed in another specimen (HE, 40).

**4.6 Spontaneous regression of HCC**

a

b

have been speculated to play a role, such as alcohol withdrawal, androgen withdrawal, and intake of herbal medicines [Gottfried et al., 1982; Lam et al., 1982; McCaughan et al., 1985]. Secondary stimulation of cytokine production by tumor necrosis or bacterial infection has also been suggested to contribute to spontaneous regression [Watanabe et al., 1994; Markovic et al., 1996]. Another important factor might be insufficient blood supply to the tumor, possibly due to rapid natural tumor growth, spontaneous arterial thrombosis, thickened fibrous capsule or gastrointestinal bleeding [Suzuki et al., 1989; Gaffey et al., 1990; Tocci et al., 1990; Imaoka et al., 1994; Nakajima et al., 2004]. A few cases of spontaneous regression of HCC after angiography have been reported [Takayasu et al., 1986; Kai et al., 2010]. The characteristic histology in these cases is coagulative necrosis of tumor cells and inflammatory granulation tissue surrounding the coagulative necrosis. Grossly, yellowwhite nodular lesions reflecting coagulative necrosis and brownish lesions reflecting inflammatory granulation tissue coexist.

### **5. Differential diagnosis by gross appearance**

Various tumors and tumor-like lesions can arise in the liver. Some of these tumors show a similar gross appearance to HCC, but some are easily discriminated from HCC on gross appearance.

### **5.1 Intrahepatic cholangiocellular carcinoma**

Intrahepatic cholangiocellular carcinoma (ICC) is also grossly classified into three types: mass-forming type; periductal infiltrating type; and intraductal growth type. Within these three types, the mass-forming type is grossly nominated as a differential diagnosis of classical HCC. This differential diagnosis is usually easily achieved based on the characteristic gross appearance of mass-forming ICC, such as a lobulated, firm and solid

Fig. 13. Cut surface of the mass-forming type of ICC.

Usual and Unusual Gross Appearance of Hepatocellular Carcinomas 169

is now considered to be derived from Hering's canal or stem cells, which have intermediate features between hepatocytes and bile duct epithelium [Shiota et al., 2001; Kojiro, 2006]. Grossly, cholangiolocarcinoma is whitish in color and solid, not encapsulated, and resembles cholangiocellular carcinoma. Histologically, tumor cells display eosinophilic cytoplasm with ovoid nuclei, and mild atypia, and adopt a tubular cordlike anastomosing pattern (so-called antler pattern). These cell cords are continuous with liver cell cords at the

HCA is a benign liver neoplasm comprising hepatocyte-like tumor cells. The incidence of HCA is about 3-4 per 100 000 population in Western countries [Rooks et al., 1979], but is

Fig. 15. a) Gross appearance of hepatocellular adenoma. Focal peliotic change is present. b) The nodule consists of tumor cells with abundant hydropic cytoplasm and little atypia

tumor boundaries, showing a replacing pattern.

**5.3 Hepatocellular adenoma (HCA)**

a

b

arranged in a trabecular pattern.

gray to gray-white mass without a fibrous capsule and often with central necrosis or scarring. Scirrhous-type HCC sometimes mimics mass-forming ICC. Intraductal growthtype HCC often mimics intraductal growth-type ICC and discrimination based on gross appearance is difficult.

### **5.2 Cholangiolocellular carcinoma**

Cholangiolocarcinoma is a rare neoplasm with a reported incidence of 0.8-1.0% among primary liver cancers [Steiner & Higginson, 1959; Shiota et al., 2001]. Cholangiolocarcinoma

Fig. 14. a) Gross appearance of cholangiolocellular carcinoma. b) A tubular cordlike anastomosing pattern (as the so-called "antler pattern") of tumor cells (HE, 100).

is now considered to be derived from Hering's canal or stem cells, which have intermediate features between hepatocytes and bile duct epithelium [Shiota et al., 2001; Kojiro, 2006]. Grossly, cholangiolocarcinoma is whitish in color and solid, not encapsulated, and resembles cholangiocellular carcinoma. Histologically, tumor cells display eosinophilic cytoplasm with ovoid nuclei, and mild atypia, and adopt a tubular cordlike anastomosing pattern (so-called antler pattern). These cell cords are continuous with liver cell cords at the tumor boundaries, showing a replacing pattern.

### **5.3 Hepatocellular adenoma (HCA)**

168 Hepatocellular Carcinoma – Clinical Research

gray to gray-white mass without a fibrous capsule and often with central necrosis or scarring. Scirrhous-type HCC sometimes mimics mass-forming ICC. Intraductal growthtype HCC often mimics intraductal growth-type ICC and discrimination based on gross

Cholangiolocarcinoma is a rare neoplasm with a reported incidence of 0.8-1.0% among primary liver cancers [Steiner & Higginson, 1959; Shiota et al., 2001]. Cholangiolocarcinoma

Fig. 14. a) Gross appearance of cholangiolocellular carcinoma. b) A tubular cordlike anastomosing pattern (as the so-called "antler pattern") of tumor cells (HE, 100).

appearance is difficult.

**5.2 Cholangiolocellular carcinoma**

a

b

HCA is a benign liver neoplasm comprising hepatocyte-like tumor cells. The incidence of HCA is about 3-4 per 100 000 population in Western countries [Rooks et al., 1979], but is

Fig. 15. a) Gross appearance of hepatocellular adenoma. Focal peliotic change is present. b) The nodule consists of tumor cells with abundant hydropic cytoplasm and little atypia arranged in a trabecular pattern.

Usual and Unusual Gross Appearance of Hepatocellular Carcinomas 171

a

b

Fig. 16. a) Gross appearance of FNH. A faint central fibrous scar is apparent in this case. b) Artery-like anomalous blood vessels are observed in benign hepatocytes (HE, 40).

HCC usually contains various degrees of histiocyte infiltration. In the authors' institution, a case of unique hepatocellular nodule with infiltration of abundant histiocytes not previously described has been encountered [Kai, 2010]. This case involved a 73-year-old Japanese man with diabetes mellitus and mild obesity without viral hepatitis.  The tumor showed characteristics of hepatocellular adenoma, but contained a focal HCC-like lesion. Similar hepatic nodules have not been

reported and most of the clinicopathological features and pathology remain unknown.

**5.5 Hepatocellular neoplasm with massive histiocyte infiltration**

lower in Japan and Asian countries. HCA predominantly occurs in females in the third and fourth decades of life, and a close etiological relationship between HCA and oral contraceptives has been described [Ishak & Rabin, 1975]. Patients taking androgens for Fanconi anemia or acquired anaplastic anemia are also at risk [Velazquez & Alter, 2004]. Nonhormonal risk factors include glycogenosis type 1 (von Gierke disease) or 3 (Forbes disease), galactosemia, tyrosinemia, familial polyposis coli, and hepatic iron overload with β-thalassemia [Shuangshoti & Thaicharoen, 1994].  Grossly, HCA is found in the normal liver as a well-demarcated, single or multiple solid tumor. Many HCAs are not encapsulated, but some are partly or entirely encapsulated. The tumor is yellowish and solid, sometimes associated with necrosis and hemorrhage and/or peliosis hepatis-like (pelioid) change and the tumor may be divided by fibrous septa. HCA developing close to the liver surface can protrude outside the liver, and is thus susceptible to rupture. Histologically, HCA consists of tumor cells with abundant hydropic cytoplasm and little atypia arranged in a trabecular pattern of one- or two-cell-thick plates, or round eosinophilic tumor cells in a thin trabecular pattern. However, because the sinusoid-like blood spaces of the tumor stroma are compressed by the swollen, hydropic tumor cells, the trabecular pattern is often obscured and the tumor takes on a solid appearance. The tumor contains many arterial and venous tumor vessels, and pelioid change is frequently observed.

#### **5.4 Focal nodular hyperplasia (FNH)**

FNH is not a true neoplasm, but rather a regenerative hyperplastic response of hepatocytes, secondary to localized vascular abnormalities with a marked female preponderance [Wanless et al., 1985; Fukukura et al., 1998]. This lesion was reported as liver cell adenoma for many years before the disease concept of FNH was defined by Edmondson in 1958 [Edmondson, 1958]. In two-thirds of cases, FNH is solitary [Wanless et al., 1989]. Most lesions are asymptomatic and discovered incidentally during laparotomy, autopsy or imaging studies for unrelated symptoms. Although it can now be precisely diagnosed by various imaging modalities, FNH is still considered one of the common lesions in the differential diagnosis of HCC. The size of FNH varies from a few millimeters to over 10 cm in diameter. Grossly, FNH is usually well demarcated, but not encapsulated. Most tumors are located close to the liver capsule, and 4.6-20% are reportedly pedunculated [Ishak, 1975; Klatskin, 1977; Luciani et al., 2002]. One of the characteristic gross features of FNH is the presence of a central fibrous scar with radiating fibrous bands dividing the tumor into lobules. The reported incidence of a central scar in FNH is 49-62.8% [Nguyen et al., 1999; Kojiro, 2006]. Histologically, classical FNH lesions are composed of benign hepatocytes arranged in a trabecular pattern of one- or two-cell-thick plates. The central scar consists of dense fibrous tissue with many artery-like or vein-like anomalous blood vessels and ductal proliferations. Hyperplastic features of hepatocytes in FNH comprise increased cellularity with an increased nuclear cytoplasmic ratio, an irregular thin trabecular pattern, and a pseudoglandular pattern. Areas showing prominent hyperplastic features mimic welldifferentiated HCC.

lower in Japan and Asian countries. HCA predominantly occurs in females in the third and fourth decades of life, and a close etiological relationship between HCA and oral contraceptives has been described [Ishak & Rabin, 1975]. Patients taking androgens for Fanconi anemia or acquired anaplastic anemia are also at risk [Velazquez & Alter, 2004]. Nonhormonal risk factors include glycogenosis type 1 (von Gierke disease) or 3 (Forbes disease), galactosemia, tyrosinemia, familial polyposis coli, and hepatic iron overload with β-thalassemia [Shuangshoti & Thaicharoen, 1994].  Grossly, HCA is found in the normal liver as a well-demarcated, single or multiple solid tumor. Many HCAs are not encapsulated, but some are partly or entirely encapsulated. The tumor is yellowish and solid, sometimes associated with necrosis and hemorrhage and/or peliosis hepatis-like (pelioid) change and the tumor may be divided by fibrous septa. HCA developing close to the liver surface can protrude outside the liver, and is thus susceptible to rupture. Histologically, HCA consists of tumor cells with abundant hydropic cytoplasm and little atypia arranged in a trabecular pattern of one- or two-cell-thick plates, or round eosinophilic tumor cells in a thin trabecular pattern. However, because the sinusoid-like blood spaces of the tumor stroma are compressed by the swollen, hydropic tumor cells, the trabecular pattern is often obscured and the tumor takes on a solid appearance. The tumor contains many arterial and venous tumor vessels, and pelioid change is frequently

FNH is not a true neoplasm, but rather a regenerative hyperplastic response of hepatocytes, secondary to localized vascular abnormalities with a marked female preponderance [Wanless et al., 1985; Fukukura et al., 1998]. This lesion was reported as liver cell adenoma for many years before the disease concept of FNH was defined by Edmondson in 1958 [Edmondson, 1958]. In two-thirds of cases, FNH is solitary [Wanless et al., 1989]. Most lesions are asymptomatic and discovered incidentally during laparotomy, autopsy or imaging studies for unrelated symptoms. Although it can now be precisely diagnosed by various imaging modalities, FNH is still considered one of the common lesions in the differential diagnosis of HCC. The size of FNH varies from a few millimeters to over 10 cm in diameter. Grossly, FNH is usually well demarcated, but not encapsulated. Most tumors are located close to the liver capsule, and 4.6-20% are reportedly pedunculated [Ishak, 1975; Klatskin, 1977; Luciani et al., 2002]. One of the characteristic gross features of FNH is the presence of a central fibrous scar with radiating fibrous bands dividing the tumor into lobules. The reported incidence of a central scar in FNH is 49-62.8% [Nguyen et al., 1999; Kojiro, 2006]. Histologically, classical FNH lesions are composed of benign hepatocytes arranged in a trabecular pattern of one- or two-cell-thick plates. The central scar consists of dense fibrous tissue with many artery-like or vein-like anomalous blood vessels and ductal proliferations. Hyperplastic features of hepatocytes in FNH comprise increased cellularity with an increased nuclear cytoplasmic ratio, an irregular thin trabecular pattern, and a pseudoglandular pattern. Areas showing prominent hyperplastic features mimic well-

observed.

differentiated HCC.

**5.4 Focal nodular hyperplasia (FNH)**

Fig. 16. a) Gross appearance of FNH. A faint central fibrous scar is apparent in this case. b) Artery-like anomalous blood vessels are observed in benign hepatocytes (HE, 40).

### **5.5 Hepatocellular neoplasm with massive histiocyte infiltration**

HCC usually contains various degrees of histiocyte infiltration. In the authors' institution, a case of unique hepatocellular nodule with infiltration of abundant histiocytes not previously described has been encountered [Kai, 2010]. This case involved a 73-year-old Japanese man with diabetes mellitus and mild obesity without viral hepatitis.  The tumor showed characteristics of hepatocellular adenoma, but contained a focal HCC-like lesion. Similar hepatic nodules have not been reported and most of the clinicopathological features and pathology remain unknown.

Usual and Unusual Gross Appearance of Hepatocellular Carcinomas 173

a

b

Fig. 18. a) Gross appearance of primary pure SCC of the liver in a patient with no history of prior liver insult. b) The nodule consists entirely of SCC tissue with or without keratinization.

Fig. 17. a) Gross appearance of hepatocellular neoplasm with massive histiocyte infiltration. The nodule was solitary, and the cut surface was whitish with a partly necrotic yellowish area. A white myxoid fibrotic area was found at the center of the nodule. b) The nodule showed a two-cell pattern: hepatocyte-like cells with rich eosinophilic cytoplasm and little nuclear atypia; and light-pink cells considered to be foam cell-type histiocytes (HE, 100).

### **5.6 Primary squamous cell carcinoma (SCC) of the liver**

Primary SCC of the liver is very rare. Adenosquamous carcinoma of the liver is also rare and reported only sporadically. Various theories have been advanced concerning the pathogenesis of SCC of the liver. Chronic inflammation of the bile duct and various

a

b

Fig. 17. a) Gross appearance of hepatocellular neoplasm with massive histiocyte infiltration. The nodule was solitary, and the cut surface was whitish with a partly necrotic yellowish area. A white myxoid fibrotic area was found at the center of the nodule. b) The nodule showed a two-cell pattern: hepatocyte-like cells with rich eosinophilic cytoplasm and little nuclear atypia; and light-pink cells considered to be foam cell-type histiocytes (HE, 100).

Primary SCC of the liver is very rare. Adenosquamous carcinoma of the liver is also rare and reported only sporadically. Various theories have been advanced concerning the pathogenesis of SCC of the liver. Chronic inflammation of the bile duct and various

**5.6 Primary squamous cell carcinoma (SCC) of the liver**

Fig. 18. a) Gross appearance of primary pure SCC of the liver in a patient with no history of prior liver insult. b) The nodule consists entirely of SCC tissue with or without keratinization.

Usual and Unusual Gross Appearance of Hepatocellular Carcinomas 175

congenital cysts of the biliary tract have been proposed as etiological factors and continuous irritation due to chronic infection, followed by metaplastic changes in the biliary epithelium, has been thought to lead to neoplasia [Clements et al., 1990; Nieweg et al., 1992; Banbury et al., 1994]. On the other hand, some investigators have suggested that adenosquamous carcinoma could arise from squamous metaplasia of adenocarcinoma cells [Barr and Hancock, 1975; Tomioka et al., 1987]. Primary SCC of the liver without history of prior liver insult suggesting development from squamous metaplasia of adenocarcinoma cells has recently been reported [Naik et al., 2009]. Gross appearance depends on the presence or absence of congenital cysts. The mass lesion in that case appeared as a solid, whitish firm mass without fibrous capsule and with a necrotic area of keratin deposits observed inside

Inflammatory pseudotumor is defined as a benign, non-neoplastic mass consisting of fibrous tissue and proliferated myofibroblasts and fibroblasts, with marked infiltration of chronic inflammatory cells, predominantly comprising polyclonal plasma cells, histiocytes, and macrophages [Coffin et al., 1995]. Lesions of known cause, such as healing abscess, tuberculosis, and parasitic infestations, and post-traumatic granulation tissue, are excluded from this category. Patients with hepatic lesions generally complain of recurrent fever, malaise, abdominal pain, vomiting, and/or diarrhea, but those clinical symptoms are not specific. The morphology of inflammatory pseudotumor apparently differs according to the stage of inflammation when the lesion is detected. Grossly, the lesion appears as a yellowish or whitish nodule with relatively clear margins and a widely varying diameter, usually within the range of 1-3 cm. Histological appearance also varies considerably, sometimes consisting of nonspecific inflammatory granulation tissue with prominent plasma cell infiltration, sometimes associated with necrotic foci, and sometimes appearing as a scar-like fibrous nodule with mild inflammatory cell infiltration [Kojiro, 2006]. Phlebitis involving peripheral portal or hepatic veins is often seen. Rarely, major hepatic and/or portal veins or the vena cava are involved by phlebitis mimicking malignant tumor [Broughan et al., 1993; Kai et al., 2007]. Lately, IgG4-related disease has been identified as an important subgroup of inflammatory pseudotumor [Zen et al., 2004, 2007]. Inflammatory myofibroblastic tumor has recently been considered as a possible neoplastic counterpart of inflammatory pseudotumor [Coffin et al., 1998], based on the finding that approximately half of inflammatory myofibroblastic tumors show an ALK gene translocation [Lawrence et al., 2000; Bridge et

This chapter documented the gross appearance and pathology of various types of tumor, along with a review of the literature. We believe that the observation of gross features with contrasting pathological and clinical findings is helpful in clarifying the nature of tumors. In rare cases, a picture of the gross specimen is necessary for case reports. Careful storage of good-quality images of gross specimens is thus important in daily clinical

**5.7 Inflammatory pseudotumor / inflammatory myofibroblastic tumor**

the nodule.

al., 2001; Chan et al., 2001].

**6. Conclusion**

work.

Fig. 19. a) A cross-section of inflammatory pseudotumor. Small, white nodules without fibrous capsules are observed. b) The tumor comprises chronic inflammatory cells, predominantly representing plasma cells (HE, 200).

a

b

Fig. 19. a) A cross-section of inflammatory pseudotumor. Small, white nodules without fibrous capsules are observed. b) The tumor comprises chronic inflammatory cells,

predominantly representing plasma cells (HE, 200).

congenital cysts of the biliary tract have been proposed as etiological factors and continuous irritation due to chronic infection, followed by metaplastic changes in the biliary epithelium, has been thought to lead to neoplasia [Clements et al., 1990; Nieweg et al., 1992; Banbury et al., 1994]. On the other hand, some investigators have suggested that adenosquamous carcinoma could arise from squamous metaplasia of adenocarcinoma cells [Barr and Hancock, 1975; Tomioka et al., 1987]. Primary SCC of the liver without history of prior liver insult suggesting development from squamous metaplasia of adenocarcinoma cells has recently been reported [Naik et al., 2009]. Gross appearance depends on the presence or absence of congenital cysts. The mass lesion in that case appeared as a solid, whitish firm mass without fibrous capsule and with a necrotic area of keratin deposits observed inside the nodule.

### **5.7 Inflammatory pseudotumor / inflammatory myofibroblastic tumor**

Inflammatory pseudotumor is defined as a benign, non-neoplastic mass consisting of fibrous tissue and proliferated myofibroblasts and fibroblasts, with marked infiltration of chronic inflammatory cells, predominantly comprising polyclonal plasma cells, histiocytes, and macrophages [Coffin et al., 1995]. Lesions of known cause, such as healing abscess, tuberculosis, and parasitic infestations, and post-traumatic granulation tissue, are excluded from this category. Patients with hepatic lesions generally complain of recurrent fever, malaise, abdominal pain, vomiting, and/or diarrhea, but those clinical symptoms are not specific. The morphology of inflammatory pseudotumor apparently differs according to the stage of inflammation when the lesion is detected. Grossly, the lesion appears as a yellowish or whitish nodule with relatively clear margins and a widely varying diameter, usually within the range of 1-3 cm. Histological appearance also varies considerably, sometimes consisting of nonspecific inflammatory granulation tissue with prominent plasma cell infiltration, sometimes associated with necrotic foci, and sometimes appearing as a scar-like fibrous nodule with mild inflammatory cell infiltration [Kojiro, 2006]. Phlebitis involving peripheral portal or hepatic veins is often seen. Rarely, major hepatic and/or portal veins or the vena cava are involved by phlebitis mimicking malignant tumor [Broughan et al., 1993; Kai et al., 2007]. Lately, IgG4-related disease has been identified as an important subgroup of inflammatory pseudotumor [Zen et al., 2004, 2007]. Inflammatory myofibroblastic tumor has recently been considered as a possible neoplastic counterpart of inflammatory pseudotumor [Coffin et al., 1998], based on the finding that approximately half of inflammatory myofibroblastic tumors show an ALK gene translocation [Lawrence et al., 2000; Bridge et al., 2001; Chan et al., 2001].

### **6. Conclusion**

This chapter documented the gross appearance and pathology of various types of tumor, along with a review of the literature. We believe that the observation of gross features with contrasting pathological and clinical findings is helpful in clarifying the nature of tumors. In rare cases, a picture of the gross specimen is necessary for case reports. Careful storage of good-quality images of gross specimens is thus important in daily clinical work.

Usual and Unusual Gross Appearance of Hepatocellular Carcinomas 177

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**Part 2**

**Surgical Treatment**

## **Part 2**

**Surgical Treatment**

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**9**

**Pure Laparoscopic Hepatectomy**

*Department of Surgery, Fujita Health University School of Medicine Banbuntane Houtokukai Hospital, Otobashi Nakagawa-ku, Nagoya Aichi*

Pure laparoscopic hepatectomy is thought to be a less invasive procedure than conventional open hepatectomy for the resection of hepatic lesions (1). Recent development of devices facilitates expansion of the indication of the procedure (2, 3). At the present moment, common advantages of laparoscopic surgery, such as early recovery and discharge with smaller postoperative pain and earlier intake, have been reported (4). However, specific advantages and/or disadvantages of pure laparoscopic hepatectomy for the settlement of

On the other hand, at the treatment of hepatocellular carcinoma (HCC) with liver cirrhosis, considerations for the control of invasive surgical stress, especially to impaired liver, should be placed besides oncological therapeutic effects. Patients with severe liver cirrhosis have various (overt and preliminary) symptoms, such as 1) deteriorations of protein synthesis and metabolism, 2) congestion of GI tract, ascites, pancytopenia due to portal hypertension and hypersplenism, 3) susceptibility to infectious diseases and hepatopulmonary syndrome (hypoxaemia) due to increased shunt vessels (5). Cirrhotic patients have high morbidity and mortality at undergoing anesthesia and surgery (6) and the risk of abdominal operations increases according to the preoperative Child's class of the patients (7). Hepatic resection for severe cirrhotic patients, even minimum, often develops refractory ascites which leads to fatal complications (8, 9). In Japan, the criteria based on 3 parameters (the presence or absence of ascites, total serum billirubin level, and indocyanine green retension rate at 15 minutes (ICG R15)) have been widely used for patient selection of hepatectomy (10). Nowadays, surgical resection, local ablation therapy or transarterial chemoembolization is adapted to each HCC patient with liver cirrhosis depending on the tumor condition and the

However, not small number of HCC patients with severe liver dysfunction exists who is not able to undergo those treatment modalities due to liver function, tumor size and/or localization, especially after repeat treatments for the disease. Furthermore, not small portion of patients need the repeat treatments for multicentric metachronous lesions occurred in chronic impaired liver. For thosee patients, "less invasive" pure laparoscopic

**1. Introduction**

liver function.

hepatectomy may provide a good option.

the indication have yet to be sufficiently discussed.

 **for HCC Patients**

Zenichi Morise

*Japan*

### **Pure Laparoscopic Hepatectomy for HCC Patients**

Zenichi Morise

*Department of Surgery, Fujita Health University School of Medicine Banbuntane Houtokukai Hospital, Otobashi Nakagawa-ku, Nagoya Aichi Japan*

### **1. Introduction**

Pure laparoscopic hepatectomy is thought to be a less invasive procedure than conventional open hepatectomy for the resection of hepatic lesions (1). Recent development of devices facilitates expansion of the indication of the procedure (2, 3). At the present moment, common advantages of laparoscopic surgery, such as early recovery and discharge with smaller postoperative pain and earlier intake, have been reported (4). However, specific advantages and/or disadvantages of pure laparoscopic hepatectomy for the settlement of the indication have yet to be sufficiently discussed.

On the other hand, at the treatment of hepatocellular carcinoma (HCC) with liver cirrhosis, considerations for the control of invasive surgical stress, especially to impaired liver, should be placed besides oncological therapeutic effects. Patients with severe liver cirrhosis have various (overt and preliminary) symptoms, such as 1) deteriorations of protein synthesis and metabolism, 2) congestion of GI tract, ascites, pancytopenia due to portal hypertension and hypersplenism, 3) susceptibility to infectious diseases and hepatopulmonary syndrome (hypoxaemia) due to increased shunt vessels (5). Cirrhotic patients have high morbidity and mortality at undergoing anesthesia and surgery (6) and the risk of abdominal operations increases according to the preoperative Child's class of the patients (7). Hepatic resection for severe cirrhotic patients, even minimum, often develops refractory ascites which leads to fatal complications (8, 9). In Japan, the criteria based on 3 parameters (the presence or absence of ascites, total serum billirubin level, and indocyanine green retension rate at 15 minutes (ICG R15)) have been widely used for patient selection of hepatectomy (10). Nowadays, surgical resection, local ablation therapy or transarterial chemoembolization is adapted to each HCC patient with liver cirrhosis depending on the tumor condition and the liver function.

However, not small number of HCC patients with severe liver dysfunction exists who is not able to undergo those treatment modalities due to liver function, tumor size and/or localization, especially after repeat treatments for the disease. Furthermore, not small portion of patients need the repeat treatments for multicentric metachronous lesions occurred in chronic impaired liver. For thosee patients, "less invasive" pure laparoscopic hepatectomy may provide a good option.

Pure Laparoscopic Hepatectomy for HCC Patients 185

Fig. 1. A case with hepatocellular carcinoma and severe liver cirrhosis treated with pure

50 years old man with type-C severe liver cirrhosis (Table 1, patient 5) developed 2 cm hepatocellular carcinoma (HCC) at the undersurface area of the segments 5, revealed in computed tomography (plain (A), arterial phase with contrast (B), venous phase (C)) and

laparoscopic hepatectomy

ultrasonography (D).

### **2. Pure laparoscopic hepatectomy for HCC patients with liver cirrhosis- our experiences**

From May 2005, 21 patients with hepatocellular carcinoma and chronic liver disease underwent pure laparoscopic hepatectomy. There were 6 out of 21 patients had severe liver cirrhosis (Child-Pugh B/C and ICG R15 of 40% or above) (Table 1). These 6 patients and the other 14 patients (Child A and ICG R15: 10.1- 27.4 (median: 13.4) %, excluded the patient with HCC and rectal carcinoma) were compared in operating time, intraoperative blood loss, day of oral ingestion started, day of drain removal, total dose of drain discharge from the operation day to post operative day 3, complication, and postoperative hospital stay.

Table 1. Patients with severe liver cirrhosis who underwent pure laparoscopic hepatectomy B: type-B viral hepatitis, C: type-C viral hepatitis, Alc: alcoholic hepatitis

For 6 patients with HCC and severe liver cirrhosis who underwent pure laparoscopic hepatectomy, the operating time was 140-341 (median: 232) minutes (for other patients with mild/moderate liver cirrhosis: 216-528 (295) minutes), the intraoperative blood loss was uncountable-213 (median: 58) ml (for other patients with mild/moderate liver cirrhosis: uncountable -696 (43) ml), the day of oral ingestion started was postoperative day 1-3 (median: 2) (for other patients with mild/moderate liver cirrhosis: day 2-3 (2) ), the day of drain removal was postoperative day 3-6 (median: 4) (for other patients with mild/moderate liver cirrhosis: day 4-6 (5)), the total dose of drain discharge from the operation day to post operative day 3 was 279-1990 (median: 820) ml (for other patients with mild/moderate liver cirrhosis: 141-1275 (416) ml), and the postoperative hospital stay was 11-21 (median: 17) days (for other patients with mild/moderate liver cirrhosis: 9-254 (20) days). One patient developed postoperative complication of cholecystitis among 6 patients with severe liver cirrhosis, and 2 patients developed postoperative complication of ileus and refractory ascites among other 14 patients. There was no post-operative mortality in both groups. These results of perioperative course were comparable without statistically significant difference in the two groups. Perioperative course of HCC patients with severe liver cirrhosis who underwent pure laparoscopic hepatectomy was favorable and comparable to that of the other HCC patients with mild/moderate liver cirrhosis. (A case is shown in figures 1-5)

Also, repeat pure laparoscopic hepatectomy (and combined treatments) for patients with liver cirrhosis and multicentric/metachronous HCCs was feasible and safe with advantage of less post-operative adhesion. (A case is shown in figures 6-10)

**2. Pure laparoscopic hepatectomy for HCC patients with liver cirrhosis- our**

From May 2005, 21 patients with hepatocellular carcinoma and chronic liver disease underwent pure laparoscopic hepatectomy. There were 6 out of 21 patients had severe liver cirrhosis (Child-Pugh B/C and ICG R15 of 40% or above) (Table 1). These 6 patients and the other 14 patients (Child A and ICG R15: 10.1- 27.4 (median: 13.4) %, excluded the patient with HCC and rectal carcinoma) were compared in operating time, intraoperative blood loss, day of oral ingestion started, day of drain removal, total dose of drain discharge from the operation day to post operative day 3, complication, and postoperative hospital stay.

Age, Background Child-Pugh TB/DB PT Alb Tumor location, Postoperative Sex liver disease (ICGR15 (%)) (mg/dl) (%) (g/dl) Tumor size (mm) Complication 64, M B,C B (42.8%) 1.3/0.3 79 3.2 S4 and 2-3, 44 Cholecyctitis

Table 1. Patients with severe liver cirrhosis who underwent pure laparoscopic hepatectomy

For 6 patients with HCC and severe liver cirrhosis who underwent pure laparoscopic hepatectomy, the operating time was 140-341 (median: 232) minutes (for other patients with mild/moderate liver cirrhosis: 216-528 (295) minutes), the intraoperative blood loss was uncountable-213 (median: 58) ml (for other patients with mild/moderate liver cirrhosis: uncountable -696 (43) ml), the day of oral ingestion started was postoperative day 1-3 (median: 2) (for other patients with mild/moderate liver cirrhosis: day 2-3 (2) ), the day of drain removal was postoperative day 3-6 (median: 4) (for other patients with mild/moderate liver cirrhosis: day 4-6 (5)), the total dose of drain discharge from the operation day to post operative day 3 was 279-1990 (median: 820) ml (for other patients with mild/moderate liver cirrhosis: 141-1275 (416) ml), and the postoperative hospital stay was 11-21 (median: 17) days (for other patients with mild/moderate liver cirrhosis: 9-254 (20) days). One patient developed postoperative complication of cholecystitis among 6 patients with severe liver cirrhosis, and 2 patients developed postoperative complication of ileus and refractory ascites among other 14 patients. There was no post-operative mortality in both groups. These results of perioperative course were comparable without statistically significant difference in the two groups. Perioperative course of HCC patients with severe liver cirrhosis who underwent pure laparoscopic hepatectomy was favorable and comparable to that of the other HCC patients

Also, repeat pure laparoscopic hepatectomy (and combined treatments) for patients with liver cirrhosis and multicentric/metachronous HCCs was feasible and safe with advantage

53, M B B (41.2%) 1.5/0.4 75 3.4 S5-6, 28 no 68, F C C (58.2%) 2.2/0.5 58 2.6 S8, 13 no 40, M Alc B (52.5%) 1.7/0.4 74 2.8 S6-7, 20 no 50, M C C (48.9%) 2.1/1.0 66 2.7 S5, 18 no 75, F C B (42.5%) 1.3/0.8 63 3.2 S3, 30 no

B: type-B viral hepatitis, C: type-C viral hepatitis, Alc: alcoholic hepatitis

with mild/moderate liver cirrhosis. (A case is shown in figures 1-5)

of less post-operative adhesion. (A case is shown in figures 6-10)

**experiences**

Fig. 1. A case with hepatocellular carcinoma and severe liver cirrhosis treated with pure laparoscopic hepatectomy

50 years old man with type-C severe liver cirrhosis (Table 1, patient 5) developed 2 cm hepatocellular carcinoma (HCC) at the undersurface area of the segments 5, revealed in computed tomography (plain (A), arterial phase with contrast (B), venous phase (C)) and ultrasonography (D).

Pure Laparoscopic Hepatectomy for HCC Patients 187

Fig. 4. Intraoperative findings of the case in figure 1 (3)

made with harmonic scalpel.

(A) After intraoperative ultrasonography, shallow cut in the surface area of the liver was

(B) In the deeper area of the liver, dissection was performed with CUSA, monopolar and

(C) The tissue with good coagulation with bipolar and monopolar was cut with scissors.

bipolar electric cautery. Thicker vessels were clipped or ligated and divided.

Fig. 2. Intraoperative findings of the case in figure 1 (1) The liver was highly atrophic with large shunt vessels in the round ligament and the hepatic flexure of the colon was migrating into the subphrenic space.

Fig. 3. Intraoperative findings of the case in figure 1 (2)

There were mild adhesions around the tumor. Dissection of the adhesion was performed, but not the mobilization of the liver. Since the liver was highly atrophic and the tumor was located deeply in the subphrenic space, mobilization of the liver was necessary for safe resection of the tumor under laparotomy. However, with laparoscopy, good direct vision and safe resection was obtained without mobilization.

The liver was highly atrophic with large shunt vessels in the round ligament and the hepatic

There were mild adhesions around the tumor. Dissection of the adhesion was performed, but not the mobilization of the liver. Since the liver was highly atrophic and the tumor was located deeply in the subphrenic space, mobilization of the liver was necessary for safe resection of the tumor under laparotomy. However, with laparoscopy, good direct vision

Fig. 2. Intraoperative findings of the case in figure 1 (1)

Fig. 3. Intraoperative findings of the case in figure 1 (2)

and safe resection was obtained without mobilization.

flexure of the colon was migrating into the subphrenic space.

Fig. 4. Intraoperative findings of the case in figure 1 (3)

(A) After intraoperative ultrasonography, shallow cut in the surface area of the liver was made with harmonic scalpel.

(B) In the deeper area of the liver, dissection was performed with CUSA, monopolar and bipolar electric cautery. Thicker vessels were clipped or ligated and divided.

(C) The tissue with good coagulation with bipolar and monopolar was cut with scissors.

Pure Laparoscopic Hepatectomy for HCC Patients 189

Fig. 6. A case with multicentric metachronous hepatocellular carcinomas and liver cirrhosis

**C D**

69 years old woman with type-C liver cirrhosis developed 2 cm HCC in the segments 3 and 1.5cm in the border of segments 2 and 3, revealed in computed tomography (upper, arterial phase with contrast (A), venous phase (B)) and underwent pure laparoscopic hepatectomy (lower, arterial phase with contrast (C), venous phase (D)) in 2008. Pathological findings of the tumors were moderately (S3) and well (S2-3) differentiated hepatocellular carcinomas.

treated repeatedly with pure laparoscopic hepatectomy

Fig. 5. Intraoperative findings (4) and resected specimen of the case in figure 1 The operation time was 140 minutes and intraoperative blood loss was 100ml. His postoperative hospital stay was uneventfull and 11 days. Pathologically, the tumor was moderately differentiated HCC in well differentiated HCC. He is alive without recurrence at 16 months after surgery

Fig. 5. Intraoperative findings (4) and resected specimen of the case in figure 1

16 months after surgery

The operation time was 140 minutes and intraoperative blood loss was 100ml. His postoperative hospital stay was uneventfull and 11 days. Pathologically, the tumor was

moderately differentiated HCC in well differentiated HCC. He is alive without recurrence at

Fig. 6. A case with multicentric metachronous hepatocellular carcinomas and liver cirrhosis treated repeatedly with pure laparoscopic hepatectomy

69 years old woman with type-C liver cirrhosis developed 2 cm HCC in the segments 3 and 1.5cm in the border of segments 2 and 3, revealed in computed tomography (upper, arterial phase with contrast (A), venous phase (B)) and underwent pure laparoscopic hepatectomy (lower, arterial phase with contrast (C), venous phase (D)) in 2008. Pathological findings of the tumors were moderately (S3) and well (S2-3) differentiated hepatocellular carcinomas.

Pure Laparoscopic Hepatectomy for HCC Patients 191

Fig. 8. Intraoperative findings of the case in figure 7 (1)

intraoperative MCT (C).

Adhesions were observed at the resected area of the liver (A) and the port site (B). There was shunt vessel formation in the port site adhesion. Two small hypovascular lesions in the

borders of segments 3 and 4, and of segments 4 and 8 were ablated with US-guided

Fig. 7. A case with multicentric metachronous hepatocellular carcinomas and liver cirrhosis treated repeatedly with pure laparoscopic hepatectomy (2)

In 2010, she developed 2.5 cm HCC at the undersurface area of the segments 6, revealed in computed tomography (plain (A), arterial phase with contrast (B), venous phase (C)) and ultrasonography. She also had two small hypovascular lesions in the borders of segments 3 and 4, and of segments 4 and 8.

Fig. 7. A case with multicentric metachronous hepatocellular carcinomas and liver cirrhosis

In 2010, she developed 2.5 cm HCC at the undersurface area of the segments 6, revealed in computed tomography (plain (A), arterial phase with contrast (B), venous phase (C)) and ultrasonography. She also had two small hypovascular lesions in the borders of segments 3

treated repeatedly with pure laparoscopic hepatectomy (2)

and 4, and of segments 4 and 8.

Fig. 8. Intraoperative findings of the case in figure 7 (1)

Adhesions were observed at the resected area of the liver (A) and the port site (B). There was shunt vessel formation in the port site adhesion. Two small hypovascular lesions in the borders of segments 3 and 4, and of segments 4 and 8 were ablated with US-guided intraoperative MCT (C).

Pure Laparoscopic Hepatectomy for HCC Patients 193

Fig. 10. Intraoperative findings of the case in figure 7 (3) and resected specimen of the case

Mirnezami et al recently reported systematic review and meta-analysis for short- and long-term outcomes after laparoscopic and open hepatic resection, with twenty-six studies met the inclusion criteria with a population of 1678 patients (60% of the patients had malignant liver tumor) (11). In their study, laparoscopic hepatectomy resulted in longer operating time, but reduced blood loss, portal clamp time, overall and liver-specific complications, and length of post-operative hospital stay. No difference was found between two groups for oncological outcomes. The benefits of laparoscopy may be particularly great for cirrhotic patients, given the potential for lower levels of parietal and hepatic injury and the preservation of venous and lymphatic collateral circulation. The safety and feasibility of the laparoscopic approach and its short-term benefits for HCC have been demonstrated by a few series to date (12-19). Tranchart et al recently reported laparoscopic resection of HCC for selected patients gave a better postoperative outcome without long- and short-term oncologic consequences from their series (42 each laparoscopic- and open-hepatectomy patients, with more than 96% Child A patients and mostly anatomical resection) (20). In our experience, perioperative course of 6 HCC patients with severe liver cirrhosis (Child B/C and ICG R15 of 40% or above) who underwent pure laparoscopic hepatectomy was favorable and comparable to that of the

Early postoperative recovery and discharge with smaller postoperative pain and earlier intake are advantages in pure laparoscopic hepatectomy for severe cirrhotic patients as the other laparoscopic surgery. On top of that, we consider that relatively small amount (median 230 ml/day) of drain discharge of severe liver cirrhosis patients, some (case 3, 5, 6 in table 1) of whom had mild or controllable ascites before surgery, was the other benefit of

The operation time was 168 minutes and intraoperative blood loss was 30ml. Her postoperative hospital stay was uneventfull 9 days. Pathologically, the tumor was moderately

differentiated HCC. She is alive without recurrence at 6 months after surgery

other HCC patients with mild/moderate liver cirrhosis.

in figure

**3. Discussion**

#### Fig. 9. Intraoperative findings of the case in figure 7 (2)

There were also adhesions at the undersurface area of the right lobe of the liver after laparoscopic cholecystectomy performed 8 years ago (A). Dissection of the adhesion, but not the mobilization of the liver, exposed the tumor (B). After intraoperative ultrasonography, shallow cut in the surface area of the liver was made with harmonic scalpel (C). In the deeper area of the liver, dissection was performed with CUSA, monopolar and bipolar electric cautery. Thicker vessels were clipped or ligated and divided (D).

Fig. 10. Intraoperative findings of the case in figure 7 (3) and resected specimen of the case in figure

The operation time was 168 minutes and intraoperative blood loss was 30ml. Her postoperative hospital stay was uneventfull 9 days. Pathologically, the tumor was moderately differentiated HCC. She is alive without recurrence at 6 months after surgery

### **3. Discussion**

192 Hepatocellular Carcinoma – Clinical Research

Fig. 9. Intraoperative findings of the case in figure 7 (2)

There were also adhesions at the undersurface area of the right lobe of the liver after

electric cautery. Thicker vessels were clipped or ligated and divided (D).

laparoscopic cholecystectomy performed 8 years ago (A). Dissection of the adhesion, but not the mobilization of the liver, exposed the tumor (B). After intraoperative ultrasonography, shallow cut in the surface area of the liver was made with harmonic scalpel (C). In the deeper area of the liver, dissection was performed with CUSA, monopolar and bipolar

**C D**

Mirnezami et al recently reported systematic review and meta-analysis for short- and long-term outcomes after laparoscopic and open hepatic resection, with twenty-six studies met the inclusion criteria with a population of 1678 patients (60% of the patients had malignant liver tumor) (11). In their study, laparoscopic hepatectomy resulted in longer operating time, but reduced blood loss, portal clamp time, overall and liver-specific complications, and length of post-operative hospital stay. No difference was found between two groups for oncological outcomes. The benefits of laparoscopy may be particularly great for cirrhotic patients, given the potential for lower levels of parietal and hepatic injury and the preservation of venous and lymphatic collateral circulation. The safety and feasibility of the laparoscopic approach and its short-term benefits for HCC have been demonstrated by a few series to date (12-19). Tranchart et al recently reported laparoscopic resection of HCC for selected patients gave a better postoperative outcome without long- and short-term oncologic consequences from their series (42 each laparoscopic- and open-hepatectomy patients, with more than 96% Child A patients and mostly anatomical resection) (20). In our experience, perioperative course of 6 HCC patients with severe liver cirrhosis (Child B/C and ICG R15 of 40% or above) who underwent pure laparoscopic hepatectomy was favorable and comparable to that of the other HCC patients with mild/moderate liver cirrhosis.

Early postoperative recovery and discharge with smaller postoperative pain and earlier intake are advantages in pure laparoscopic hepatectomy for severe cirrhotic patients as the other laparoscopic surgery. On top of that, we consider that relatively small amount (median 230 ml/day) of drain discharge of severe liver cirrhosis patients, some (case 3, 5, 6 in table 1) of whom had mild or controllable ascites before surgery, was the other benefit of

Pure Laparoscopic Hepatectomy for HCC Patients 195

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pure laparoscopic hepatectomy for these patients in our series. Pure laparoscopic hepatectomy might have possible advantage of minimal postoperative drain discharge (ascites) which leads to lower risk of disturbance in water/electrolyte balance and hypoproteinemia. These disorders could trigger fatal liver failure. There are also reports which described little development of postoperative ascites on laparoscopic hepatectomy (14, 17). In case of pure laparoscopic hepatectomy for severe cirrhotic patients, this feature could be the most remarkable specific advantage for postoperative course. Patients who undergo hepatectomy are exposed three different types of stresses, 1) general surgical stress for whole body depends on operating time, amount of bleeding etc, 2) reduced liver function due to resected liver volume, 3) injury for liver parenchyma and environment around the liver by surgical procedure (destruction of the collateral blood and lymphatic flow caused by laparotomy and mobilization of the liver and, also, mesenchymal injury caused by compression of the liver). We consider that the reduction of the third type of stress mentioned above with pure laparoscopic hepatectomy leads to lowering the risk of refractory ascites, resulting in reduced risk of successive complications and smooth recovery for HCC patients with severe liver cirrhosis.