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The Oncologist, Vol. 12, No. 3, 320-324, March 2007; doi:10.1634/theoncologist.12-3-320
© 2007 AlphaMed Press

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Hepatobiliary

A Case of an {alpha}-Fetoprotein-Producing Intrahepatic Cholangiocarcinoma Suggests Probable Cancer Stem Cell Origin

Kenji Ishikawaa, Atsushi Sasakia, Naotsugu Haraguchia, Yasuji Yoshikawab, Masaki Moria

Departments of aSurgical Oncology and bPathology, Medical Institute of Bioregulation, Kyushu University, Beppu, Japan

Key Words. Alpha-fetoprotein • Intrahepatic cholangiocarcinoma • Chronic hepatitis • Hepatitis virus C Cancer stem cells • Immunohistochemical analysis

Correspondence: Masaki Mori, M.D., Ph.D., F.A.C.S., Department of Surgical Oncology, Medical Institute of Bioregulation, Kyushu University, 4546 Tsurumibaru, Beppu 874-0838, Japan. Telephone: 81-977-1650; Fax: 81-977-1651; e-mail: mmori{at}beppu.kyushu-u.ac.jp

Received August 23, 2006; accepted for publication January 1, 2007.


   ABSTRACT
Top
 Abstract
Introduction
 Case Report
 Discussion
 Disclosure of Potential...
 References
 
Recent evidence suggests that some cancers may originate from cancer stem cells, which may derive from carcinogenesis of normal stem cells. A hepatic progenitor cell population, which gives rise to hepatocytes and cholangiocytes, has been suggested in humans, though whether these cells can give rise to malignant tumors has not been confirmed. We report here a case of an {alpha}-fetoprotein (AFP)-producing intrahepatic cholangiocarcinoma (ICC) in an 81-year-old woman with chronic hepatitis C viral infection, suggesting malignant transformation of hepatic stem cells as a mechanism for hepatic neoplasia. Abdominal computed tomography revealed a low-density mass with surrounding enhancement measuring 5 cm x 5 cm in segments IV and VIII of the liver. The preoperative serum levels of tumor markers were 1.7 ng/ml of carcinoembryonic antigen, 22 mAU/ml of protein induced by vitamin K absence or antagonist II, 43.4 U/ml of carbohydrate antigen 19–9, and 1,560 ng/ml of AFP. Following central bisegmentectomy of the liver, serum AFP levels decreased dramatically. Histologically, the tumor cells showed indistinct glandular structures with abundant fibrous stroma. Immunohistochemical analysis demonstrated that the neoplastic cells reacted strongly to antibodies against AFP and cytokeratin (CK) 7. In addition, cancer cells showed partially positive reaction to anti-CK14, a liver stem cell marker, and to anticluster designation (CD) 133, a hematopoietic stem cell marker, and negative reaction to antihepatocyte paraffin (HepPar) 1. These data may indicate that the tumor was derived from a normal liver stem cell that underwent oncogenic transformation.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Case Report
 Discussion
 Disclosure of Potential...
 References
 
Intrahepatic cholangiocarcinoma (ICC), a malignant tumor arising from peripheral intrahepatic bile duct epithelium, occurs more rarely than hepatocellular carcinoma (HCC). HCC accounts for more than 90% of all primary liver cancers in the hepatitis C virus (HCV) endemic area, while ICC accounts for approximately 5% of primary liver cancers and {alpha}-fetoprotein (AFP)-positive ICC accounts for less than 1%, as determined by the Liver Cancer Study Group of Japan [1]. HCC tumor markers are AFP and protein induced by vitamin K absence or antagonist II (PIVKA II), while ICC tumor markers are carcinoembryonic antigen (CEA) and carbohydrate antigen 19–9 (CA19–9) [2]. Elevation of serum AFP to levels higher than 20 ng/ml is seen in approximately 80% of HCC patients and 20% of ICC patients [1]; however, only a few ICC patients show an elevation of serum AFP to levels higher than 1,000 ng/ml.

Three fundamental types of ICC, classified on the basis of macroscopic shape, were established by the Liver Cancer Study Group of Japan [3]: mass forming (MF), periductal infiltrating (PI), and intraductal growth (IG). The MF type forms a definite mass with a relatively clear margin. Additionally, MF type ICC frequently invades into the portal vein and forms intrahepatic metastases, similar to HCC [4]. The PI type extends mainly longitudinally along the bile duct, often resulting in dilatation of the peripheral bile duct. The IG type is a papillary or granular tumor that grows into the bile duct.

Several factors have been reported as etiologic factors of ICC, including liver fluke infection, primary sclerosing cholangitis, and hepatolithiasis [5]. Although cirrhosis and hepatitis viral infection also seem to be risk factors for ICC, the relation between these diseases and ICC is not as clear as that between cirrhosis and HCC [5]. However, recent evidence suggests that infection with hepatitis virus type B (HBV) and/or type C (HCV) is involved in the pathogenesis of ICC [1, 6–8].

The mechanism of ICC carcinogenesis remains unclear. Recently, some researchers have suggested that some cancers may originate from cancer stem cells, which may be derived from carcinogenesis of normal stem cells [9–11]. It has been suggested that hepatocytes and cholangiocytes arise from the same pool of hepatic precursor cells [12]. Carcinogenesis of such hepatic precursor cells may cause ICC. We present here a case of AFP-producing ICC derived from a liver infected by HCV.


   CASE REPORT
Top
 Abstract
 Introduction
 Case Report
Discussion
 Disclosure of Potential...
 References
 
An 81-year-old woman was seen for a follow-up visit at a local hospital for chronic hepatitis related to HCV 1 year postdiagnosis. Although she had no symptoms, abdominal computed tomography (CT) revealed a tumor in the liver. Thereafter, she was referred to our hospital. Serum levels of total bilirubin, albumin, CEA, and PIVKA II were normal at the time of admission to our hospital as follows: total bilirubin, 0.82 mg/dl (normal, 0.3–1.2); albumin, 4.3 g/dl (normal, 4.0–5.0); CEA, 1.7 ng/ml (normal, 0–5); PIVKA II, 22 mAU/ml (normal, <40). However, serum levels of AFP and CA19–9 were abnormally elevated: AFP, 1,560 ng/ml (normal, <10.0); and CA19–9, 43.4 U/ml (normal, <40). The retention rate of indocyanine green at 15 minutes (ICG-R15) was 8.7% (normal, 0–10), and activity time of prothrombin was 96.4% (normal, ≥70).

Abdominal CT revealed a low-density mass, measuring 5 cm by 5 cm, in segments IV and VIII of the liver. The tumor did not show enhancement by contrast medium during the arterial phase (Fig. 1A) but did show peripheral enhancement during the portal phase (Fig. 1B). The central area of the tumor showed mosaic enhancement during the late phase (Fig. 1C). The CT scan also revealed small enhanced tumors, considered intrahepatic metastases, measuring about 5 mm in diameter, in segments IV and VIII of the liver. CT revealed neither enlarged lymph nodes at the hepatoduodenal ligament nor ascites. The portal vein was intact until the third branch.


Figure 1
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  		Figure 1. Abdominal computed tomography (CT). CT scans show a low-density mass, measuring 5 x 5 cm, in segments IV and VIII of the liver. The tumor did not show enhancement during the arterial phase (A), but did show peripheral enhancement during the portal phase (B). The central area of the tumor showed mosaic enhancement during the late phase (C).

 
Preoperatively, the tumor was diagnosed as ICC or HCC of poorly differentiated or schirrhous types. The patient underwent central bisegmentectomy of the liver without lymph node dissection because of her advanced age and poor heart function. After the surgery, the serum AFP level decreased to 97.0 ng/ml. No postoperative adjuvant chemotherapy was performed because of her advanced age. As of 6 months postoperatively, she is alive with recurrent tumors in the liver remnant.

Macroscopically, the tumor was whitish and hard (5.5 x 5 x 7 cm) with a well-demarcated margin (Fig. 2). Neither extensive necrosis nor hemorrhage was found in the central area of the tumor. Small intrahepatic metastases were present around the main tumor.


Figure 2
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  		Figure 2. The resected liver specimen. Macroscopically, a whitish, hard tumor (5.5 x 5 x 7 cm) with a well-demarcated margin was found in the resected liver. Neither extensive necrosis nor hemorrhage was found in the central area of the tumor. Intrahepatic metastases were found around the main tumor.

 
For microscopic study, a paraffin-embedded fixed specimen was sliced into 5 µm sections. Hematoxylin-eosin staining and immunohistological staining using antibodies to AFP; cytokeratin (CK) 7, 14, and 20; cluster designation (CD) 133; and hepatocyte paraffin (HepPar) 1 were performed on seven serial sections. Microscopically, cuboidal cancer cells with chromatin-rich nuclei had proliferated invasively, forming indistinct glandular structures (Figs. 3A and 4A). Immunohistochemically, cancer cells reacted diffusely positively to antibodies against AFP (Fig. 3B) and CK7 (Fig. 3C). In addition, cancer cells reacted partially positively to antibodies against CK14, CK20, and CD133 (Fig. 3D–F). "Partially positive" reaction means that only some of the cancer cells reacted positively. In other regions, cancer cells also reacted positively to antibodies against AFP and CK7 (Fig. 4B and C), whereas the cancer cells in these regions reacted negatively to antibodies against CK14, CK20, and CD133 (Fig. 4D–F). In both regions, the cancer cells reacted negatively to antibody against HepPar1, a monoclonal antibody specific for hepatocytes (Figs. 3G and 4G). So, we diagnosed this case as an AFP-producing ICC.


Figure 3
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  		Figure 3. Histological and immunohistological staining of the intrahepatic cholangiocarcinoma. Hematoxylin and eosin staining (A) and immunohistological staining for AFP (B), CK7 (C), CK14 (D), CK20 (E), CD133 (F), and HepPar1 (G). The tumor cells reacted diffusely positively to AFP and CK7, partially positively to CK14, CK20, and CD133, and negatively to HepPar1. (Original magnification x200.)

 

Figure 4
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  		Figure 4. Histological and immunohistological staining in other regions of the intrahepatic cholangiocarcinoma. Hematoxylin and eosin staining (A) and immunohistological staining for AFP (B), CK7 (C), CK14 (D), CK20 (E), CD133 (F), and HepPar1(G). The tumor cells showed positive staining for AFP and CK7, diffusely, and negative staining for CK14, CK20, CD133, and HepPar1. (Original magnification x200.)

 

   Discussion
Top
 Abstract
 Introduction
 Case Report
 Discussion
Disclosure of Potential...
 References
 
AFP is a normal fetal serum glycoprotein that is synthesized and secreted by fetal hepatocytes, gastrointestinal cells, and yolk sac cells [13]. Usually, synthesis of AFP stops at birth; therefore, its presence in the serum after 1 year of age is associated with pathological conditions [13]. Elevation of serum AFP has been reported in association with some human cancers, predominantly in association with HCC and nonseminomatous germ cell tumors [14], and rarely with carcinomas of the stomach [15, 16], colon [17], gallbladder [18], ovary [19], pancreas [20], lung [21], kidney [22], duodenum [23], prostate [16], and urinary bladder [24]. AFP-producing ICC is extremely rare, and according to the report of the Liver Cancer Study Group of Japan, serum AFP levels higher than 1,000 ng/ml are seen only in 6.3% of ICC cases [1]. The mechanism of AFP production in cancer cells remains unclear.

Recently, some researchers have suggested that some cancers may originate from cancer stem cells, which may form via carcinogenesis of normal stem cells. It has been suggested that hepatocytes and cholangiocytes arise from hepatic precursor cells [12]. It was demonstrated that hepatic progenitor cells, also called oval cells, strongly express AFP mRNA and produce AFP during differentiation [25, 26]. Our data may indicate that one mechanism for the development of ICC involves the neoplastic transformation of oval cells and that the oval cell precursor retains its ability to produce AFP through the process of malignant transformation. Interestingly, our group also previously reported that the hepatoma cell line HuH7 consists of two major cell populations [27]: a side population (SP) of cells that may contain abundant stem cells and a main population (MP) of cells consisting of differentiated hepatoma cells. SP cells show characteristics of both hepatocytes and cholangiocytes. In addition, SP cells show positive staining for CK14, a liver stem cell marker, and CD133, a hematopoietic stem cell marker. We performed immunohistological staining for CK14 and CD133 on eight cases of HCC and five cases of non AFP-producing ICC. No case showed positive staining for CK14 and CD133 (data not shown). In the case presented in this report, cancer cells showed diffusely positive staining for AFP and CK7 and partially positive staining for CK14, CK20, and CD133. In other regions of the same tumor sections, cancer cells were also positive for AFP and CK 7 diffusely but were negative for CK14, CK20, and CD133. While the tumor sample presented in this case displayed heterogeneous expression of such antigens, this finding may indicate that progenitor cells such as oval cells may exist in humans and that AFP-producing ICC results from the carcinogenesis of hepatic oval cells. Another interpretation of our data is that dedifferentiation to oval cells occurs in ICC cells [28].

Hepatitis viral infection and cirrhosis are generally considered to be unrelated to the mechanism of ICC carcinogenesis [29]. However, recent evidence suggests a possible etiological role of HCV infection in ICC. Tomimatsu et al. [6] reported frequent HCV infection (30.8%) among 13 cases of ICC. Additionally, Kobayashi et al. [7] reported that the risk for development of ICC in patients with HCV-related cirrhosis is about 1,000 times as high as that in the general population in Japan. Furthermore, Yamamoto et al. [8] speculated that HCV-infected proliferating cholangioles in patients with chronic hepatitis C might be associated with the development of MF type ICC. Indeed, Uchida et al. [30] demonstrated that HCV core antigen is present in proliferating bile duct epithelia but not in normal bile duct epithelium. Such proliferating bile duct epithelia might derive from oval cells. It is possible that chronic HCV infection induces the activation of oval cells, and that such continued activation induces oval cells to develop into cholangiocytes or hepatocytes with the acquisition of certain genetic changes associated with carcinogenesis.

Here, we report a case of AFP-producing ICC in a patient with chronic hepatitis related to HCV. This case is very interesting because the tumor may be derived from carcinogenesis of an oval cell of the liver.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
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 Abstract
 Introduction
 Case Report
 Discussion
 Disclosure of Potential...
References
 
The authors declare no potential conflicts of interest.


   REFERENCES
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 Abstract
 Introduction
 Case Report
 Discussion
 Disclosure of Potential...
 References
 

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