Hey-Chi Hsu

Growth rate of asymptomatic hepatocellular carcinoma and its clinical implications.

The growth rate of 31 asymptomatic hepatocellular carcinomas (diameter less than or equal to 5 cm) discovered in 28 patients by a prospective screening program was determined by real-time ultrasonography over 36-860 days. Except for one tumor that shrank on follow-up, the doubling time ranged from 29 to 398 days, with a median of 117 days, an arithmetic mean of 136 days, and a geometric mean of 110 days. In 17 tumors with more than two measurements, the growth rate remained exponential in nine, declined in growth in seven, and showed an initial lag period in one. Doubling time correlated with initial tumor diameter but was independent of the patients age, sex, hepatitis B surface antigen status, tumor location, liver function tests, stage of liver cirrhosis, histologic type, or grade of malignancy. Although initial alpha-fetoprotein levels did not correlate well with growth rate, in 14 patients with an exponential increase of serum alpha-fetoprotein, the alpha-fetoprotein doubling time was closely related to the tumor doubling time. Based on the above data, the median detectable subclinical period of hepatocellular carcinoma was deduced to be 3.2 yr, and the suitable screening interval for its early detection in our area was 4-5 mo.

ELASTIC MODULUS MEASUREMENTS OF HUMAN LIVER AND CORRELATION WITH PATHOLOGY

Viral hepatitis causes fibrosis in the liver and may change mechanical properties of the liver. To evaluate the impact of fibrosis on elastic properties of human liver and to investigate potential benefits of ultrasonic elasticity imaging, 19 fresh human liver samples and 1 hepatic tumor (focal nodular hyperplasia) sample obtained during operations were studied. Simple 1-D estimates based on the cyclic compression-relaxation method were performed. Elastic modulus values were derived from the predetermined strain (controlled by a step motor system) and the stress values (measured by an electronic balance). Each specimen subsequently received histologic examination and a grade of liver fibrosis was scored from 0 to 5. Results show that the elastic modulus values were on the order of several hundreds to thousands of Pascals. The elastic modulus generally increased with the fibrosis grade, although some discrepancies existed at the middle grades of fibrosis (scores 1 to 3). The correlation between the fibrosis score and the elastic modulus was significant (p < 0.01) based on the statistical analysis using the Pearson correlation method. In addition, the relation between the elastic modulus and the fibrosis grade generally exhibited a quadratic trend. It was concluded that severity of fibrosis had a good correlation with stiffness of the liver. Results also indicated that the elasticity imaging of the liver may provide significant clinical values if the elastic modulus can be accurately measured.

β-Catenin Mutations Are Associated with a Subset of Low-Stage Hepatocellular Carcinoma Negative for Hepatitis B Virus and with Favorable Prognosis

To better understand the role of beta-catenin mutation in hepatocellular carcinoma (HCC), we correlated the gene mutation with hepatitis virus B (HBV) and hepatitis virus C (HCV) status and the clinicopathological features in 366 patients with resected primary unifocal HCC. beta-Catenin mutations were also analyzed in 55 patients with multifocal HCC (68 tumors). Of the whole series, 57 (13.1%) of 434 tumors examined had beta-catenin mutations, 34 occurred at the serine/threonine residues of the GSK-3beta region of beta-catenin. Outside the GSK-3beta phosphorylation site, codons 32 and 34 were two mutational hot spots (17 tumors). The non-HBV-related HCC that was predominantly HCV related had a higher frequency of mutation (P: < 0.00001) and more frequent mutations at codon 45 than HBV-related HCC. HBV-related HCC had a younger mean age (P: < 0.00001), and higher male-to-female ratio (P: < 0.003) and positive familial history of HCC (P: < 0.014). Among 366 unifocal HCCs selected for clinicopathological analysis, beta-catenin mutations were associated with grade I (P: = 0.005) and stage I and II HCC (P: < 0.0001), and a better 5-year survival rate (P: = 0. 00003). These findings suggest mechanisms for beta-catenin mutations differ between HBV-related and non-HBV-related HCCs, and that beta-catenin mutation is a favorable prognostic factor related to low stage. beta-Catenin mutation was associated with nuclear expression of the protein (P: < 0.00001), but we failed to detect point or large fragment deletion mutation in 39 HCCs with nuclear beta-catenin expression, presumably wild-type protein. HCCs expressing mutant nuclear beta-catenin had a better 5-year survival rate (P: < 0.007), suggesting that mutant and wild-type nuclear beta-catenin proteins are not functionally equivalent and deserve more studies for further clarification.

Overexpression and Amplification of Aurora-A in Hepatocellular Carcinoma

Purpose: Aurora-A/STK15/BTAK, a centrosome-associated serine/threonine kinase, has been shown to induce chromosomal instability, leading to aneuploidy and cell transformation. The purpose of this study was to investigate the expression and amplification of Aurora-A in hepatocellular carcinoma (HCC). Experimental Design: Aurora-A mRNA levels were measured in 224 HCCs and 199 paired nontumorous liver tissues by reverse transcription-PCR. Aurora-A mRNA and protein levels of 8 were also measured by reverse transcription-PCR and Western blot hybridization in 8 liver cancer cell lines. Amplification of Aurora-A was determined by Southern blot hybridization in 99 cases. Results: Aurora-A was overexpressed in 137 of 224 (61%) HCCs and all 8 of the cell lines. Overexpression of Aurora-A was associated with high-grade (grade II-IV), and high-stage (stage IIIB-IV) tumors, p53 mutation, infrequent β-catenin mutation, and poor outcome. Aurora-A overexpression and p53 mutation acted synergistically toward poor prognosis. Amplification of Aurora-A was detected only in 3 HCCs. Conclusion: The results show that Aurora-A is overexpressed frequently in HCC, and correlated with high grade and high stage, indicating that overexpression of Aurora-A plays a role in the development and progression of HCC.

High α‐fetoprotein level correlates with high stage, early recurrence and poor prognosis of hepatocellular carcinoma: Significance of hepatitis virus infection, age, p53 and β‐catenin mutations

α‐Fetoprotein (AFP) is often elevated in hepatocellular carcinoma (HCC). This study was to elucidate the significance and related factors of AFP elevation in HCC in 781 unifocal HCCs receiving curative hepatectomy. We showed that high AFP (> 200 ng/ml), which was associated with AFP mRNA expression in HCC (p = 0.00001), correlated with major clinicopathologic factors. Younger age (≤ 55 years; p = 0.00001), hepatitis B surface antigen (HBsAg) in serum (p = 0.00001), p53 mutation (p = 0.008), large tumor (p = 0.00001), vascular invasion (p = 0.00001) and early tumor recurrence (p = 0.00001) were significant associates of high AFP, while anti‐HCV in serum and β‐catenin mutation in HCC had less frequent high AFP (p = 0.013 and < 0.0001, respectively). We also showed that HCC with high AFP had a lower 10‐year survival (p < 0.0001), particularly in large HCC (p < 0.0001). At univariate analysis, high AFP (p < 0.0001), HBsAg positivity (p = 0.05), p53 mutation (p = 0.0004), liver cirrhosis (p = 0.0094), large tumor (p = 0.0003), vascular invasion (p < 0.0001) and early recurrence (p < 0.0001) were significant unfavorable prognostic factors. In Cox proportional hazards regression analysis, high AFP remained a borderline significance (OR = 1.2; CI = 1.0–1.4) after adjustment for the effect of tumor size and tumor stage (p = 0.0821). Furthermore, the detection of AFP mRNA in the liver of AFP mRNA‐positive HCC was associated with more frequent early recurrence (p = 0.0026) and might be a useful marker of intrahepatic spread. We therefore conclude that AFP elevation, more than a coincidental epiphenomenon, appears to contribute to vascular invasion and HCC progression and help to identify subsets of HCC patients with increased risk for early recurrence and poor prognosis after hepatectomy.

Overexpression of osteopontin is associated with intrahepatic metastasis, early recurrence, and poorer prognosis of surgically resected hepatocellular carcinoma

Intrahepatic metastasis via portal vein spread is an important feature and a crucial unfavorable prognostic factor of hepatocellular carcinoma (HCC). To identify the molecular factors for tumor progression, the authors used differential display (DD) to analyze aberrant gene expression in HCC. The goal of the current study was to elucidate the clinicopathologic and prognostic significance of osteopontin (OPN) in HCC progression.

Early detection of hepatocellular carcinoma by real-time ultrasonography. A prospective study

For early detection of hepatocellular carcinoma (HCC), real‐time ultrasonography (US) was performed prospectively in 528 patients, including 236 with cirrhosis, 81 with chronic hepatitis, 168 asymptomatic hepatitis B surface antigen carriers, and 43 with a family history of HCC. Simultaneous measurement of serum alpha‐fetoprotein (AFP) level was also done. In addition, 233 patients had regular controls at 3‐ to 6‐month intervals, with an average follow‐up period of 1.4 years. On initial screening, a total of 17 patients were found to have HCC: 13 in the cirrhotic group, 3 in the HCC family group, and 1 in the asymptomatic carriers. Of these HCCs, 7 were smaller than 3 cm, 6 were between 3 to 5 cm, and 4 were larger than 5 cm. In patients with tumors smaller than 5 cm, the AFP levels were normal in 46.2%, between 20 to 400 ng/ml in another 46.2%, and only 7.6% were over 400 ng/ml. On follow‐up, another seven patients, all in the cirrhotic group, were found to have HCCs varying from 1.6 to 4.7 cm; three of them had normal serum AFP level. The authors conclude that real‐time US is more sensitive than AFP assay in early detection of HCC, and the high‐risk subjects should receive this procedure at regular intervals.

Expression of the c-kit protein is associated with certain subtypes of salivary gland carcinoma

The c-Kit protein, a receptor type tyrosine kinase that plays an important role in the development of hematopoietic cells, melanocytes, and germ cells, is expressed in mastocytosis, gastrointestinal stromal cell tumors (GISTs), germ cell tumors, and several other tumors. Gain-of-function mutations in exon 11 and exon 17 have been shown as a mechanism of c-kit activation in some tumors. To study the role of c-kit in salivary gland carcinomas, we analyzed the c-kit protein expression in 79 carcinomas of major and minor salivary glands by immunohistochemistry. Although varying in intensity of staining, c-kit expression was identified very often in adenoid cystic carcinomas (20/25), lymphoepithelioma-like carcinomas (6/6) and myoepithelial carcinomas (2/2), but not in other types of salivary gland carcinoma (0/46), P<0.00001. By DNA sequencing, genetic alteration of c-kit juxtamembrane domain (exon 11) and tyrosine kinase domain (exon 17) was not found in all the three types of salivary carcinoma that had c-kit protein expression. In conclusion, c-kit protein overexpression is involved in the pathogenesis of certain types of salivary gland carcinoma, but mutation of the gene is not the mechanism of c-kit activation.

Serum α-Fetoprotein in the Early Stage of Human Hepatocellular Carcinoma

The serum alpha-fetoprotein levels of 17 patients with cancers less than or equal to 3 cm in size were studied using radioimmunoassay to determine alpha-fetoprotein response in the early stage of human hepatocellular carcinoma. The levels were normal in 6 patients (35%), and elevated to 645 +/- 1140 micrograms/L (mean +/- SD) in the remaining patients. The levels were not correlated with tumor size. In 10 surgically resected patients, 5 had elevated levels of alpha-fetoprotein that returned to normal after surgery. The levels and tumor sizes were serially observed for 3-26 mo in the remaining 7 patients not surgically treated. In 1 patient alpha-fetoprotein levels were persistently normal and in the other 6 patients, the levels tended to increase with a median doubling time of 60-75 days (range 30-223 days). Despite continued tumor growth, a spontaneous fall in serum alpha-fetoprotein levels was encountered in 4 patients; in 2 patients the levels even fell within the normal range. After this fall, the levels increased drastically in 4 patients. We concluded that in the early stage of hepatocellular carcinoma, serum alpha-fetoprotein level is frequently normal, and thus determination of serum alpha-fetoprotein levels only is not a reliable indicator in the early detection of human hepatocellular carcinoma. A spontaneous fall in the level of alpha-fetoprotein is not an uncommon finding in the early stages of this cancer and cannot be used to rule out the diagnosis of hepatocellular carcinoma.

Prognostic histologic features of resected small hepatocellular carcinoma (HCC) in Taiwan: a comparison with resected large HCC

The morphologic features and their prognostic significance were analyzed in 83 surgically resected hepatocellular carcinomas (HCC): 44 of them were of ⩽5 cm in diameter and 39 were larger. This study demonstrated a high prevalence of tumor capsule in both small and large HCCs, 86.4% and 84.6%, respectively. In small HCC, the capsule formation was significantly higher in the liver with cirrhosis (96.9%, or 31/32) than without (58.3%, or 7/12) (P < 0.003). In both small and large HCCs, the most important histologic parameter influencing the tumor recurrence was the liver invasion. In the small but not in the large HCC, the invasion through the capsule also correlated well with recurrence (P < 0.05). None of the 19 patients whose HCC were confined by a tumor capsule and did not invade the liver, had a recurrence during the 10 months or longer follow‐up period. The significantly more favorable outcome of smaller HCC was related to the lower frequencies of liver invasion (P < 0.001), portal vein involvement (P < 0.01), and satellite formation (P < 0.01). Tumor recurrence did not correlate with the tumor grading, presence of clear cells, liver cell dysplasia, or host inflammatory response. The HBsAg positivity in tumor cells was significantly higher in small (40.9%, or 18/44) than in large HCC (10.3%, or 4/39), suggestive of a gradual loss of the cytoplasmic expression of HBsAg in the tumor cells during the growth of HCC.