PO Box 2345, Beijing 100023, China World J Gastroenterol 2004;10(17):2472-2477

Fax: +86-10-85381893 World Journal of Gastroenterology

E-mail: [email protected] www.wjgnet.com Copyright © 2004 by The WJG Press ISSN 1007-9327

• LIVER CANCER •
Thrombocytosis: A paraneoplastic syndrome in patients with
hepatocellular carcinoma
Shinn-Jang Hwang, Jiing-Chyuan Luo, Chung-Pin Li, Cheng-Wei Chu, Jaw-Ching Wu, Chiung-Ru Lai, Jen-Huei Chiang,
Gar-Yang Chau, Wing-Yiu Lui, Chun-Chung Lee, Full-Young Chang, Shou-Dong Lee

Shinn-Jang Hwang, Department of Family Medicine, Taipei Veterans Furthermore, the expression of TPO mRNA was found to
General Hospital and National Yang-Ming University School of be more in tumor tissues than in non-tumor tissues of liver
Medicine, Taiwan, China in an HCC patient with thrombocytosis.
Shinn-Jang Hwang, Jiing-Chyuan Luo, Chung-Pin Li, Cheng-
Wei Chu, Jaw-Ching Wu, Full-Young Chang, Shou-Dong Lee,
CONCLUSION: Thrombocytosis is a paraneoplastic syndrome
Division of Gastroenterology, Department of Medicine, Taipei
Veterans General Hospital and National Yang-Ming University School of HCC patients due to the overproduction of TPO by HCC.
of Medicine, Taiwan, China It is frequently associated with a large tumor volume and
Chiung-Ru Lai, Department of Pathology, Taipei Veterans General high serum α-fetoprotein.
Hospital and National Yang-Ming University School of Medicine,
Taiwan, China Hwang SJ, Luo JC, Li CP, Chu CW, Wu JC, Lai CR, Chiang JH,
Jen-Huei Chiang, Department of Radiology, Taipei Veterans General Chau GY, Lui WY, Lee CC, Chang FY, Lee SD. Thrombocytosis:
Hospital and National Yang-Ming University School of Medicine, A paraneoplastic syndrome in patients with hepatocellular
Taiwan, China carcinoma. World J Gastroenterol 2004; 10(17): 2472-2477
Gar-Yang Chau, Wing-Yiu Lui, Department of Surgery, Taipei http://www.wjgnet.com/1007-9327/10/2472.asp
Veterans General Hospital and National Yang-Ming University School
of Medicine, Taiwan, China
Chun-Chung Lee, Central Laboratory, Shin Kong Wu Ho-Su
Memorial Hospital, Taiwan, China INTRODUCTION
Shinn-Jang Hwang, Taipei Municipal Yang-Ming Hospital, Taipei, Hepatocellular carcinoma (HCC) is the most common malignancy
Taiwan, China in Taiwan. During its clinical course, patients may manifest a
Supported by a grant from Taipei Veterans General Hospital, Taiwan,
variety of paraneoplastic syndromes, including hypercholes-
No. VGH88-299
Correspondence to: Shinn-Jang Hwang, M.D., F.A.C.G., Superintendent terolemia, hypoglycemia, hypercalcemia, and erythrocytosis[1].
Office, Taipei Municipal Yang-Ming Hospital, 105 Yu-Sheng Street, According to our previous reports, the prevalence of paraneoplastic
Shih-Lin, Taipei, 111, Taiwan, China. [email protected] syndromes was 11.4-12.1% for hypercholesterolemia, 2.8-5.3% for
Telephone: +886-2-28353461 Fax: +886-2-28347528 hypoglycemia, 1.8-4.1% for hypercalcemia, and 2.5-3.1% for
Received: 2004-02-06 Accepted: 2004-02-24 erythrocytosis[2-6]. Thrombocytosis has been found in children
with hepatoblastoma and other malignancies[7-9]. The prevalence
and clinical significance of thrombocytosis in adult patients
Abstract with HCC have not been previously reported.
Human thrombopoietin (TPO), also known as megakaryocyte
AIM: Hepatocellular carcinoma (HCC) patients manifest a growth factor, is known to play a key role in the development
variety of paraneoplastic syndromes. Thrombocytosis was of megakaryocytes [10,11] . TPO is secreted principally by
reported in children with hepatoblastoma. The aims of this hepatocytes and bone marrow stromal cells[10-14]. In addition,
study were to evaluate the prevalence and clinical significance the expression of TPO gene has been found in both rat and
of thrombocytosis in HCC patients and its relationships with human hepatoma cell lines [15,16]. The relationships between
serum thrombopoietin (TPO). serum TPO levels and platelet counts in HCC patients,
especially those associated with thrombocytosis, are of clinical
METHODS: We retrospectively reviewed clinical, biochemical interest. Our aim was to evaluate the prevalence of thrombocytosis
and image data of 1 154 HCC patients. In addition, we in Chinese patients with HCC in a retrospective study. The
measured platelet count and serum TPO in HCC patients clinical, biochemical and image characteristics of HCC patients
with and without thrombocytosis, in patients with cirrhosis, with thrombocytosis were evaluated. Moreover, in order to
chronic hepatitis and healthy subjects in a cross-sectional study. evaluate the role of serum TPO in the manifestations of
thrombocytosis in HCC patients, serum TPO levels were
RESULTS: Thirty-one (2.7%) of 1 154 HCC patients had measured in HCC patients (with and without thrombocytosis),
thrombocytosis (platelet count >/=400 K/mm 3 ). HCC cirrhotic patients, chronic hepatitis patients, and normal
patients with thrombocytosis were significantly younger, subjects in a cross-sectional study.
had a higher serum α-fetoprotein, higher rate of main portal
vein thrombosis, larger tumor volume, shorter survival,
and were less likely to receive therapy than HCC patients MATERIALS AND METHODS
without thrombocytosis. Multivariate logistic regression The clinical, laboratory and image data were retrospectively
analyses showed that tumor volumes >/=30% and serum reviewed in 1 269 consecutive patients diagnosed with HCC at
α-fetoprotein >/=140 000 ng/mL could significantly predict the Division of Gastroenterology, Taipei Veterans General
thrombocytosis. HCC patients with thrombocytosis had a Hospital, from January 1991 to December 1994[6]. Of these
significantly higher mean serum TPO than those without, patients, 1 253 were enrolled in this study who met the following
as well as patients with cirrhosis, chronic hepatitis and criteria: (1) histological proof of HCC; or (2) at least two typical
healthy subjects. Platelet count and serum TPO dropped HCC image findings (ultrasonography, computerized
significantly after tumor resection in HCC patients with tomography, celiac angiography or magnetic resonance
thrombocytosis and re-elevated after tumor recurred. imaging) along with a serum AFP of more than 20 ng/mL. Among
 Hwang SJ et al. Hepatocellular carcinoma and thrombocytosis 2473

the 1 253 patients, data of 1 197 cases were analyzed after for HCC were homogenized with a pestle. Total RNA was
excluding the patients with incomplete examination and data purified by TRIZOL reagent (Invitrogen and Life Technologies,
for analysis. Finally, data of 1 154 patients were selected for Rockville, MD, USA). Reverse transcription-polymerase chain
analyses in this study after excluding 43 patients with an reaction (RT-PCR) was carried out to amplify 1 µg RNA using
evidence of acute infections or gastrointestinal bleeding. the First-strain cDNA Synthesis Kit (Amersham Pharmacia
Patients with polycythemia vera were also excluded. Biotech, Buckinghamshire, UK) according to the instruction
We defined hypercholesterolemia in HCC patients as a serum manual. PCRs were carried out with 35 cycles at 94 °C for 1 min, at
cholesterol level greater than 250 mg/dL (two standard 60 °C for 1 min and at 72 °C for 1 min. Primer sequences were
deviations above the mean value of age-and-sex-matched sense: 5’-TGCGTTTCCTGATGCTTGTAG-3’ and anti-sense: ’-
healthy controls); hypoglycemia as plasma glucose less than AACCTTACCCTTCCTGAGACA-3’[12]. β-actin was used as
60 mg/dL; hypercalcemia as corrected serum calcium level more an internal standard and diethyl pyrocarbonate-treated water
than 11.0 mg/dL; and erythrocytosis as a hemoglobin level was used as a negative control. The analysis of PCR products
greater than 16.7 gm/dL or hematocrit greater than 50% as in was performed by 20 g/L agarose gel electrophoresis. For
our previous reports [2-6]. Thrombocytosis was defined as quantitation of TPO mRNA, the PCR product was purified by
having a platelet count greater than 400 K/mm3. the QIAquick Gel Extraction Kit (QIAGEN GmbH, Hilden,
To compare the serum TPO level, 18 consecutive HCC Germany) and A260 nm was measured to estimate the DNA
patients with thrombocytosis and 72 age-sex-tumor volume contents. Serial dilutions of this standard were then included
matched HCC patients without thrombocytosis were in the following real-time PCR along with samples to produce a
consecutively collected in a cross-sectional study from January standard curve. Quantitative PCR was performed in 20 µL reaction
1999 to December 2000. In addition, 42 age-sex-matched cirrhotic capillaries using 1×DNA master SYBR Green I mix (Roche
patients and 66 chronic hepatitis patients were randomly Diagnostics, Indianapolis, IN, USA), bovine serum albumin,
selected for comparison. The etiologies of chronic hepatitis 4 mmoL/L MgCl2, 200 µmoL/L dNTPs, 0.4 U InViTaq-polymerase
and cirrhosis were either viral hepatitis B or hepatitis C, which (InViTec; Berlin, Germany), 1 µL cDNA and 0.5 µmoL/L of the TPO-
were all confirmed by liver biopsies. None of the patients with specific sense and antisense primers. DNA amplification, data
chronic hepatitis or cirrhosis received interferon or other anti- collection and analyses were performed with LightCycler
viral treatments before blood sampling. Alcoholic patients were (Roche Diagnostics)[19]. The program was optimized and
not enrolled. Patients were also excluded if an acute infection performed finally as denaturation at 94 °C for 30 s followed by 40
or gastrointestinal bleeding was noted during enrollment. In cycles of amplification (at 94 °C for 0.1 s, 60 °C for 0.1 s, 72 °C
addition, 62 healthy subjects who received their annual physical for 20 s). The temperature ramp rate was 20 °C/s. At the end of
examinations at Taipei Veterans General Hospital, and whose each extension step, the fluorescence of each sample was measured
age and sex were matched with the aforementioned 90 HCC to allow the quantification of the PCR product. After the PCR
patients, were randomly selected as healthy controls. Sera of was completed, the melting curve of the product was measured
the aforementioned subjects or patients were stored in aliquots by a temperature gradient from 60 °C to 96 °C at 0.2 °C/s with
at -70 °C until analyzed. continuous fluorescence monitoring to produce a melting
The underlying liver cirrhosis in HCC patients was diagnosed profile of the primers. For normalization, the amount of TPO
histologically or by characteristic image findings with the amplicon was divided by the amount of β-actin amplicon of
presence of ascites or esophageal varices. Patients with the respective sample.
cirrhosis were given a score from 5-15 according to Child-Pugh’s All data are expressed as mean±SD. Results were compared
classification[17]. Tumor volume was calculated from computerized between groups using the Chi-square test, Fisher’s exact test,
tomographic films and was expressed as percentages of tumor Student’s t-test, Mann-Whitney test or cross tabulation
volume in total liver volume. The grade of differentiation and depending on the type of data analyzed. Survival adjusted for
arrangement of tumor cells were assessed by a liver pathologist therapy was analyzed using the Kaplan-Meier method and was
according to the classification of Edmondson and Steiner[18]. compared using the log rank method. Univariate and multivariate
The pathologist was not given any clinical information logistic regression using SPSS software (SPSS Inc., Chicago,
pertaining to the biopsy specimens. IL, USA) were performed to evaluate the predictive values of
All clinical data of HCC patients including age, sex, Child- the patients’ clinical, laboratory, and tumor features associated
Pugh’s scores, liver biochemistries (measured by a Hitachi with thrombocytosis. For all tests, only the results with P values
Model 736 automatic analyzer, Tokyo, Japan), prothrombin time, less than 0.05 (two-tail test) were considered to be statistically
and complete blood counts were recorded when an HCC was significant.
first diagnosed or at the time the thrombocytosis developed.
Serum α-fetoprotein (AFP) was measured by a commercial kit
(ELSA2-AFP, CIS bio-international, Cedex, France). Anti-HCV RESULTS
was measured by a second-generation enzyme immunoassay Among the 1 154 HCC patients (1 005 males, 149 females, mean age
kit (Abbott Laboratories, Chicago, IL, USA). The serum markers 62.0±12.0 years, with a range of 13 to 84 years), 735 (63.7%) patients
of hepatitis B surface antigen (HBsAg) and antibody to hepatitis were HBsAg positive, 180 (15.6%) were anti-HCV positive,
D virus (Abbott Laboratories) were recorded. Distant metastases 49 (4.3%) were positive for both HBsAg and anti-HCV and
to extrahepatic regional lymph nodes or other organs were 19 (1.6%) were positive for HBsAg and antibody to hepatitis D
evaluated by image studies. Methods of therapy for HCC, virus. The mean serum AFP level was 43 983±163 097 ng/mL
including surgical resection of tumors, transcatheter arterial (median 268 ng/mL, range 3-1 892 500 ng/mL).
chemoembolization (TACE), sono-guided percutaneous ethanol Totally, 243 (21.1%) of 1 154 patients had paraneoplastic
injection, or systemic chemotherapy and survival times were syndromes during the clinical course of HCC, in which 175 had
recorded in each patient. This study was approved by Taipei a single paraneoplastic manifestation and 68 had multiple
Veterans General Hospital, Taiwan. paraneoplastic manifestations. Thirty -one (2.7%) of 1 154 HCC
Serum TPO levels were quantitatively measured by a solid patients had thrombocytosis (mean platelet count 484±87 K/mm3,
phase enzyme immunoassay (Quantikine, R&D systems, range 403-688 K/mm3). The prevalence of hypercholesterolemia
Abingdon, UK). For the expression of TPO mRNA, an equal amount was 12.6%, hypoglycemia was 5.4%, hypercalcemia was 4.4%,
of fresh frozen tumors and non-tumor parts of the liver samples and erythrocytosis was 3.2%. Eighteen of 31 HCC patients
from a patient with thrombocytosis who received lobectomy with thrombocytosis had other paraneoplastic manifestations
 2474 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol September 1, 2004 Volume 10 Number 17

(11 with hypercholesterolemia, 1 with hypoglycemia, 4 with all selected as independent variables in logistic regression
hypercholesterolemia and hypoglycemia, 1 with hypercholes- analyses, with the presence of thrombocytosis as the dependent
terolemia and hypercalcemia, and 1 with hypercholesterolemia, variable. The continuous variables were transformed to
hypoglycemia and hypercalcemia). Nine -hundred and eleven categorical variables with the cut-off points determined by the
HCC patients were free of paraneoplastic manifestations. Receiver Operating Characteristic Curve. In an univariate
In comparison of the clinical, laboratory data and tumor analysis, an age <60 years, serum AFP >140 000 ng/mL, tumor
features between HCC patients with thrombocytosis and those volume >30% of the total liver volume, bilobar tumor involvement,
without, HCC patients with thrombocytosis were significantly and MPV tumor thrombosis were significantly correlated with
younger in age, had a higher mean serum AFP level, higher rate the presence of thrombocytosis (Table 2). In a stepwise
of main portal vein (MPV) thrombosis, bilobar tumor involvement, multivariate analysis, a tumor volume >30% (odds ratio: 9.901,
larger tumor volume, were less likely to receive or be suitable 95% confidence interval: 2.187-44.402, P = 0.003) and serum
for HCC therapy, and had a shorter survival time than those AFP >140 000 ng/mL (odds ratio: 2.660, 95% confidence interval:
without thrombocytosis (Table 1). HCC patients with 1.090-6.487, P = 0.031) were significant predictive variables
thrombocytosis tended to have more extra-hepatic metastases associated with the presence of thrombocytosis in HCC patients.
than those without thrombocytosis (32% vs 19%, P = 0.059). In the cross-sectional study, the mean platelet count was
There were no significant differences in sex distribution, viral 440±43 K/mm3 (range 401 to 540 K/mm3) in 18 HCC patients
etiologies, mean Child-Pugh’s scores, and rates of cirrhosis with thrombocytosis, 174±100 K/mm3 (range 102 to 396 K/mm3)
between the two groups. Two hundred and fifty-eight (22%) in 72 HCC patients without thrombocytosis, 72±29 K/mm3 (range
of 1 154 HCC patients and 10 (32%) of 31 patients with 18 to 154 K/mm3) in 41 patients with cirrhosis, 178±48 K/mm3
thrombocytosis had their HCC tissues for histological analyses. (range 105 to 300 K/mm3) in 66 patients with chronic hepatitis,
Among the 10 HCC patients with thrombocytosis, tumor cell and 212±35 K/mm3 (range 125 to 347 K/mm3) in 62 healthy
arrangement showed a trabecular pattern in 7 patients, and a controls. The mean serum TPO level was significantly higher in
mixed trabecular and acinar pattern in 3. Tumor cell HCC patients with thrombocytosis (404±196 pg/mL), when
differentiation revealed grade I in 3 patients, grade II in 4 patients, compared with HCC patients without thrombocytosis
and grade III in 3 patients. The tumor cell arrangement and (181±85 pg/mL), cirrhotic patients (103±34 pg/mL), chronic
differentiation showed no significant differences between hepatitis patients (113±46 pg/mL), and healthy subjects
patients with and without thrombocytosis. (123±53 pg/mL) (Figure 1). Serum TPO levels were positively
Age, sex, viral hepatitis markers, serum AFP, MPV thrombosis, correlated with platelet counts among 90 HCC patients (r = 0.474,
metastases, bilobar tumor involvement, and tumor volumes were P<0.001). Among the10 HCC patients with thrombocytosis, the

Table 1 Comparison of clinical and laboratory data, and tumor features between hepatocellular carcinoma (HCC) patients with
and without thrombocytosis

HCC patients with HCC patients without P value

thrombocytosis (n = 31) thrombocytosis (n = 1 123)

Age (yr) 52±19 62±12 <0.001

Sex (male: female) 25:6 980:143 0.201
HBV: HCV: HBV+HCV-related 24:1:0 711:179:49 0.114
Mean Child-Pugh’s scores 7.2±2.1 6.8±2.2 0.336
Mean platelet counts (k/mm3) 484±87 154±80 <0.001
Median (range) 441 (403-688) 133 (11-396)
Mean α-fetoprotein (ng/mL) 251 042±458 734 57 879±212 788 <0.001
Median (range) 4 859 (4-1 892 500) 252 (3-1 621 700)
Cirrhosis (+:-) 23:8 913:210 0.445
MPV tumor thrombosis (+:-) 11:20 171:952 ` 0.005
Tumor metastases (+:-) 10:21 196:927 0.059
Tumor volumes % 63.5±21.2 30.7±26.9 <0.001
Both lobes with tumor involvements (+:-) 22:9 439:684 <0.001
Therapy for HCC (+:-) 10:21 583:540 0.048
Mean survival (d) 144±208 373±521 <0.001
Median (range) 77 (4-852) 147(2-3 666)

Data were expressed as mean±SD. HBV: hepatitis B virus, HCV: hepatitis C virus, MPV: main portal vein.

Table 2 Significant predictive variables in association with thrombocytosis in hepatocellular carcinoma patients using univariate
logistic analyses

Variables Odds ratio 95% confidence interval P value

Age <60 yr 2.905 1.415-5.967 0.004

α-fetoprotein >140 000 ng/mL1 6.802 3.515-14.663 <0.001
Tumor volume >30%1 16.1 3.757-68.333 <0.001
Both lobes with tumor involvements 3.788 1.732-8.306 <0.001
Main portal vein tumor thrombosis 3.408 1.435-6.467 0.004

1
Significantly predictive variables in multivariate analyses.
 Hwang SJ et al. Hepatocellular carcinoma and thrombocytosis 2475

mean platelet count dropped significantly from 420±48 K/mm3 and AFP fell significantly after a surgical removal of the
to 278±38 K/mm3 (P<0.001) after a surgical tumor resection or tumors. However, all re-elevated when the tumor recurred
TACE, while the mean platelet count for HCC patients with 18 wk after surgical treatments.
thrombocytosis, who did not receive any therapy, showed a
progressive increase during follow up. B L T N
Figure 2 illustrates the relationship of platelet counts with
the serum levels of cholesterol, AFP and TPO in a 36-year-old
HCC male patient. The serum AFP level was 164 000 ng/mL and
serum HBsAg was positive. Image studies revealed a large
tumor mass over the right lobe of liver with an invasion to the 588 bp
left medial segment. Hypercholesterolemia (serum cholesterol
level 602 mg/dL) and thrombocytosis (platelet count 403 K/mm3)
were noted at the diagnosis of HCC. He received a surgical
removal of tumors followed by chemotherapy with 5-fluorouracil
for 12 wk. Platelet counts, serum levels of cholesterol and AFP 318 bp
dropped significantly after a surgical removal of the tumors.
However, all re-elevated when the tumor recurred 18 wk after Figure 3 Analyses of thrombopoietin from tumor tissues (T)
surgical treatment. Serum TPO level was 360 pg/mL before and non-tumor liver tissues (L) in a hepatocellular carcinoma
surgery (platelet count 403 K/mm3), and fell to 130 pg/mL one patient with thrombocytosis using reverse-transcription poly-
week after operation (platelet count 120 K/mm3). Figure 3 shows merase chain reaction. β-actin was used as an internal stan-
the qualitative expression of TPO mRNAs using RT-PCR in dard (B). N: negative control. Results from an agarose gel elec-
tumor and matched non-tumor tissues of the resected liver trophoresis showed a more intense density of thrombopoietin
samples. The expression of TPO mRNAs was more intensive in band from tumor tissues when compared with non-tumor
tumor than in the non-tumor liver tissues. The results from the liver tissues.
quantitative real-time PCR also revealed that the concentration
of TPO mRNAs in tumors was 1.92 times greater than in matched DISCUSSION
non-tumor liver tissues.
Common paraneoplastic syndromes seen in HCC patients
800
include hypercholesterolemia, hypoglycemia, hypercalcemia,
and erythrocytosis[1]. Thrombocytosis has been reported in
Serum TPO level (pg/mL)

600
children with hepatoblastoma[7-9]. The prevalence of thrombo-
cytosis in HCC patients has not been previously reported. Our
results showed that 2.7% of HCC patients hadthrombocytosis
400 which was defined as a platelet count >400 K/mm3. The
prevalence of thrombocytosis might be underestimated because
200
most HCC patients were associated with liver cirrhosis, and
thrombocytopenia was frequently seen in these patients.
0 The clinical significance of thrombocytosis in HCC patients
h sis ut tic is hy
w i t c y t o i t h o t o s i s irrhonts patit e a l t ols were similar to HCC patients with other paraneoplastic syndromes,
C o w y C tie he H ntr
H Cromb H C C mboc p a ic c o
th thr
o ron ts including hypercholesterolemia, hypoglycemia, hypercalcemia,
Ch tien
po and erythrocytosis[2-6]. Large tumor volumes, high serum AFP,
high rates of MPV tumor thrombosis, low rates of receiving
Figure 1 Distribution of serum thrombopoietin levels in hepa- therapy and poor prognosis have been identified in HCC
tocellular carcinoma (HCC) patients with and without patients with thrombocytosis. According to the multivariate
thrombocytosis, patients with cirrhosis, chronic hepatitis and logistic regression analyses, HCC patients with thrombocytosis
healthy subjects. The mean serum thrombopoietin level in were characterized by a large tumor volume and high serum
HCC patients with thrombocytosis was significantly higher
AFP. HCC patients with thrombocytosis seemed to have a
than in HCC patients without thrombocytosis, patients with
cirrhosis, chronic hepatitis and healthy subjects. similar life expectancy as HCC patients with erythrocytosis or
hypercholesterolemia, and had a better prognosis than patients
with hypoglycemia and hypercalcemia which were usually
Chemotherapy
pre-terminal events[6].
1 700 700 Before the cloning of TPO, the cause of thrombocytosis in
Cholesterol (mg/mL)

AFP
platelet (/mm3)×1 000

1 500 600 children with hepatoblastoma and in other malignancies remains

Platelet
AFP (mg/mL),

800 500 unclear. TPO, a glycoprotein hormone, is a potent stimulator of

Cholesterol 400 the growth and maturation of megakaryocytes and platelet
600
300 production[10,11]. The main sites of TPO production are the liver
400 200 and, to a lesser degree, the kidneys, bone marrow and spleen.
200 100 Messenger RNA transcripts of TPO have been found mainly in
0 0 the liver and released into circulation[14-16]. Most TPO is bound
6 8 10 13 0
18 3 26 with and degraded by circulating platelets and megakaryocytes
Time (wk) in the bone marrow, and the serum level is low. Circulating TPO
Trisegmentectomy
levels are inversely correlated with the number of TPO receptors
(c-Mpl-molecules) in regulating megakaryocytopoiesis and
Figure 2 Clinical course of a hepatocellular carcinoma pa- platelet production. When thrombocytopenia develops, binding
tient with thrombocytosis. The serum alfa-fetoprotein (AFP)
receptors decrease and serum TPO levels increase. Elevated
level was 164 000 ng/mL before tumor resection. Hypercho-
lesterolemia (serum cholesterol level: 602 mg/dL) and throm- TPO levels stimulate megakaryocytopoiesis and result in
bocytosis (platelet count: 403 K/mm3) were noted before increased platelet production[20-25]. This reverse relationship of
operations. The platelet counts, serum levels of cholesterol platelet counts and serum TPO levels has been found in patients
 2476 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol September 1, 2004 Volume 10 Number 17

with hematological diseases[26,27]. Patients with cirrhosis were 9 Komura E, Matsumura T, Kato T, Tahara T, Tsunoda Y,
frequently associated with low platelet counts. However, serum Sawada T. Thrombopoietin in patients with hepatoblastoma.
TPO levels in cirrhotic patients were found to be lower than Stem Cells 1998; 16: 329-333
10 de Sauvage FJ, Hass PE, Spencer SD, Malloy BE, Gurney AL,
chronic hepatitis patients or normal subjects due to inadequate
Spencer SA, Darbonne WC, Henzel WJ, Wong SC, Kuang WJ,
TPO production by the diseased livers[28-32]. Serum TPO of cirrhotic Oles KJ, Hultgren B Jr, Solberg LA, Goeddel DV, Eaton DL.
patients increased after orthotopic liver transplantation, which Stimulation of megakaryocytopoiesis and thrombopoiesis by
was followed by an increase in platelet counts[14,33-35]. Our results the c-Mpl ligand. Nature 1994; 369: 533-538
revealed serum TPO levels were significantly lower in cirrhotic 11 Lok S, Kaushansky K, Holly RD, Kuijper JL, Lofton-Day CE,
patients than in patients with chronic hepatitis or normal Oort PJ, Grant FJ, Helpel MD, Burkhead SK, Kramer JM, Bell
controls, and were consistent with previous reports. LA, Sprecher CA, Blumberg H, Johnson R, Prunkard D, Ching
Despite the fact that hepatoma cells have been found to AFT, Mathewes SL, Bailey MC, Forstrom JW, Buddle MM,
express TPO in vitro and in animal models, the mechanisms by Osborn SG, Evans SJ, Sheppard PO, Presnell SR, O’hara PJ,
Hagen FS, Roth GJ, Foster DC. Cloning and expression of murine
which thrombocytosis develops in HCC patients have not been
thrombopoietin cDNA and stimulation of platelet production
studied. According to our results, HCC patients with thrombocytosis in vivo. Nature 1994; 369: 565-568
had a significantly higher mean serum TPO level than HCC 12 Sungaran R, Markovic B, Chong BH. Localization and regula-
patients without thrombocytosis. In addition, the platelet tion of thrombopoietin mRNA expression in human kidney,
counts and serum TPO levels in HCC patients with thrombocytosis liver, bone marrow and spleen using in situ hybridization. Blood
dropped after a surgical removal of the tumor or TACE, and re- 1997; 89: 101-107
elevated when a tumor recurred. Changes of platelet counts 13 Nomura S, Ogami K, Kawamura K, Tsukamoto I, Kudo Y,
and serum TPO levels were parallel to the changes of serum Kanakura Y, Kitamura Y, Miyazaki H, Kato T. Cellular local-
AFP. Furthermore, by using the reverse transcription and real- ization of thrombopoietin mRNA in the liver by in situ
hybridization. Exp Hematol 1997; 25: 565-572
time PCR methods, we demonstrated that the expression of
14 Martin TG, Somberg KA, Meng YG, Cohen RL, Heid CA, de
TPO mRNA was more in tumor tissues than in matched non- Sauvage FJ, Shuman MA. Thrombopoietin levels in patients
tumor tissues in an HCC patient with thrombocytosis. Our with cirrhosis before and after orthotopic liver transplantation.
results were consistent with the report that thrombocytosis in Ann Intern Med 1997; 127: 285-288
patients with hepatoblastoma was related to increased TPO 15 Shimada Y, Kato T, Ogami K, Horie K, Kokubo A, Kudo Y,
production in tumors[9]. Thus, we speculate that thrombocytosis Maeda E, Sohma Y, Akahori H, Kawamura K, Miyazaki H.
in HCC patients is due to the overproduction of TPO by tumors, Production of thrombopoietin (TPO) by rat hepatocytes and
and mostly in patients with a large tumor burden. The hepatoma cell lines. Exp Hematol 1995; 23: 1388-1396
mechanisms of thrombocytosis in HCC patients are similar to 16 Hino M, Nishizawa Y, Tagawa S, Yamane T, Morii H, Tatsumi
N. Constitutive expression of the thrombopoietin gene in a
those for other paraneoplastic manifestations. Hypoglycemia
human hepatoma cell line. Biochem Biophys Res Commun 1995;
has been related to the overproduction of insulin-growth-factor 217: 475-481
II with insulin-like activities[2]. The cause of hypercalcemia has 17 Pugh RN, Murray-Lyon M, Dawson JL, Pietroni MC, Williams
been related to overproduction of a parathyroid-related protein R. Transection of esophagus for bleeding esophageal varices.
which interacts with parathyroid hormone receptors [4,36]. Br J Surg 1973; 60: 646-649
Elevation of serum erythropoietin has been seen in HCC patients 18 Okuda K, Kondo Y. Primary carcinomas of the liver. In:
with erythrocytosis[5,37]. Haubrich WS, Schaffner F, Berk JE, eds. Gastroenterology Vol-
In conclusion, thrombocytosis is one of the paraneoplastic ume 3. 5th ed. Philadelphia : W. B. Saunders 1995: 2462-2463
syndromes in patients with HCC, due to the overproduction of 19 Wittwer CT, Ririe KM, Andrew RV, David DA, Gundry RA,
Balis UJ. The LighCyclerTM a microvolume multisample fluo-
TPO by HCC. HCC patients with thrombocytosis are associated
rimeter with rapid temperature control. Biotechniques 1997; 22:
with a large tumor volume and high serum AFP level. 176-181
20 McCarty JM, Sprugel KH, Fox NE, Sabath DE, Kaushansky K.
Murine thrombopoietin mRNA levels are modulated by plate-
REFERENCES
let count. Blood 1995; 86: 3668-3675
1 Okuda K, Kondo Y. Primary carcinomas of the liver. In: Haubrich 21 Nichol JL, Hokom MM, Hornkohl A, Sheridan WP, Ohashi H,
WS, Schaffner F, Berk JE, eds. Gastroenterology Volume 3.5th Kato T, Li YS, Bartley TD, Choi E, Bogenberger J. Megakaryo-
ed. Philadelphia: W. B. Saunders 1995: 2467-2468 cyte growth and development factor. Analyses of in vitro effects
2 Wu JC, Daughaday WH, Lee SD, Hsiao TSY, Chou CK, Lin HD, on human megakaryopoiesis and endogenous serum levels
Tsai YT, Chiang BN. Radioimmunoassay of serum IGF-I and during chemotherapy-induced thrombocytopenia. J Clin Invest
IGF-II in patients with chronic liver diseases and hepatocellu- 1995; 95: 2973-2978
lar carcinoma with or without hypoglycemia. J Lab Clin Med 22 Cohen-Solal K, Villeval JL, Titeux M, Lok S, Vainchenker W,
1988; 112: 589-594 Wendling F. Constitutive expression of Mpl ligand transcripts
3 Hwang SJ, Lee SD, Chang CF, Wu JC, Tsay SH, Lui WY, Chiang during thrombocytopenia or thrombocytosis. Blood 1996; 88:
JH, Lo KJ. Hypercholesterolemia in patients with hepatocellu- 2578-2584
lar carcinoma. J Gastroenterol Hepatol 1992; 7: 491-496 23 Stoffel R, Wiestner A, Skoda RC. Thrombopoietin in thromb-
4 Yen TC, Hwang SJ, Wang CC, Lee SD, Yeh SH. Hypercalcemia ocytopenic mice: evidence against regulation at the mRNA
and parathyroid hormone-related protein in hepatocellular level and for a direct regulatory role of platelets. Blood 1996;
carcinoma. Liver 1993; 13: 311-315 87: 567-573
5 Hwang SJ, Lee SD, Wu JC, Chang CF, Lu CL, Tsay SH, Lo KJ. 24 Emmons RV, Reid DM, Cohen RL, Meng G,Young NS, Dunbar
Clinical evaluation of erythrocytosis in patients with hepato- CE, Shulman NR. Human thrombopoietin levels are high when
cellular carcinoma. Chin Med J 1994; 53: 262-269 thrombocytopenia is due to increased platelet destruction. Blood
6 Luo JC, Hwang SJ, Li CP, Hsiao LT, Lai CR, Chiang JH, Lui WY, 1996; 87: 4068-4071
Chang FY, Lee SD. Paraneoplastic syndromes in patients with 25 Eaton DL, de Sauvage FJ. Thrombopoietin: the primary regu-
hepatocellular carcinoma in Taiwan. Cancer 1999; 86: 799-804 lator of megakaryocytopoiesis and thrombopoiesis. Exp
7 Nickerson HJ, Silberman TL, McDonald TP. Hepatoblastoma, Hematol 1997; 25: 1-7
thrombocytosis, and increased thrombopoietin. Cancer 1980; 26 Tahara T, Usuki K, Sato H, Ohashi H, Morita H, Tsumura H,
45: 315-317 Matsumoto A, Miyazaki H, Urabe A, Kato A. A sensitive
8 Shafford EA, Ritchard JP. Extreme thrombocytosis as a di- sandwich ELISA for measuring thrombopoietin in human
agnostic clue to hepatoblastoma. Arch Dis Child 1993; 69: serum: serum thrombopoietin levels in healthy volunteers and
171-174 in patients with haemopoietic disorders. Br J Haematology 1996;
 Hwang SJ et al. Hepatocellular carcinoma and thrombocytosis 2477

93: 783-788 levels in patients with chronic hepatitis and liver cirrhosis,
27 Tomita N, Motomura S, Sakai R, Fujimaki K, Tanabe J, Fukawa and its relationship with circulating thrombocyte counts.
H, Harano H, Kanamori H, Ogawa K, Mohri H, Marata A, Hepatogastroenterology 2002; 49: 1645-1648
Kodama F, Ishigatsubo Y, Tahara T, Kato T. Strong inverse 33 Goulis J, Chau TN, Jordan S, Mehta AB, Watkinson A, Rolles
correlation between serum TPO level and platelet count in K, Burroughs AK. Thrombopoietin concentrations are low in
essential thrombocythemia. Am J Hematol 2000; 63: 131-135 patients with cirrhosis and thrombocytopenia and are restored
28 Peck-Radosavljevic M, Zacherl J, Meng YG, Pidlich J, Lipinski after orthotopic liver transplantation. Gut 1999; 44: 754-758
E, Langle F, Steininger R, Muhlbacher F, Gang A. Is inadequate 34 Faeh M, Hauser SP, Nydegger UE. Transient thrombopoietin
thrombopoietin production a major cause of thrombocytope- peak after liver transplantation for end stage liver disease. Br J
nia in cirrhosis of the liver? J Hepatol 1997; 27: 127-131 Haematol 2001; 112: 493-498
29 Sezai S, Kamisaka K, Ikegami F, Usuki M, Urabe A, Tahara T, 35 Tsukahara A, Sato Y, Yamamoto S, Szuki S, Nakatsuka H,
Kato T, Miyazaki H. Regulation of hepatic thrombopoietin Watanabe T, Kameyama H, Hatakeyama K. Thrombopoietin
production by portal hemodynamics in liver cirrhosis. Am J levels and peripheral platelet counts following living related
Gastroenterol 1998; 93: 80-82 donor liver transplantation. Hepatogastroenterology 2003; 50:
30 Wolber EM, Ganschow R, Burdelski M, Jelkmann W. Hepatic 227-230
thrombopoietin mRNA levels in acute and chronic liver failure 36 Suva LJ, Winslow GA, Weltenhall RE, Hammonds RG, Moseley
of childhood. Hepatology 1999; 29: 1739-1742 JM, Difenbach-Jagger H, Rodda CP, Kemp BE, Rodriguez H,
31 Giannini E, Borro P, Botta F, Fumagalli A, Malfatti F, Podesta Chen EY. A parathyroid hormone-related protein implicated in
E, Romagnoli P, Testa E, Chiarbonello B, Polegato S, Mamone malignant hypercalcemia: cloning and expression. Science 1987;
M, Testa R. Serum thrombopoietin levels are linked to liver 237: 893-896
function in untreated patients with hepatitis C virus related 37 Kew MC, Fisher JW. Serum erythropoietin concentrations in
chronic hepatitis. J Hepatol 2002; 37: 572-577 patients with hepatocellular carcinoma. Cancer 1986; 58:
32 Koruk M, Onuk MD, Akcay F, Savas MC. Serum thrombopoietin 2485-2488

Edited by Zhu LH and Xu FM