Hepatoid Adenocarcinoma of the Stomach: Biological Significance of Hepatic Transdifferentiation in Adenocarcinoma Cells

Hepatoid Adenocarcinoma of the Stomach: Biological Significance of Hepatic Transdifferentiation in

Adenocarcinoma Cells

Hiroshi Ishikura

, Yana Supriatna

, Kazuhiro Kojima

, Shigeyuki Kamata

, Hiroki Nakaya

, and Takashi Kishimoto

Clinicopathology of Hepatoid Adenocarcinoma of the Stomach

Hepatoid adenocarcinoma was first described in the stomach as a subtype of alpha- fetoprotein (AFP)-producing gastric cancers with distinct clinicopathological prop- erties [1,2]. Hepatoid adenocarcinoma of the stomach is defined as a primary gastric carcinoma with areas of functionally and structurally distinctive foci of hepatocellu- lar differentiation. It usually arises as a circumscribed mass in the antrum to body of the stomach in middle-aged to elderly persons (Fig. 1).

Tumor cells in a certain type of AFP-producing gastric carcinomas were once regarded as poorly differentiated or undifferentiated due to the sheetlike arrangement of tumor cells with abundant, eosinophilic cytoplasm. However, after the recognition of a liverlike trabecular arrangement, as well as production of a set of liver-specific proteins [2,3], the sheetlike proliferation of AFP-producing gastric carcinoma has now come to be regarded as a morphological mimicry of the liver structure (Figs. 2–5).

Most importantly, hepatoid adenocarcinomas exhibit hepatocellular differentiation, usually in association with well-differentiated tubular or papillary adenocarcinoma [2,4]. Adenocarcinoma cells are either of intestinal epithelial type with production of mucin, or of clear cell type with a primitive appearance. Adenocarcinoma cells usually can be found in the mucosal layer of the gastric wall (Fig. 6), which strongly indicates the emergence of hepatoid cells from adenocarcinoma through neometaplasia or transdifferentiation. Rare cases of hepatoid adenocarcinoma, however, have no obvious adenocarcinoma components; in such cases, de novo development has not been excluded. Transition between adenocarcinoma and hepatoid cells is gradual in some cases (Fig. 7) and abrupt in others.

139

1Department of Molecular Pathology,²Department of General Surgery, Chiba University Grad- uate School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba, Japan

3Department of Gastroenterology and Hematology, Hokkaido University Graduate School of Medicine, Sapporo, Japan

e-mail: hishik@med.m.chiba-u.ac.jp

Hepatocellular differentiation in a hepatoid adenocarcinoma includes structural mimicry of liver tissue, for example, trabecular arrangement with sinusoid-like vas- culature and formation of bile (Fig. 8), glycogen, and bile canaliculus-like structures (Fig. 9), and production of liver-specific proteins such as AFP [1,2], albumin [3,5], transferrin [2], PIVKA-II [6], and Hep-par 1 [7,8] antigens. Periodic acid-Schiff (PAS) staining-positive hyaline globules are numerous in some cases (Fig. 10). The hepato- cellular phenotype in a hepatoid adenocarcinoma is determined by a collective con- sideration of the afore-mentioned factors; the mere presence or absence of AFP production does not necessarily characterize phenotype. Thus, not all AFP-producing gastric carcinomas are hepatoid adenocarcinoma, whereas there are many hepatoid adenocarcinomas that do not produce AFP (Fig. 11) [4]. The nonhepatoid, AFP- producing tumors include enteroblastic gastric tumors [9], medullar tumors with gastrointestinal tract-specific AFP [10], and yolk sac tumors [11]. The biological behavior of hepatoid adenocarcinomas is definitively worse than that of other types of AFP-producing gastric cancers, both clinically [4] and experimentally [12].

A striking feature exhibited by hepatoid adenocarcinoma is its invasion of vessels, usually extending to veins. This invasion can readily be seen by light microscopy as expanded veins in the gastric wall that are filled with tumor cells (Fig. 12). In addi- tion, intravenously invading tumor cells can even be seen grossly as tumor thrombi from the serosal surface in occasional hepatoid adenocarcinoma cases (Fig. 13). Hepa- toid adenocarcinoma of the stomach, therefore, is characterized by almost inevitable blood-borne metastasis to the liver, which results in an extremely poor prognosis of patients with hepatoid adenocarcinoma [4]. In a study with a limited number of cases, hepatoid adenocarcinoma metastasizes to the liver even if its invasion is limited to the submucosal layer of the gastric wall [13,14].

In rare circumstances, tumor thrombi were in direct continuity with those in the portal vein. Considering that morphology of liver metastatic foci also resembles that of hepatocellular carcinoma, the presence of portal vein thrombi that are contiguous to gastric veins causes a diagnostic problem; it is difficult to differentiate between hepatocellular carcinoma metastatic to the stomach and hepatoid adenocarcinoma metastatic to the liver [15]. Our previous study revealed that the absence of liver cir- rhosis and the presence of intramucosal adenocarcinoma favor the origin of the stomach, that is, hepatoid adenocarcinoma metastatic to the liver [15]. In contrast, hepatocellular carcinoma metastatic to the stomach is characterized by the gross appearance of a gastric submucosal tumor in association with liver cirrhosis or hep- atitis virus-associated hepatitis. Before the introduction of hepatoid adenocarcinoma, several case studies had concluded the former cases were rare examples of hepato- cellular carcinoma metastatic to the stomach [15].

Hepatoid Adenocarcinoma as a Ubiquitously Occurring Carcinoma with Hepatocellular Transdifferentiation

Hepatoid adenocarcinoma was first described in the stomach, followed by reports of it in a variety of other organs, with gastric primaries being most common [16–19].

Extragastric hepatoid adenocarcinoma has been found in the digestive organs, includ-

ing the esophagus [20,21], duodenum [22], large intestine [23], gallbladder [24], and

pancreas [25]. It has also been found in the lung [26,27]. In addition, the genitouri- nary organs are one of the most common sites of origin; it has been described in the renal pelvis [28], urinary bladder [29], uterus [30–32], fallopian tube [33], and ovary [34–37]. In all these organs except the ovary, hepatoid adenocarcinomas almost always arise from the mucosa. Hepatoid carcinoma of the ovary was classified into the mis- cellaneous tumor category in the WHO classification in 2003, but the proposal that it belongs in the common epithelial category has been made by several investigators [37–39].

Hepatoid Adenocarcinoma as a Model for Transdifferentiation in Adenocarcinoma

Phenotypic microheterogeneities are well known in a variety of human and animal tumors. For example, the occurrence of argentaffin/argyrophil cells in otherwise common-type gastric adenocarcinoma cells has been shown [40]. This finding may be due to maturation or transdifferentiation of adenocarcinoma cells with a ques- tionable clinicopathological impact; the occurrence has not clearly been related to an altered prognosis. In contrast, the emergence of transdifferentiated oat cell-type neu- roendocrine tumor cells in gastric adenocarcinoma may profoundly alter the clinico- pathology [41]. Similarly, the emergence of transdifferentiated hepatocellular tumor tissues in gastric adenocarcinoma has a great impact on the biology of gastric adenocarcinoma through frequent blood-borne metastases to the liver [2,4]. This biological modification seen in hepatocellular transdifferentiation in hepatoid ade- nocarcinoma warrants intensive study of its genesis and regulation.

Several cell lines of AFP-producing gastric cancers have been cultured [42–44].

These lines provide an opportunity to study phenotypic regulation of gastric carci- noma at the molecular level. Currently, some of them have been shown to represent hepatocellular transdifferentiation based on the production of a set of liver-specific proteins (Supriatna et al. 2005, manuscript in preparation).

Liver-Enriched Nuclear Factors, Hepatogenesis, and Hepatic Transdifferentiation in Carcinoma Cells

We showed that FU97 cells, an AFP-producing gastric adenocarcinoma cell line, pro- duced a set of liver-specific proteins (Supriatna et al. 2005, manuscript in prepara- tion). In addition, transcriptional factors positively regulate transcription of the AFP gene in FU97 cells, which is indicated by the fact that FU97 cells expressed the herpes simplex tymidine kinase (HSVtk) gene through the function of the AFP promoter/enhancer sequence [45]. It is probable that other genes for liver-specific proteins may also be expressed by a mechanism identical or similar to it.

Cooperative expressions of multiple master transcriptional factors have been

shown in multiple organogenesis sites such as the lung [46]. In hepatogenesis, several

important transcriptional factors, collectively called liver-enriched nuclear factors,

exert their role on structural and functional development of the liver [47,48]. Based

on the fact that transdifferentiation of adenocarcinoma toward the hepatocellular

phenotype might be homologous to liver bud emergence in the primitive digestive tract at the duodenum, some of the liver-enriched nuclear factors might well be involved in the genesis of hepatoid adenocarcinoma, as suggested by the HSVtk autocidal experiment [45].

Our previous data indicated that HNF-4a mRNA is expressed in the gastric hepa- toid adenocarcinoma tissues [3]. However, gastric adenocarcinoma tissues without distinctive hepatocellular transdifferentiation also expressed similar amounts of HNF-4a mRNA, indicating that this liver-enriched nuclear factor may not be the sole factor responsible for hepatic transdifferentiation in gastric carcinomas [3]. With regard to the lung, upregulation of HNF-4a mRNA was specifically seen in hepatoid adenocarcinoma but not in the normal lung and conventional lung carcinoma tissues.

Furthermore, HNF-4a was specifically stained in the nuclei in the transdifferentiated but not in the conventional areas of the hepatoid adenocarcinoma of the lung, indicating an organ-specific role of liver-enriched nuclear factors in the genesis of hepatoid adenocarcinoma (Kishimito et al. 2005, manuscript in preparation).

Hepatic transdifferentiation has been shown in neoplastic and nonneoplastic rodent pancreas cells [49]. In rodent models, regenerated pancreas contained liver cells [50,51]. A recent experiment demonstrated a C/EBP-b-rendered transdifferenti- ation of rat pancreatic carcinoma cells into tumor cells with a hepatocellular pheno- type [52]. This observation exemplifies that a single, or a few, master transcriptional protein(s) indeed have the potential to transdifferentiate tumor cells into another phe- notype, in this case, into a hepatocellular phenotype. The transcriptional regulatory region of the mdr-1/p-glycoprotein gene contains a C/EBP-b site [53]. This finding adds to the possible importance of liver-enriched nuclear factors in the treatment of transdifferentiated tumor cells.

Conclusions

Hepatoid adenocarcinoma is an extrahepatic carcinoma with functionally and struc- turally distinctive foci of hepatocellular differentiation. The usual presence of adeno- carcinoma indicates the emergence of hepatocellular transdifferentiation from adenocarcinoma cells. Hepatoid adenocarcinoma most commonly occurs in the stomach, but other gastrointestinal and genitourinary organs are also common sites of origin. Master transcriptional regulators, particularly liver-enriched nuclear factors, might be involved in the transdifferentiation process, as well as in the biology of hepatoid adenocarcinoma tissues.

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Fig. 1. Hepatoid adenocarci- noma of the stomach. A 5¥ 8 cm, circumscribed mass with ulcera- tion is seen in the body of the stomach in a 76-year-old man.

Serum alpha-fetoprotein (AFP) was 7600 ng/ml

Fig. 2. Sheetlike proliferation of polygonal carcinoma cells with abundant, eosinophilic cyto- plasm in a hepatoid adenocarci- noma. This pattern was once regarded as “patternless,”

resulting in the designation of this area as poorly differentiated or undifferentiated carcinoma.

Thin networks of microvessels give an appearance of vague thick trabeculae

Color Plates

Fig. 3. Sheetlike proliferation of polygonal carcinoma cells with abundant, eosinophilic cyto- plasm in a hepatoid adenocarci- noma. Anastomosing networks of capillaries are dilated in several areas, giving the appear- ance of a distinct middle trabec- ular pattern

Fig. 4. Sheetlike proliferation of polygonal carcinoma cells with abundant, eosinophilic cyto- plasm in a hepatoid adenocar- cinoma. Capillary networks are incomplete. Middle-to-thin trabeculae are conspicuous

Fig. 5. Cords and sheets of hyperchromatic carcinoma cells in a hepatoid adenocarcinoma.

Glandlike spaces are filled with mucin. Cytoplasm of these tumor cells is abundant and eosinophilic, giving the appear- ance of hepatoid cells

Fig. 7. Close apposition of adenocarcinoma (right) and hepatoid cells (left). There is a transition between these areas at the middle upper portion of the figure

Fig. 8. Production of bile pigment in a hepatoid adenocar- cinoma. Only a limited number of cases show bile production, but this finding demonstrates a clear-cut hepatocellular differentiation

Fig. 6. Adenocarcinoma seen in the mucosal layer of a hepatoid adenocarcinoma of the stomach.

Note the transition of tall, columnar, mucin-producing adenocarcinoma cells to hepa- toid cells with large eosinophilic cytoplasm

Fig. 9. Canalicular structures in a hepatoid adenocarcinoma. These structures are intermingled in the hepatoid portion, and positive for polyclonal anticarcinoembryonic antigen antibodies.

Given that these antibodies cross-react with biliary glycoprotein, the morphological finding in combination with this immunohistochemical finding suggests a development of bile canaliculi in hepatoid adenocarcinoma cells, even in the absence of visible bile pigment

Fig. 10. Periodic acid-Schiff (PAS)-positive hyaline globules in a hepatoid adenocarcinoma

Fig. 11. Hepatoid adenocarci- noma with no obvious AFP pro- duction. There was no elevation in the patient’s serum AFP level.

Immunostaining for AFP was negative. Vascular permeation was extensive, and multiple liver metastases were seen.

The histopathological pattern indicated a hepatoid adenocarcinoma

Fig. 12. Extensive vascular involvement seen in the primary gastric wall in a hepatoid adeno- carcinoma

Fig. 13a,b. Gross appearance of hepatoid adenocarcinoma with wormlike tumor thrombi within veins in the gastric serosa. a A mucosal view of the primary gastric hepatoid adenocarci- noma. b A serosal view. Adipose tissues surrounding the veins were removed. Note the expanded, wormlike veins radiating from the primary site a

b