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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.

Background Aspiration biopsy cytology of thyroid tumors has been used more frequently in recent times to differentiate between malignant and benign lesions. Chromatin patterns of the tumor cell nuclei are one of most important factors for cytologic diagnosis. The interpretation of nuclear chromatin patterns is subjective and more difficult than that of nuclear size or shape. In the present report, we investigated how to detect underlying chromatin characteristics of benign and malignant thyroid tumor cells by means of texture and factor analyses. Methods We employed a computer-aided system in which light microscopy was combined with an image processor and monochrome camera. Using this system, 100 randomly selected cells in a Papanicolaou stained, aspiration biopsy cytologic smear in each case of 39 benign and malignant thyroid tumor cases were digitized. We applied two-dimensional and higher texture analyses with the use of co-occurrence and run-length matrices to analyze the chromatin patterns. Factor analysis was used to determine whether a large number of independent variables actually measured one or more underlying common variables. Results According to parameters with high factor-loading values, the morphologic chromatin characters were classified into three categories according to heterogeneity, contrast, and homogeneity of chromatin patterns. On the basis of analyses with these morphologic categories, nuclei of papillary carcinoma showed higher contrast of chromatin patterns than did those of the benign group. Moreover, there was a variety of contrasting chromatin patterns among cells in each papillary carcinoma case in comparison with the benign group. In contrast, follicular carcinomas showed a significant difference in the standard deviation of factor 3, which indicated more monotonous chromatin patterns among cells in each follicular carcinoma case than in each benign case. Conclusion We believe that this technique, using texture and factor analyses, is useful in the detection of underlying characteristics of nuclear chromatin patterns in aspiration biopsy cytology. Cytometry 49:91–95, 2002. © 2002 Wiley-Liss, Inc.

The study was conducted to clarify the cytocidal effect of combination therapy consisting of administration of acridine orange (AO), which is a photosensitizer, and radiation therapy using in vitro and in vivo mouse osteosarcoma models. The results revealed that AO combined with low-dose X-ray irradiation of about 1–5 Gy had a strong cytocidal effect on the cultured mouse osteosarcoma cells regardless of their chemosensitivity, and that this combination therapy inhibited growth of the in vivo mouse osteosarcoma by induction of tumor necrosis. This effect was inhibited by L-histidine, but not by mannitol. These findings suggested that AO might be excited by X-rays and kill osteosarcoma cells through the release of singlet oxygen, which is toxic to living cells. This mechanism is similar to that of photodynamic therapy with AO.

Thirty-four untreated hepatocellular carcinomas (HCCs) with known growth rates were classified into 5 groups on a tumor profile chart based on their doubling time (DT), Ki-67-positive index (Ki-67-PI), apoptotic index (Apo-I), and histologic grade. The slow-growing HCCs (DT > 100 days) consisted of well-differentiated tumors with slight cell kinetic imbalance and were divided into groups A and B. Group A had Apo-I values

Objective Since most chondrocytes in articular cartilage are in the resting phase (G0) of the cell cycle, it has been difficult to investigate their cell kinetics using 3H-thymidine autoradiography, or immunohistochemistry. In the present study, DNA cytofluorometry, which is useful to analyse the cell kinetics even for such inactive cell populations as in the G0 phase, was applied to human chondrocytes of the articular cartilages under normal aging and pathologic conditions such as osteoarthritis (OA), rheumatoid arthritis (RA), and aseptic necrosis (AN). Design The human articular cartilages for the study were obtained from autopsy and surgical materials. Fifty joints were used for the study of aging, 54 for the study of OA, 20 for studying RA, and 10 for AN study. The isolated chondrocytes were quickly prepared from fresh articular cartilages, using a combination method of enzymatic digestion with papain and collagenase, followed by mechanical cell separation by churning and homogenization. Results The DNA histograms obtained by cytofluorometry with propidium-iodide staining showed that most chondrocytes had diploid DNA content (2c) in all cartilages studied, suggesting that they were in the G0 phase. However, there were a few chondrocytes having tetraploid DNA content (4c) in the normally aged articular cartilages, and there were some cells having DNA content between 2c and 4c in the diseased cartilages. The former cells were considered to be G0-phase cells of the 4c chondrocytes, while the latter cells were considered to be in the DNA synthetic (S) phase or G2-phase of the 2c chondrocytes. The frequency of 4c chondrocytes in aged cartilage was significantly increased, compared to that in the young cartilage. In contrast to the normal cartilage, the frequency of S- and G2-phase cells, which was expressed as the S– G2 index, in diseased cartilages (OA, RA and AN) was significantly high ( P 0.0001). In OA cartilage, the S–G2 index was much higher in the severe or moderate stage than in the mild stage, suggesting that the chondrocytes in clusters may actively proliferate. Conclusion These results showed that in normal articular cartilages most chondrocytes are in the G0 phase, while some became 4c polyploid cells, and that these G0-phase chondrocytes had a potential to proliferate under diseased conditions.

A long-term survival case of multiple hepatocellular carcinoma (HCC) with metachronous metastasis to a lymph node is reported. The patient, a 66-year-old woman, had two primary HCC nodules, one each in the left and right hepatic lobes, which were resected. She developed a lymph node lesion and a secondary HCC 45 and 62 months after the first operation, respectively. She has been well for the 7 years since the first operation despite undergoing hepatic resection for HCC twice as well as lymph node resection. Clonal analysis, based on the methylation pattern of the X chromosome-linked androgen receptor gene, suggested that the two primary tumors were multicentric and that the lymph node lesion had arisen by metastasis from the primary tumor in the right hepatic lobe.

A recent study of multidrug resistance (MDR) 1 gene transfected osteosarcoma cells found a cause-effect relationship between increased expression of P-glycoprotein (P-gp) and a low aggressive phenotype. However, several experimental and clinical studies have observed contradictory findings in that P-gp expression has been associated with tumour progression. In the present study, we characterized P-gp-positive and P-gp-negative single-cell clones of a murine osteosarcoma, to further investigate the relationship between P-gp expression and changes in cell phenotype. Although these clones were all selected by doxorubicin (DOX) exposure, they were heterogeneous with respect to MDR1 gene expression. The P-gp-positive clones revealed MDR phenotype, whereas the P-gp-negative clones showed no resistance to drugs. Morphological and functional analysis showed that both the P-gp-positive and P-gp-negative clones were more differentiated than the parent cells in terms of enhanced activity of cellular alkaline phosphatase, an increase in well-organized actin stress fibres and enhanced osteogenic activity. Moreover, these subclones all displayed a decrease in malignant potential such as oncogenic activity, tumour growth rate and metastatic ability, regardless of their P-gp status. These results indicate that the observed osteoblastic differentiation and less aggressive phenotype in DOX-selected osteosarcoma cells may not only be explained by the direct effect of P-gp, and accordingly, consideration of the effect of DOX, as well as P-gp, appears to be important. © 2000 Cancer Research Campaign

Clonal analysis has shown that hepatocellular carcinoma arises from a single cell. However, the clonality of precancerous lesions and adjacent nonneoplastic tissues is not clear. We analyzed a human androgen receptor locus to elucidate the clonal state of liver tissues including post-hepatitic lesions associated with hepatocarcinogenesis. The analysis was based on a restriction fragment length polymorphism involving an androgen receptor locus on the X chromosome, taking advantage of physiologic random inactivation by methylation of 1 of 2 X chromosomes in females during embryogenesis. Clonality was assessed in 79 randomly located tissue samples microdissected from noncirrhotic liver, including a total of 40 morphologically normal sites in 4 normal livers and 39 sites from a single HCV-infected liver. In addition, 51 regenerative nodules, 4 areas of adenomatous hyperplasia, and 18 hepatocellular carcinomas were sampled. All samples were obtained from livers involved by various neoplasms. Eight of forty samples (20.0%) from the four normal livers and 20 of the 39 samples (51.3%) from the single HCV-infected liver showed a monoclonal pattern. Moreover, 30 of 51 regenerative nodules (58.9%) showed a monoclonal pattern. No histologic differences were evident between mono- and polyclonal nodules. On the other hand, the 18 carcinomas and 4 areas of adenomatous hyperplasia all were monoclonal. Mean calculated monoclonal areas of normal liver and liver with chronic hepatitis were 1.1 and 3.3 mm(2). Our results suggest that areas representing a single clone of hepatocytes are present in normal liver, and these progressively expand as changes advance from chronic hepatitis to hepatocellular carcinoma.

In this study, we observed and analyzed the sub-micron motion of interphase chromosomes in living cells, labeled with the fluorescent thymidine analogue, Texas-Red dUTP. Our approach has an advantage in that chromosomes can be analyzed with regard to the nuclear architecture. We calibrated the observed motion of fluorescence-labeled chromatin by eliminating the rotational and translational movement of living nuclei that could significantly affect chromatin motion. Mathematical analyses of chromatin motion showed that: (1) interphase chromatin in living nuclei moves randomly, and the motion is limited within a small sub-region; (2) chromatin near the nuclear envelope moves in a more limited area than does centrally located chromatin; and (3) closely situated chromatin domains move independently of each other. Random and constrained chromatin motion in living nuclei supports the concept that interphase chromatin fibers are loose, flexible and floating in the nuclear matrix, and that chromatin anchors to the backbone of chromosomes. Moreover, that the random motions of DNA domains are independent of each other suggests that interphase chromatin arranges without structurally rigid continuity. This active motion of chromatin is consistent with dynamic biological processes, requiring chromosome motility and interactions. Additionally, the dynamic properties of interphase chromosomes may be significant in the interpretation of acquired chromosomal aberrations.

It was recognized early on that the digitization of medical information would advance the efficiency of diagnostic technology. However, the digitization of image data, which makes up the majority of medical information, is dependent on advances in technologies such as input, processing, transmission, storage, and display. Insufficient advances in such technologies has effectively limited the digitization of image data for medical use. The result of this has been non-networked systems or LANs confined to a single hospital. Such isolated systems integrate only portions of digital medical images such as x-ray computer tomography (CT), magnetic resonance (MR), and computed radiography (CR). Fortunately, recent advances in the areas of super high definition image I/O, high-quality encoding, super high speed transmission, and high-capacity storage has turned the tide in favor of the digitization and networking of all medical information. This paper will focus on the digitization and networking of medical image information used within hospitals and provide a multifaceted study of the technologies necessary for these advances. This will allow us to discuss the present state of related technical developments and the level that has been attained so far. In addition, we have targeted image information that demands the highest level of quality (radiological and pathological images) for application in medical diagnosis using super high definition images, the image technology being developed by the authors of this paper. We will cover the concrete issues and approaches to solutions that must be investigated when building and networking a digital system.

Overcoming multidrug resistance (MDR) is an urgent issue to improve the prognosis of osteosarcoma patients. In this study, we undertook to clarify the effect of photodynamic therapy (PDT) with acridine orange (AO) on the MDR mouse osteosarcoma (MOS / ADR1) cell line, by comparing the outcome with the effect on a chemosensitive osteosarcoma (MOS) cell line. Cultured cells of MOS and MOS / ADR1 cell lines were exposed to AO at various concentrations for various times, followed by long- or short-term (10 or 1 min) illumination with blue light (466.5 nm) for excitation. Living cells were counted by means of the trypan blue exclusion test. The results showed that AO rapidly bound to DNA, RNA and lysosomes of living MOS and MOS / ADR1 cells and also that most tumor cells in both cell lines died rapidly (viability ratio to untreated cells: 1/1000) within 48 h under conditions of continuous or 15-min flash exposure to AO at concentrations above 1.0 microg/ml plus 10-min illumination with blue light. Even after flash exposure to AO at concentrations above 1.0 microg/ml plus 1-min illumination, the viability of MOS/ADR1 cells decreased to a viability ratio of less than 1/ 1000 within 72 h. Based on these results, we concluded that AO with photo-excitation has a strong cytocidal effect, not only on chemosensitive mouse osteosarcoma cells, but also on MDR mouse osteosarcoma cells. These results suggested that photodynamic therapy with AO may be a new approach to treating MDR human osteosarcomas.

We analyzed the DNA ploidy alterations after preoperative chemotherapy in 30 patients with non-metastatic osteosarcomas of the extremities. All of the patients received intensive chemotherapy with doxorubicin, cisplatin and methotrexate as well as wide tumor resection. DNA ploidy was determined by DNA cytofluorometry using isolated and smeared cells from biopsied and resected tumors after preoperative chemotherapy. The results showed that 12 diploid and nine non-diploid osteosarcomas did not change their ploidy pattern, but nine non-diploid tumors changed to a diploid pattern with the disappearance of the aneuploid cells. The nine patients with altered ploidy tumors had a better histologic response to chemotherapy and a better prognosis than the patients with non-altered tumors especially diploid tumors (P = 0.0138). Therefore, we conclude that a decrease in aneuploid cells after chemotherapy is closely correlated with a good prognosis in half of the cases of aneuploid osteosarcoma. These results also suggest that aneuploid cells are more chemosensitive than diploid cells in human osteosarcomas.

An analysis of the chromosomal aberrations and DNA ploidy in the interphase nuclei of seven human osteosacomas was preformed by double-target fluorescence in situ hybridization (FISH) and DNA cytofluorometry. The FISH study of the numerical aberrations in chromosomes 1 and 17 or the structural aberrations in chromosome arm 1p or 17p was carried out by using four locus specific DNA markers, with one pair consisting of 1q12 and 1p36 and the other pair consisting of the 17 cemtromere and 17p13.3. There was no significant differences in the percentage of deletions in chromosome 1 and 17 between osteosarcomas and normal tissues. However, all seven tumors studied had extra copies. Cells with more than three probe signals were regarded as having chromosome polysomy. The percentage of polysomy of chromosome 1 was 20.0-64.0%, and chromosome 17 was 28.0-60.0%. The DNA ploidy patterns of hyperdiploid cells showing a greater DNA content than diploid cells were obtained by DNA cytoflurometry. Five of the seven tumors were non-diploid, and the remaining two were diploid. The percentage of polysomy was correlated with the percentage of hyperdiploid cells in each tumor. Thus, these findings indicated that the DNA ploidy changes were closely correlated with aberrations in the chromosome copy number in osteosarcomas.

DNA ploidy analysis by DNA cytofluorometry was performed on 41 tumors obtained from 37 patients with primary giant cell tumor of bone (GCT). Histologically, 26 of the tumors from primary or recurrent lesions were evaluated as grade I, and 13 tumors as grade II. Among the 33 primary GCT patients, 4 patients had local recurrence or pulmonary metastasis. The DNA ploidy pattern and the percentage of hyperdiploid cells showing a greater DNA content than diploid cells, were obtained from DNA cytofluorometry. All of the 33 primary tumors were diploid. Of 6 recurrent tumors, 4 were diploid and 2 were euploid-polyploid. One of the two pulmonary metastatic tumors was diploid, but another that demonstrated a malignant transformation to malignant fibrous histiocytoma was aneuploid. The percentage of hyperdiploid cells was significantly different between primary and recurrent tumors (P = 0.0188) and between grade I and grade II tumors (P = 0.0052), while there was no difference between primary tumors in the cases that recurred or metastasized and those that did not. Thus, these data indicate that cell proliferative activity is closely correlated with biological aggressiveness and histological grading, although DNA ploidy is not useful for predicting prognosis.

In this study, we analysed the DNA ploidy of osteosarcomas at biopsy and attempted to clarify the relationship between DNA ploidy pattern and prognosis. Thirty patients with non-metastatic osteosarcoma of an extremity were studied. All underwent intensive chemotherapy with doxorubicin, cisplatin and methotrexate, in addition to wide tumor resection. DNA ploidy was detected by DNA cytofluorometry, using isolated and smeared cells of biopsied tumor tissue. Twelve tumors showed a diploid ploidy pattern and 18 showed a non-diploid pattern such as aneuploidy (15 tumors) and euploid-polyploidy (3 tumors). The event-free survival rate at 9 years was 63.5% in non-diploid osteosarcoma patients and 13.3% in diploid osteosarcoma patients. There was a statistically significant difference between the two groups (P = 0.0278). These results lead us to conclude that a non-diploid osteosarcoma may be more sensitive to chemotherapy than a diploid tumor.

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.

There are several problems on the practical use of telemedicine, for example, the difficulties involved in promoting communication between medical facilities, uncooperative clinicians, and the absence of high-speed circuits and high-resolution CRT. From the Japanese point of view, we suggest ways to resolve these problems. We will analyze and propose scenarios for realizing successful communications among medical institutions, medical communication and its characteristics, barriers to the promotion of communications among medical institutions, second-opinion centers, and separate satellites and separate circuits. We also mention the World Wide Web for teleconsultation, provision of assistance to people with data handicaps via a communications satellite, and assistance to programs designed for training telemedicine specialists. Using a communication satellite, we offer programs that explain preventive medicine, support activities for nursing at home, explain the risks of fast food, and support activities for the handicapped and women in a simple manner to computer illiterates.

Analysis of six human osteosarcoma cell lines was performed by using double-target fluorescence in situ hybridization (FISH). FISH was applied to interphase nuclei, not to metaphase chromosomes. In this study, numerical aberrations of chromosomes 1 and 17 or structural chromosomal aberrations of chromosome arm 1p or 17p, in which it has been suggested that there are one or more tumor suppressor genes in various malignant tumors, were examined with this technique. All six of the human osteosarcoma cell lines studied had extra copies of chromosomes 1 and 17. A high frequency of deletions (>60%) in chromosome 1 was found in two cell lines and deletions of chromosome 17 were found in one cell line.

BACKGROUND. Although chemosensitivity to antiosteosarcoma agents is the most important prognostic factor in human osteosarcoma, none of the many chemosensitivity tests reported previously are reliable and clinically useful. In this study, the authors investigated the reliability and clinical availability of doxorubicin (Adriamycin; Adria Laboratories, Columbus, OH) binding assay (ABA) as a new chemosensitivity test for osteosarcoma. METHODS. Doxorubicin (adriamycin [ADM]) binding ability (%AB) to nuclear DNA in isolated osteosarcoma cells was assessed by ABA in 14 patients with primary osteosarcoma who were treated with preoperative chemotherapy containing ADM and 6 patients with relapsed osteosarcoma after intensive chemotherapy. Histologic responses to preoperative chemotherapy were evaluated by percentage of tumor necrosis (%necrosis). RESULTS. Four of the 14 patients with primary osteosarcoma had %AB > 80% (97.3 ± 3.7%) and demonstrated good histologic responses (>90% of %necrosis) to preoperative chemotherapy, whereas the remaining 10 patients had %AB < 80% (38.9 ± 21.0%) and demonstrated poor responses. Patients with recurrent osteosarcoma that was clinically evaluated to be resistant to previous chemotherapy also had low %AB (34.2 ± 28.3%). CONCLUSIONS. Because the results of the current study revealed that ABA is useful for predicting chemosensitivity to chemotherapy with ADM as well as chemotherapy without ADM for patients with osteosarcoma, and because ABA technically is simple and results can be assessed rapidly, the authors conclude that ABA is a clinically useful chemosensitivity test for patients with osteosarcoma.

To prove the relationship between chromosomal aberration and DNA ploidy in human malignant fibrous histiocytoma (MFH), fluorescence in situ hybridization (FISH) and DNA cytofluorometry were performed in this study. For FISH study, the nucleus of each tumor cell was isolated from paraffin-embedded tissue of nine MFHs. Five chromosome-specific DNA probes (1p36, 1q12, 8q21.3, 11 centromere, and 17 centromere) were hybridized on cell nuclei. Cells with more than three probe signals were regarded as chromosome polysomy. All of the tumors analyzed by FISH had extra copies. The average percentage of polysomy in all tumors was high, ranging from 10.2% to 49.2%. The DNA ploidy patterns, and the percentage of hyperdiploid cells showing a greater DNA content than diploid cells, were obtained from DNA cytofluorometry. Three of nine were diploid patterns and six were non-diploid patterns, and the percentage of hyperdiploid cells in all tumors was high, ranging from 9.1% to 61.9%. The percentage of polysomy could be correlated with the percentage of hyperdiploid cells in each cell. In this study, we found that the DNA ploidy change was closely correlated with aberrations of chromosome copy number in MFH. In addition, the alterations of specific chromosome copy number could be detected in MFH showing diploid cells. Thus, these data indicate that FISH and DNA cytofluorometry are available as a cytogenetic tool for the analysis of interphase nuclei of bone and soft tissue tumors including MFH.

To evaluate the role of inducible nitric oxide synthase (iNOS) and proinflammatory cytokines in acute respiratory distress syndrome (ARDS), we immunostained alveolar macrophages (AMs) in the bronchoalveolar lavage fluid (BALF) collected from patients with ARDS, patients in the operating room whose lung function were normal, and non-ARDS patients who were intubated and artificially ventilated for one week in the intensive care unit. We observed the significant expression of iNOS, interleukin-6 (IL-6) and interleukin-8 (IL-8), in AMs obtained from BAL in ARDS patients by the immunocytochemical approach. By comparison, statistically significant elevation was detected in IL-8 levels in BALF supernatant from the ARDS patients compared with the control and the non-ARDS patients. These results suggest that the expression of proinflammatory cytokines and iNOS in AMs as determined by immunocytochemical technique may be an important predictor of the early stage of lung injury in ARDS. We suggest that NO and proinflammatory cytokines produced by AMs might play a pivotal role in acute lung injury. The immunocytochemical approach could be the most sensitive predictor for activated AMs in ARDS patients.

Recently, interphase cytogenetics using fluorescent in situ hybridization (FISH) was performed on various kinds of solid tumor, and their inherent karyotypic heterogeneities were revealed. Concerning this heterogeneity, we evaluated both the exhibiting number of chromosome 17 and nuclear DNA content on an identical nucleus by means of computer-controlled auto-scanning stage in order to demonstrate the alteration in number of chromosome 17 among cytofluorometrically distinct subpopulations.We investigated 8 lesions of surgically resected colorectal carcinomas, which were classified as aneuploid in quantitative DNA analysis and also exhibited an increase of 17-aneusomy nuclei. We used paraffin-embedded archival blocks. First, we prepared isolated cell specimens, and memorized the position of the cells on a glass slide using computer-controlled auto-scanning stage. Next, the specimens were stained with propidium iodide, and the fluorescent intensity was evaluated as nuclear DNA content in the order of cell position data. And lastly, FISH was performed with (peri) centromerespecific DNA probes for chromosome 17, and we enumerated the number of signals in a nucleus also according to cell position data. Then, we compared the distribution of number of chromosome 17 among cytofluorometrically distinct subpopulations. Three of 8 lesions showed a single GO+G1 peak, and the rest exhibited plural GO+G1 peaks in DNA profile. And 4 of 5 lesions, which showed plural GO+G1 peaks, presented a peak at the DNA value of (near) 2c. We could detect an alteration in the distribution of number of chromosome 17 between diploid peak and aneuploid peaks in 4 of 4 lesions which presented a peak at the DNA value of (near) 2c. However, we could ot find a difference in the distribution of number of chromosome 17 between GO+G1 peak and G2+M peak. These observations indicate that the distribution of number of chromosome 17 reflects an endoreduplication of genome content, yet, it does not alter in accordance with the phase of cell cycle. It is necessary to evaluate nuclear DNA content simultaneously in order to assess an essential cytogenetic change.

私たちの教室で治療した胃癌症例中で多発胃癌は5.6%あるが, 早期胃癌の中では多発のものは8.6%を占めて近年増加傾向にある. この多発早期胃癌は単発早期胃癌と比較して高齢者に多く, 隆起型, 分化型 (高分化腺癌) の頻度が高く, 占居部位は大半が胃体部, 幽門部で幽門側切除が可能であった. 私たちは多発早期胃癌の悪性度評価法として, 各病巣の癌細胞増殖動態を顕微測光法で解析してきた. 予後良, 好例では各病巣のDNAプロイディ・パターンはdiploidで増殖動態は類似し, 病巣間で著差を認めなかった. 一方, 予後不良例ではS-G2期細胞の増加, 多倍体化などの進行胃癌で見られる増殖動態を示し, これらの所見は病巣間でばらつきを示した. したがって, DNAプロイディ・パターンは多発早期胃癌の悪性度の指標になりうると考えられた. また, リンパ節転移の頻度は単発例と差がなく, 単発早期胃癌と同様な進行度評価を適用しうると考えられた.

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.

To elucidate the role of proliferating cell nuclear antigen (PCNA) in vivo, PCNA immunohistochemistry combined with 3H-thymidine autoradiography was performed on 2 types of regenerating rat livers. One group of rats underwent 2/3 partial hepatectomy (PH), and the other group 1/3 PH. After 3H-thymidine labeling, the livers were fixed with paraformaldehyde (PFA) and methanol, and paraffin-embedded. In methanol-fixed tissues, the serial changes of the PCNA-positive index (PCNA-PI) corresponded well with those of the 3H-thymidine-labeling index (3H-thymidine-LI) after each PH. In PFA-fixed tissues, however, the PCNA-PI was 26% before PH, when the 3H-thymidine-LI was below 1%. Furthermore, the 1/3 PH group, despite a continued low 3H-thymidine-LI, showed a rise in PCNA-PI to a level comparable to that in the 2/3 PH group. From these results, we propose 3 phases of PCNA expression. In PFA-fixed tissues, PCNA that was detectable before PH was considered pooled in G0-phase nucleoplasm (Phase I), and some or many of the PCNA-positive cells failed to enter the S-phase in the 1/3 PH group, reflecting the increased PCNA not bound to DNA replicons (Phase II). In methanol-fixed tissues, PCNA was detected only in the S-phase, indicating its direct involvement in DNA synthesis (Phase III). Therefore, determining PCNA-PI in methanol-fixed tissues is useful to evaluate proliferative activity, whereas that in PFA-fixed tissues must be assessed with great caution.

To analyze EGF binding activity in liver regeneration at cell cycle level, we performed the double measurement of nuclear DNA content and cellular bound EGF content in freshly isolated rat hepatocytes from normal and regenerating liver after 67% hepatectomy using autostaging cytofluorometry. The data demonstrated that the bound EGF content of resting cells increased in proportion to their DNA content, while cycling cells had significantly consistently lower bound hEGF throughout cell cycle. These changes are supposed to reflect ‘down-regulation’ of EGF receptor at the cell cycle level, and the data suggested a major role of endogenous EGF in cell proliferation during liver regeneration.

症例は67歳女性.呼吸困難,乾性咳,発熱にて入院した.現病歴,胸部X線写真, CTなどより特発性間質性肺炎と診断した.抗生物質,ステロイドの投与にていったん症状の改善を認めたが,入院中再び呼吸困難が増悪し,ステロイドの増量にもかかわらず呼吸不全にて死亡した.剖検では肺炎所見を認めず,死因は急速に進行した肺線維化による呼吸不全と右心不全によるものと考えられた.経過中,補体分解産物iC3, Bb, c4dおよびC3a, C5aを経時的に測定できた.剖検所見と補体の動きからその病態に補体の活性化がかかわっていたことが示唆された.

Bromodeoxyuridine (BrdU) immunohistochemistry has recently been introduced for the visualization of DNA-synthesizing nuclei. To determine the optimal conditions for detecting BrdU-incorporated nuclear DNA in formalin- or ethanol-fixed, paraffin-embedded human gastric tissues, we tested several different pretreatment procedures of hydrolysis with HCl and enzymatic digestion with actinase before the immunohistochemical staining. Three cases of surgically resected stomachs were perfused with artificial blood containing BrdU and fixed in formalin, ethanol or with their combination for different durations. The results of BrdU-immunohistochemical stainings were checked and the labeling indices were compared. As for hydrolysis, treatment with 2N HCl at 25°C for 90min was sufficient to detect the immunoreactivity in the stomach irrespective of fixative conditions tested. However, to obtain the most suitable stainings an additional enzymatic digestion was needed, and the optimal condition with actinase was different according to the fixation time in formalin- and/or ethanol-fixed tissues. There are also a few differences in the demonstrability of nuclear BrdU among various kinds of cells, i.e. normal epithelial cells, immunoblasts in the lymphoid follicle and cancer cells. Our method could extend the range of application of BrdU immunohistochemistry for cell kinetic studies using human surgically resected organs.

私達の近年の開発研究に基づく顕微測光と画像解析の結合解析法を応用し, 顕微蛍光測光法による細胞増殖動態の解析単独では鑑別の困難な, 甲状腺の腺腫様過形成5例濾胞腺腫12例および濾胞癌12例を検索した.単離採取した一つひとつの細胞について, 細胞測光による核DNA定量と細胞核の形態解析 (面積, 周囲長, 核DNA濃度, 形状係数など) を対応させて行った.各症例について細胞周期の全ての時期の細胞 (全細胞) 群に対して形態解析を行うとともに, 細胞周期に基づく形状変化の影響を除外して客観的に形態学的特性を評価するために, 2倍体細胞 (G0-G1期細胞) 群に限定した解析も行った.その結果, 検索した腺腫様過形成, 濾胞腺腫および濾胞癌の核DNA量分布パタンは類似していたが, 2倍体細胞の核形態の不規則さは, 腺腫様過形成や腺腫よりも腺癌において有意に高度であることが定量的に示された.以上より, 本法が, 甲状腺腫瘍の良・悪性鑑別に, 細胞測光法や画像解析法単独による解析よりも, 有用であると考えられた.

The modified Bodian method with protargol (silver protein) is ordinarily used to detect nerve fibers. With this technique, applied to decalcified rat bone sections, the bone canaliculi were clearly stained black with good contrast to the bone matrix in both lamellar and woven bone. In addition, the connections between the bone canaliculi and other canaliculi, osteoblasts, osteoclasts, and chondrocytes were easily detectable. We found that the bone canaliculi of woven bone were fewer in number and ran more irregularly than those of lamellar bone. We believe that this staining method for bone canaliculi in decalcified bone is superior to previously reported methods and may be useful in studies on bone pathology.

Objective Although it is well known that binuclear cells commonly appear among the chondrocytes of normal cartilages as well as among neoplastic chondrocytes of chondrosarcomas, the mechanism of binucleation is still unclear. Therefore, this study was undertaken to clarify the mechanism of binucleation in chondrocytes, using primary culture cells of growth plate cartilage. Design These chondrocytes were exposed to acridine orange (AO) which is a fluorescent dye for differentiating certain DNAs and RNAs in nuclei and cytoplasm, and which inhibits mitosis. After exposure to 0.5μg/ml AO, for 0, 6, 24, 48, and 96h, the following parameters were investigated: (1) cell growth rate (GR); (2) frequency of hyperdiploid cells (%HDC) by DNA cytofluorometry; (3) mitotic index (MI); (4) BrdU labeling index (LI); (5) frequency of binuclear cells (%BNC). Results Compared with the control cells, which were cultured in AO-free medium, the GR was remarkably inhibited at 24h. MI was also decreased from 6 to 24h, and LI decreased at 48h. However, these parameters were recovered at 96h. The %HDC was increased from 6 to 96h, and the %BNC was also increased to a maximum of six times that of the control cells at 96h. Discussion These results suggested that the binuclear cells observed among the cultured chondrocytes may be formed from G2 arrested cells by amitotic nuclear division, but not by mitosis without cytoplasmic division or cell fusion.

We recently found that silver impregnation staining with protargol (silver protein), that is, a modified Bodian method, is useful for histologically identifying the details of bone canaliculi structure, using thin sections of decalcified bone tissues. With this staining method, we conducted the present study to assess the development of bone canaliculi during the process of intramembranous ossification using a fracture-like stimulation model of the rat femur. After making a drill-hole in the cortex of the rat femur, decalcified thin sections were obtained after 3, 5, 7, and 14 days by the standard paraffin-embedding procedure. Silver staining for bone canaliculi was performed using our previously reported technique. The results showed that woven bone covered the fracture surface of the cortex after 5 days, then immature lamellar bone attached to the woven bone after 7 days, and finally the lamellar bone matured and became thick with appositional growth after 14 days. The osteocytes in the woven bone appeared at an early stage of bone repair and developed a few canaliculi that were short and irregularly distributed in the osteoid matrix, while the osteocytes in the lamellar bone at a late stage formed many bone canaliculi that were long and regularly distributed in mature bone matrix. Therefore, we concluded that woven bone osteocytes may be necessary for induction of the lamellar bone osteocytes followed by active appositional growth of the lamellar bone at the early stage of bone repair, and also that both bone tissues could be clearly distinguished from one another based on the pattern of development of bone canaliculi by the osteocytes, as seen with the use of our sensitive staining method.

Acridine orange (AO) has unique biological actions enabling tumor visualization (fluorovisualization) and a strong cytocidal effect (photodynamic therapy: AO-PDT) under illumination with blue light. Accordingly, in this study, we attempted to develop a new surgical technique for total tumor cell elimination using these photodynamic reactions with AO in a mouse osteosarcoma model. The results showed that local tumor recurrence was significantly inhibited (23%) in the group treated with curettage under fluorovisualization and AO-PDT, compared to that (80%) in the control group treated with curettage alone under ordinary light. Therefore, we concluded that the combination of curettage under fluorovisualization and AO-PDT may be useful for total tumor cell elimination with minimum damage to normal tissue in musculoskeletal sarcomas.

Background: There are two general categories of drug resistance: acquired and intrinsic. The mechanisms involved in acquired drug resistance have been extensively studied, and several mechanisms have been described. However, the mechanisms responsible for intrinsic drug resistance have not been elucidated, to our knowledge. The purpose of the present study was to investigate the cytological and biochemical differences between acquired and intrinsic drug resistance in osteosarcoma cells. Methods: We previously isolated a clonal cell line (MOS/ADR1) to study acquired resistance in osteosarcoma by exposure of parental murine osteosarcoma cells (MOS) to doxorubicin. In the present study, we cloned a new, intrinsically resistant cell line (MOS/IR1) by single-cell culture of MOS cells and we investigated the differences in cell phenotype and the mechanisms of resistance in both of these resistant clones. Results: The MOS/ADR1 and MOS/IR1 cells were sevenfold and fivefold more resistant to doxorubicin than the parental murine osteosarcoma cells. Morphologically, the MOS/ADR1 cell line was composed of polygonal cells, whereas the MOS/IR1 cell line consisted of plump spindle cells with long cytoplasmic processes. The MOS/IR1 cells showed a much lower level of alkaline phosphatase activity than did the MOS/ADR1 and MOS cells. There were no substantial differences in the cellular DNA content or the doubling time among these three lines. Overexpression of the P-glycoprotein involved in the function of an energy-dependent drug-efflux pump was detected in the MOS/ADR1 cells but not in the MOS/IR1 cells. After the cells were incubated with doxorubicin for one hour, the two resistant lines had less accumulation of the drug than did the parent line (p < 0.05). The addition of a P-glycoprotein antagonist, verapamil, or the depletion of cellular adenosine triphosphate resulted in a marked increase in the accumulation of doxorubicin in the MOS/ADR1 cells (p < 0.05) but not in the MOS/IR1 cells. The MOS/ADR1 cells were found to exhibit cross-resistance only to substrates for P-glycoprotein (such as doxorubicin, vincristine, and etoposide), whereas the MOS/IR1 cells were resistant to all of the drugs studied (including cisplatin and methotrexate). The degree of drug resistance in the MOS/IR1 cells was found to be associated with the molecular weight of the drugs (p < 0.05). Permeabilization of the plasma membrane by saponin increased both the accumulation of doxorubicin (p < 0.05) and the cytotoxic activity of this drug in all lines, but the effects were most pronounced in the MOS/IR1 cells. Conclusions: Taken together, this data suggests that reduced drug accumulation in the MOS/IR1 cells may be due to the effect of decreased permeability of the plasma membrane on the transport of drugs from the extracellular environment into the cytosol of the cell and that this may be the mechanism responsible for intrinsic resistance to multiple drugs in the MOS/IR1 cell line. Clinical Relevance: Current drug treatment for human osteosarcoma may include multiple chemotherapeutic agents, such as doxorubicin, cisplatin, and methotrexate. These drugs exhibit different cytotoxic actions and, thus, the mechanisms of resistance to individual drugs vary. Clinical resistance to multidrug chemotherapy may be observed in tumors that recur after repetitive chemotherapy and in previously untreated tumors. In the former group, a tumor cell may express multidrug resistance by combining several different mechanisms due to its exposure to various drugs. In the latter group, however, this is not likely. Decreased intracellular drug accumulation due to reduced permeability of the plasma membrane, found in the MOS/IR1 cells, is one possible mechanism and may explain the intrinsic resistance to multidrug chemotherapy for the treatment of osteosarcoma. Further study regarding the resistance mechanism in the MOS/IR1 cells may help to overcome the intrinsic drug resistance in osteosarcoma.

We previously reported that the doxorubicin binding ability detected by the doxorubicin (adriamycin) binding assay was closely correlated with the chemosensitivity of human osteosarcomas. In this study, we undertook to clarify the relationship between P-glycoprotein positivity (%PPG) and doxorubicin binding ability (%DB) in human osteosarcomas in order to determine which is a more sensitive index of histologic response to chemotherapy. Ten primary osteosarcomas were analyzed by the doxorubicin binding assay and by immunofluorescence to detect cellular P-glycoprotein positivity. Three good responders to chemotherapy containing doxorubicin showed a %DB greater than 90% (average: 96.43%), whereas the seven poor responders had values less than 80% (average: 35.31%). The difference between the two groups was statistically significant (P = 0.0167). However, the average %PPG of the three good responders was 6.73%, whereas the %PPG of the seven poor responders was 14.27%. There was no significant difference in %PPG between the two groups (P = 0.3051). No negative correlation between the %DB and the %PPG of all osteosarcomas (r = 0.536, P = 0.1104) was found, although there was a trend that those tumors with a high %PPG showed a low %DB. These results suggest that osteosarcomas showing a low %DB and %PPG with poor response to chemotherapy, may have multidrug resistance mechanisms other than P-glycoprotein. Therefore, we conclude that doxorubicin binding ability, which reflects all of the doxorubicin-resistant mechanisms, was more sensitive than P-glycoprotein positivity in predicting the chemosensitivity of human osteosarcoma.

Primary and pulmonary metastatic and pulmonary metastatic tumors (two synchronous and seven metachronous metastases) in nine patients with osteosarcomas were studied by DNA cytofluorometry. All patients were treated with both pre and postoperative chemotherapy. The results showed that all five diploid osteosarcomas and three of the four aneuploid tumors did not markedly change their ploidy pattern after preoperative chemotherapy, and had almost the same ploidy patterns as the pulmonary metastatic lesions. Those eight tumors showed poor histologic response and chemoresistance by the doxorubicin binding assay. Only one aneuploid osteosarcoma showing good histologic response and chemosensitivity changed its ploidy pattern to diploid, with the disappearance of aneuploid tumor cells and its synchronous pulmonary metastatic tumor also showed conversion to a diploid pattern with massive tumor necrosis. It is evident that those tumors showing no change in their ploidy pattern after chemotherapy were resistant to the chemotherapy. Therefore, we conclude that regardless of whether the pulmonary metastatic tumors were synchronous or metachronous, they showed the same change in their ploidy pattern as well as their chemosensitivity as the primary human osteosarcoma from which they were derived.

It has been recognized early on that digitizing medical information makes diagnostic technology more advanced and efficient. In order to convert image information, which comprises the majority of all medical information, into digital data, various technologies including those for input, processing, transmission storage, and display need to develop at roughly the same pace. To data, there have been few cases where this has been done. However, recent major advances in high-resolution image input/output, image encoding, super-fast transmission, high-capacity storage, and other technologies have intensified the drive towards digitizing and networking all medical information. This paper will show that the spread of super-high-speed networks capable of transmitting large amounts of data in a short time is indispensable for accurate medical diagnosis, and that this will make it possible to realize an integrated medical information syste. A target application for the medical image diagnosis of the Super High Definition imags being developed by the authors of this paper is telepathology, which particularly demands high-quality images. In this paper, we will study, among other things, the concrete issues crucial to building and networking a digital system and the approach to resolving such issues. We will also report on the building of our experimental system that fulfills such demands as well as discuss a pathological microscopic image transmission system with image quality that will not lower diagnostic accuracy and fast response and good operability that will not make diagnosticians feel impatient. Finally, we will discuss a test in which we remotely operated a microscope over an ATM line to prove that it is possible to capture, transmit, and display a still super-high-definition digital image with a resolution of 2,048 X 2,048 pixels in about 5 seconds.

PURPOSE: The efficacy of the reduction time (tau) after castration as a prognostic factor was examined by comparing to Gleason score. MATERIALS AND METHODS: The change of prostatic volume after castration was observed from the castration to 3 months after in 24 cases of prostatic cancer. Prostatic volume was examined by transrectal ultrasonography of the prostate. Survival curves was calculated by Kaplan-Meier method. Differences among survival curves were analyzed using Cox-Mantel test. RESULTS: tau had a close relationship to the prognosis of each case (Wilcoxon test: p < 0.05, Cox-Mantel test: p < 0.05). Gleason score had a weak relationship to prognosis (Wilcoxon test: N. S., Cox-Mantel test: p < 0.05). CONCLUSIONS: tau was efficient as prognostic factor compared to Gleason score.

Study objective The objective of this study was to evaluate the role of inducible nitric oxide synthase (iNOS) and proinflammatory cytokines in alveolar macrophages (AMs) in the pathogenesis of ARDS following sepsis. Setting ICU in a university hospital. Design Prospective exploratory, open-labeled study was carried out. Patients A total of 24 patients were investigated: 8 patients diagnosed as having ARDS following sepsis (ARDS group); 8 patients under general anesthesia in the operating room whose lung functions were normal (control group); and 8 patients who were intubated and artificially ventilated for 1 week in the ICU whose lung functions were not deteriorated without fulfilling the ARDS criteria and whose general state fulfilled the sepsis criteria (long-term ventilation group, or LTV group). Measurements and results The expression of iNOS, interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-8 (IL-8) in AMs obtained from RAL fluid (RALF) was determined by the immunofluorescent technique. We observed the significant expression of iNOS, IL-6, and IL-8 only in the ARDS group. Meanwhile, NOx (the sum of NO 2 – + NO 3 – ) was elevated in the BALF supernatant, and IL-6 and IL-8 levels in both the BALF supernatant and the serum were also elevated in the ARDS group. No significant expressions were detected in the control and the LTV group. Conclusions The result that iNOS was detected only in ARDS patients following sepsis suggests that iNOS together with proinflammatory cytokines produced by AMs might play a pivotal role in the pathogenesis of acute lung injury and be useful for monitoring disorders in the lung in such conditions.

We have previously reported that P-glycoprotein (Pgp)-overexpressing multidrug resistant (MDR) osteosarcoma cells were functionally more differentiated than their parent cells. The present study showed that in the parent cells, the actin filaments were sparsely distributed or were diffusely spread throughout the cytoplasm, whereas the MDR osteosarcoma cells exhibited a remarkable increase in well-organized actin stress fibers. Furthermore, dihydrocytochalasin B, a specific inhibitor of actin polymerization, dramatically disrupted this network of stress fibers, increased the intracellular accumulation of doxorubicin (DOX) and modified the resistance against DOX. These results indicate that the organization of actin filaments associated with cellular differentiation may be involved in the expression of Pgp function in the MDR osteosarcoma cells.

Digitizing high-quality microscopic images and developing input/output technology for displaying those results is critical to tele-pathology in which pathological microscopic images are transferred to remote locations where they are diagnosed by specialists. This paper will discuss the results achieved by directly digitizing pathological microscopic images at a 2k by 2k resolution, and then using a super high definition imaging system to analyze their signals and evaluate compression performance. We will start off by digitizing samples that a pathologist will actually use in making a diagnosis, and then analyze their color distribution and spatial frequencies characteristics by comparing them to general images. This will make it apparent that such pathological images characteristically contain high spatial frequency in their chrominance components. We will also discuss the evaluation results of color differences for L.a.b space and compression ratios achieved when using JPEG to encode pathological images. We will also present a subjective evaluation of the influence sub- sampling of chrominance components has on image quality.© (1997) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

We have been studying a medical tele-education support system by an individual tutoring system, called CALAT, and a super high definition (SHD) image processing system, called SuperFM-III.Now, we are in a trial operation to use the SuperFM-III for a super high definition image controlviewer on the CALAT client side, and have created the courseware of the pathological images. Inthis paper, we show the concept and the implementation of this system.KEY WORDS: Super High Definition image, Tele-education, Medical-education 1. INTRODUCTION Recently, the research on the tele-education support system which uses the characteristic ofthedistributed hypermedia environment of WWW (World-Wide Web) is becoming active. However,there have been few systems where the individual adaptation mechanism operates to select the appropriate content that the system can actively present according to the student's levelof understanding.In NTT (Nippon Telegraph and Telephone Corporation), an individual tutoring system CAIAT(Computer Aided Learning and Authoring environment for Tele-education)1 which uses WWW isbeing investigated. In CALAT, the individual adaptation mechanism is achieved by using anintelligent education support system CAIRNEY (CAl expeRt system for NEw technologY)2 for theWWW server side. CAIRNEY, which has been developed in NTf, is an intelligent CAl system ofa standalone type. The system has the courseware more than 200 titles.Also, NTT is researching on the SuperFM-III (Super Frame Memories III system)3' 4) The

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. Cancer 1996;77:271-7.