Novel formula for cell kinetics in xenograft model of hepatocellular carcinoma using histologically calculable parameters.

The growth rate of tumors should be assessed in terms of both tumor cell proliferation and death. The former is considered to be determined by growth fraction and cell-cycle time, whereas the latter is mainly determined by apoptosis, especially in tumors with a low level of necrosis. While most hepatocellular carcinomas (HCCs) in a relatively early stage contain only a small amount of necrosis, the growth rate supposedly depends mainly on growth fraction, cell-cycle time, and apoptosis. However, their quantitative relationship remains unknown. We have derived a novel theoretical formula for determining this relationship in nonnecrotic HCC, using Ki-67-positive index, apoptotic score, and a correction factor, all calculable by histological assessment without injecting labeling agents. Furthermore, we confirmed the reliability of this formula, using a xenograft model of human HCC with less than 15% necrosis. In this model the values of cell-cycle time calculated from the formula were very close to those estimated by a conventional double-labeling method and showed high correlations. Since our novel formula can clarify the cell kinetics without cumbersome labeling procedures, it is expected to be clinically applicable to HCC with a small portion of necrosis, using the radiographically measured growth rate and the histologically assessed cell kinetic parameters.

Activated hepatic stellate cells participate in liver fibrosis in a patient with transfusional iron overload.

We describe liver fibrosis caused by iron overload after a long history of blood transfusion in a patient with chronic renal failure. Pertinent laboratory data were: serum (s)-Fe 148 microg/dl; unsaturated iron binding capacity (UIBC) 14 microg/dl; s-ferritin 9350 ng/ml; human leukocyte antigen (HLA) A2, A24, B39, B55, Cw1, Cw7. Computed tomography revealed a high density in the liver, and laparoscopy revealed a brown liver. Liver histology showed bridging fibrosis from portal tracts. A heavy iron deposit was seen in Kupffer cells as well as in hepatocytes surrounded by fibrosis around the portal tracts. Immunocytochemistry revealed alpha-smooth muscle actin in many stellate cells distributed along the fibrotic area, and electron microscopy revealed infiltrating myofibroblastic stellate cells coexisting with collagen fibers around degenerated hepatocytes containing iron deposits. The findings are consistent with the notion that stellate cells play an important role in liver fibrogenesis in both genetic and transfusional iron overload hemochromatosis.

Angiogenesis in hepatocellular carcinoma as evaluated by CD34 immunohistochemistry.

To clarify the relationship between angiogenesis and hepatocarcinogenesis on progression of hepatocellular carcinoma (HCC), we quantitatively evaluated angiogenesis by CD34 immunohistochemistry in liver cirrhosis (LC), adenomatous hyperplasia (AH), and HCC, and proliferative activity estimated by Ki-67 immunohistochemistry. Angiogenesis was evaluated by CD34 immunohistochemistry using monoclonal antibody HPCA-2, and tumor proliferative activity was evaluated using monoclonal antibody MIB-1. We used an image analysis system to assess the microvessel density as the area percentage of the endothelial area. Angiogenesis was generally observed in HCC and there was no significant difference among all clinical stages and histological grades of HCC. On the other hand, the staining of CD34 was partly observed in sinusoids of AH, although no positive staining was seen in any sinusoids of LC. The proliferative activity was significantly correlated with the clinical stage and histological grade of HCC. Our results indicate that the quantitation of angiogenesis does not provide significant prognostic information in HCC, but that it may have diagnostic value in distinguishing HCC from non-HCC. Meanwhile, AH, which is not morphologically diagnosed as cancer, shows positive staining for CD34, suggesting that some portion of AH contains cancerous characteristics.

Cytogenetic analyses of hepatocellular carcinoma by in situ hybridization with a chromosome-specific DNA probe.

BACKGROUND: Numerical chromosome analysis has been established in solid tumors by using in situ hybridization (ISH) with a chromosome-specific probe. We analyzed human hepatocellular carcinoma (HCC) by ISH for chromosome 17 and investigated the correlation of its copy number with histologic malignancy, proliferative activity, p53 mutation, and DNA ploidy. METHODS: Chromosome 17 was hybridized with a pericentromere-specific DNA probe directly on the tumor cells isolated from paraffin blocks of 25 surgically resected HCCs. Proliferative activity was measured by Ki-67 immunohistochemistry, p53 mutation was analyzed by p53 immunohistochemistry, and DNA ploidy was estimated by cytofluorometry. RESULTS: Forty-four percent of the 25 HCCs showed numerical abnormality of chromosome 17. Many disomic cases had a less malignant histology, whereas many polysomic cases had a more malignant histology. The Ki-67 positive index of polysomic cases was higher than that of disomic cases. In 22 cases (88.0%), the copy number of chromosome 17 was well matched with DNA ploidy. However, the numerical abnormality of chromosome 17 did not show a significant correlation with p53 mutation. Two of four HCCs that showed histologic heterogeneity were also heterogenous on ploidy pattern and the copy number of chromosome 17. Conversely, there was one case in which only ISH could demonstrate heterogeneity, although the other features exhibited homogeneity. CONCLUSIONS: Numerical chromosome abnormalities correlated with the increase of histologic malignancy proliferative activity, and DNA ploidy. Moreover, ISH analysis was useful in assessing the intratumoral heterogeneity in HCC, especially when current methods failed to detect it. Thus, ISH provides information on important biologic features, such as malignant potential and intratumoral heterogeneity, in HCC.

Identification of apoptotic hepatocytes in situ in rat liver after lead nitrate administration.

Apoptosis plays a major role in the regression of mitogen (lead nitrate)-induced hepatic hyperplasia. We compared the in situ end-labeling (ISEL) technique with the conventional detection of apoptotic bodies in this process. In hematoxylin and eosin (H&E) sections, apoptosis is usually recognizable by the presence of apoptotic bodies (apoptosis phase 2). Although the early phase of apoptosis (apoptosis phase 1) can be detected as a prekaryorrhectic appearance in H&E sections, it is difficult to detect and is easily overlooked. On the other hand, ISEL presents intense staining mainly in phase 1 and weak or negative staining in phase 2. Thus, simultaneous investigation by these two methods in two serial sections is the most reliable way to calculate the incidence of apoptosis and gives us precise information on the stages of apoptosis in situ. Since the colorized signals of ISEL are much easier to detect than apoptotic bodies in H&E sections, ISEL is particularly useful for liver tissues, where the incidence of apoptosis is low.

Evaluation of hepatic proliferative activity in chronic liver diseases and hepatocellular carcinomas by proliferating cell nuclear antigen (PCNA) immunohistochemical staining of methanol-fixed tissues.

The proliferative activity of chronic liver diseases and hepatocellular carcinomas (HCCs) was studied by PCNA immunohistochemistry. Human liver tissues were obtained by surgical operation or needle biopsy, and PCNA was detected by immunohistochemistry. PCNA-labelling indices (PCNA-LIs) of methanol-fixed tissues corresponded with the incidence of S-phase cells previously reported, whereas paraformaldehyde-fixed tissues showed extremely high PCNA-LIs in all specimens. Therefore, methanol-fixed tissues were used for evaluation. The PCNA-LIs of the methanol-fixed tissues were: normal liver 0.78 +/- 0.38%, chronic persistent hepatitis 1.06 +/- 0.86%, chronic aggressive hepatitis 2A 1.01 +/- 0.50%, chronic aggressive hepatitis 2B 4.20 +/- 1.79%, inactive cirrhosis 0.81 +/- 0.49%, active cirrhosis 1.96 +/- 0.93%, HCC of Edmondson's type I 4.83 +/- 1.98%, type II 6.65 +/- 1.69%, and type III 38.7 +/- 30.6%. PCNA-positive cells showed little specific distribution; in periportal areas in chronic hepatitis, at the margins of pseudolobules in cirrhosis, and throughout the tumor in HCC. These findings indicated that proliferative activity increased during the progression of chronic hepatitis, but that it decreased at the stage of cirrhosis. In chronic liver diseases, the PCNA-LIs reflected hepatitis activity. HCC showed higher proliferative activity than liver cirrhosis, and the histological grade was correlated with the PCNA-LI.

[Disease-free survival in a patient with squamous cell lung carcinoma following complete response with the combination chemotherapy of cisplatin and etoposide].

Disease-free survival is reported in a patient with non-small cell lung carcinoma successfully treated with the combination chemotherapy of cisplatin and etoposide (CDDP-ETOP). An 80-year-old woman was diagnosed as having squamous cell carcinoma of the lung with bone metastasis. Chest X-ray showed a tumor (64 x 68 mm in size) in the S1/4 segment of the right lobe, and CT scan revealed no apparent metastasis of hilar lymphnode. In March 1989, the patient achieved a complete response (CR) after two courses of CDDP-ETOP therapy, followed by ETOP(100mg) every two weeks and daily UFT (400 mg) for 10 months. Subsequently, a single course of CDDP-ETOP was administered as a consolidation therapy. The patient is alive with no evidence of recurrent disease for 3 years and half after achieving CR.