CASE REPORT

Primary AdenosquamousCarcinoma of the Liver WhichProduces Granulocyte-Colony-Stimulating Factor andParathyroid HormoneRelated Protein: Association

with Leukocytosis and HypercalcemiaTatsuhiko Hayashi, Akira Mizuki, Takuya Yamaguchi, Tasuku Hasegawa, Takamu Kunihiro,

Nobuhiro Tsukada, Kentaro Matsuoka*, Hideki Orikasa* and Kazuto Yamazaki*

Abstract

A 55-year-old man was admitted to our hospital withfever and vomiting. Abdominal computed tomography (CT)revealed multiple low density masses in the liver. A diagno-sis of primary adenosquamouscarcinoma of the liver wasconfirmed by histological examination of a necropsy speci-men. The present case showed leukocytosis and hypercal-cemia with high levels of serum granulocyte-colony-stimu-lating factor (G-CSF) and parathyroid hormone relatedprotein (PTHrP). Recent studies have shown that G-CSFand PTHrPare responsible for the paraneoplastic syn-dromes with leukocytosis and hypercalcemia. The tumorcells demonstrated positive cytoplasmic immunohistochem-istry staining with anti-G-CSF and anti-PTHrP antibod-ies. This result suggested that the tumor produced G-CSFand PTHrP.(Internal Medicine 40: 631-634, 2001)

Key words: primary liver tumor, paraneoplastic syndrome,cytokine

Introduction

Adenosquamous carcinoma (ASC) is a tumor composed ofboth squamouscell carcinoma and adenocarcinoma compo-nents. Primary adenosquamouscarcinoma of the liver is rare.All the reported cases were in an advanced stage and had apoor prognosis. On the other hand, it is well known that theparaneoplastic syndromesof leukocytosis and hypercalcemiasometimesoccur in malignancies. Thecoexistence of leuko-cytosis and hypercalcemia in certain cancers has been reported( 14-1 8). Granulocyte-colony-stimulating factor (G-CSF) and

parathyroid hormone related protein (PTHrP) are responsiblefor these types of paraneoplastic syndromes. Wereport here arare case of primary adenosquamouscarcinoma of the liverassociated simultaneously with leukocytosis and hypercalce-mia, with some discussion of the literature.

Case ReportA 55-year-old manwas admitted to our hospital for a low-grade fever and vomiting on June 5, 1999. He had no history ofblood transfusion. His family history was not contributory. Onphysical examination, his body temperature was 37.8°C, hisblood pressure was 140/72 mmHg,and his pulse rate was 74/min and regular. There was no jaundice. Cervical and supra-clavicular lymph nodes were not palpable. Abdominal exami-nation revealed a tender liver, extending seven finger-breadthsbelowthe right costal margin. Therewas no ascites.Table 1 shows the laboratory values on admission. Erythro-cyte sedimentation rate (ESR) was 106 mm/h and C-reactiveprotein (CRP) was 17 mg/dl. Total white blood cell (WBC)count was 27,500/jil, absolute neutrophil (segment and bands)count (ANC) was 23,500/|il (85.5%). Blast and immature cellswere not found in the peripheral blood. Blood chemistry waswithin the normal range except for elevated aspartate

amionotransferase (AST) and alkaline phosphatase (ALP).Hepatitis B viral surface antigen and hepatitis C viral antibodywere negative. The levels of serum a-fetoprotein (AFP) andcarcinoembryonic antigen (CEA) were within the normal range.The levels of serum carbohydrate antigen 19-9 (199.7 U/ml,normal range <37 U/ml) and squamous cell carcinoma relatedantigen (6.6 ng/ml, normal range <1.5 ng/ml) were moderatelyhigh. Protein induced by vitamin K antagonist-II (PIVKA-II)was also elevated to 45 mAU/ml(normal range <40 mAU/ml).There was markedly elevated ANC,although no evidence ofactive infectious disease was found. His serum G-CSFwas el-

From the Department of Internal Medicine and *the Department of Pathology, Tokyo Saiseikai Central Hospital, TokyoRecieved for publication June 15, 2000; Accepted for publication December 1 8, 2000Reprint requests should be addressed to Dr. Nobuhiro Tsukada, the Department of Internal Medicine, Tokyo Saiseikai Central Hospital, 1 -4- 1 7 Mita, Minato-

ku, Tokyo 108-0073

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Hayashi et al

Tablel. Laboratory Data on Admission

Erythrocyte sedimentation rate 1 06 mm/h Blood ChemistryHematology Total protein 6. 9 g/dl

White blood cell 27,500/^,1 Albumin 2.9 g/dlSegment 84% Sodium 1 35 mEq//Band 1.5% Potassium 3.6 mEq//Lymphocyte 5 % Chloride 95 mEq//Monocyte 6.5% Calcium 1 2.6 mg/dlEosinophil 3% Blood urea nitrogen 1 2 mg/dlBasophil 0% Creatinine 0. 6 mg/dl

Red blood cell 390x l 04/|al Total bilirubin 0.8 mg/dlHemoglobin 8.5 g/dl Aspartate aminotransferase 36 IU//Hematocrit 26.3 % Alanine aminotransferase 24 IU//Platelet 493x 1 OVjxl Lactate dehydrogenase 246 IU//

Serology Alkaline phosphatase 654 IU//C-reactive protein 1 7 mg/dl y-Glutamyltranspeptidase 45 IU//Hepatitis B viral surface antigen (-) Cholinesterase 1 ,6 1 0 IU//Hepatitis C viral antibody (-) OthersTumor marker G-CSF 79 pg/mloc-fetoprotein 1.2 ng/ml PTHrP l O PMol//Carcinoembryonic antigen 1.4 ng/ml Intact PTH 6 pg/mlCarbohydrate antigen 19-9 199.7 U// Interleukin- l a 10.5 pg/mlSSC 6.6 ng/ml Interleukin- l p <10 pg/mlPIVKA-II 45 mAU/ml Interleukin-6 95.7 pg/ml

G-CSF: granulocyte-colony-stimulating factor, PTHrP: parathyroid hormone related protein, intact PTH: intactparathyroid hormone, SSC: squamous cell carcinoma related antigen, PIVKA-II: protein induced by vitamin Kantagonist -II.

evated to 79 pg/ml (normal range <6-21 pg/ml). The serumlevel of interleukin la (10.5 pg/ml, normal range <7.8 pg/ml)and interleukin 6 (95.7 pg/ml, normal range <4 pg/ml) werehigh, while interleukin lp (<10 pg/ml) was within the normalrange. On August 2, 1999, his serum level of calcium was el-evated to 12.6 mg/dl and the serum level of PTHrP was high( 10 PMol//, normal range <0.6 PMol//), while serum intact PTHwas low (6 pg/ml, normal range 10-65 pg/ml).Computed tomograghy (CT) showed multiple nodules lo-cated in the anterior and medial segment of the liver (Fig. 1). Afine-needle biopsy of the liver tumor revealed squamous cellcarcinoma. Since there wasno evidence of other primary sites,a diagnosis of primary squamous cell carcinoma of the liverwas made. However, because of the rapid development of theliver tumor, the patient died of liver failure 64 days after ad-mission to the hospital. Uponautopsy, the liver weighed 4,300g, and showed a solid whitish tumor, 5 cm in diameter, locatedin the anterior segment of the liver, with diffuse small nodulesinvading into two lobes (Fig. 2). Histologic examination re-vealed that the liver tumor was composedmainly of squamouscell carcinoma and partly of focal areas of adenocarcinoma(Fig. 3). No carcinomas were found in the gallbladder, stom-ach, pancreas or other organs. Finally, a diagnosis of primaryASCwas confirmed. In addition, the tumor was immunostainedusing antisera against G-CSF (Fig. 4) and PTHrP (Fig. 5). Sometumor cells were positively stained. This demonstrated that thetumor produced G-CSF and PTHrP.

Figure 1. Abdominal computed tomography scan showed mul-tiple low density masses in the anterior and medial segment ofthe liver.

Discussion

Adenosquamouscarcinoma (ASC) is a tumor which is com-posed of elements of both adenocarcinoma and squamous cellcarcinoma. Since the description of Pianzola and Drut in 1971(1), only 34 cases (including the present case) of a primary

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G-CSF and Hepatic Adenosquamous Carcinoma

Figure 2. Uponautopsy, the liver demonstrated a solid whitishtumor, 5 cm in diameter, located in the anterior segment, with

diffuse small nodule invasion into two lobes.

Figure 3. Microscopic findings of the tumor obtained from theautopsy showed both squamous cell carcinoma and adenocarci-nomacomponents. HEstain, x75.

liver tumor with similar composite histology of areas of ad-enocarcinoma and squamouscell carcinoma, such as ASC,mucoepidermoidcarcinoma or adenoacanthoma, have beenreported (2-4). Among those 34 cases, ASC was found in only26 cases, including the present case, with definitive histologi-cal studies. This histologic type of hepatic ASCis not consis-tently found in a particular site within the liver and shows noevidence of being related to hepatitis virus. ASC typically de-velops in the gallbladder, the extrahepatic bile duct, or the pan-creas, but rarely in the liver. The diagnosis of primary ASCof

the liver should be madewhenboth malignant squamousandglandular elements are present, and primary cancers of the gall-bladder, pancreas, extrahepatic bile duct, or lungs should be

ruled out. ASCof the liver is currently assumed to originate

Figure 4. Immunostaining of the tumor tissue with monoclonalanti -G-CSF antibodies demonstrated positive cytoplasmic stain-ing of the tumor cells. Avidin-biotin peroxidase complex method,xl50.

Figure 5. Immunostaining of the tumor tissue with monoclonalanti-PTHrP antibodies demonstrated positive cytoplasmic stain-ing of the tumor cells. Avidin-biotin peroxidase complex method,xl50.

from the intrahepatic bile duct. This tumor is considered to bea subtype of cholangiocarcinoma,whichis not a commontypeof liver malignancy. However, the histogenesis of the tumor isstill largely unknown. Someinvestigators support the theorythat the tumor might develop by transformation of pre-existingadenocarcinoma in the transitional areas between adenocarci-noma and squamous cell carcinoma (5, 6). Concerning thepathogenesis, somereports have demonstrated that ASCis as-sociated with hepatolithiasis (7, 8) or hydatid cysts (1, 9) whichhave been proposed as etiological factors. But the present casedid not have these complications.It is well known that both leukocytosis and hypercalcemiaare considered to be paraneoplastic syndromes associated with

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Hayashi et al

a variety of malignancies. Recently, it has been reported thatG-CSFis responsible for this kind of paraneoplastic syndrome.More than 200 cases of G-CSF-producing tumors have beenreported since Asano et al (10) reported the first case of a pa-tient with lung cancer in 1977. The present case had associatedleukocytosis, even in the absence of active infectious diseases,and had an elevated level of serum G-CSF. Positive staining ofthe tumor cells by immunohistochemistry with monoclonal anti-G-CSF antibodies revealed that the tumor cells produce G-CSF.Concerning the biological relationship of G-CSF and tumorgrowth, it has been reported that G-CSFalso stimulatesnonhematopoietic tumor cells. G-CSF-producing tumor cellsexpressed a G-CSFreceptor which induces tumor cell growthby G-CSFvia the autocrine pathways (1 1). For this reason G-CSF-producing tumors usually grow rapidly and the prognosisis extremely unfavorable. Recent studies have shownthat theG-CSF producing tumor is a multi-cytokine-producing tumorwhich secretes IL-1 and IL-6 in addition to G-CSF (12). IL-1levels may exceed those of G-CSFand IL-6. The present casealso showed elevated serum levels of IL-1 and IL-6. This sug-gests that IL-1 mayplay an important role in the regulation ofG-CSF and IL-6 production in this G-CSF producing hepatictumor.

Furthermore, hypercalcemia also occurred in the presentcase. His serum PTHrPlevels were elevated. PTHrPhas beenidentified as a major causative peptide for hypercalcemia inmalignancies (13). The immunohistochemistry study of thepresent tumor tissue demonstrated positive staining for PTHrP.This suggests that the tumor produces PTHrPwhich is respon-sible for hypercalcemia. The concomitant existence of leuko-cytosis and hypercalcemia has been reported in cancers of theoral mucosa (14), thyroid (15), lungs (16), gallbladder (17),and cholangiocarcinoma (18). It is still uncertain whether leu-kocytosis and hypercalcemia are complicated randomly or ifthere are some factors commonlyregulating them. IL-l is aosteoclast-activating factor which causes hypercalcemia syn-ergistically with PTHrP (19). IL- 1 is also a hemopoetin- 1 , whichpotentiates G-CSF-induced leukocytosis. Sato et al (19) re-ported that IL-1a has dual effects on the development of leu-kocytosis and hypercalcemia. However, the detailed mecha-nism of hypercalcemia-leukocytosis syndromeis not clear. Inorder to elucidate it, further investigation will be necessary.

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