Masami Sakurai

Clonal analysis of high-grade squamous intra-epithelial lesions of the uterine cervix

We previously reported that invasive squamous cell carcinomas of the uterine cervix are of monoclonal composition. In the current study, we extended our previous work to determine the clonal composition of cases of high‐grade squamous intra‐epithelial lesion (HSIL). Clonal analysis targeting the HUMARA locus was performed on cervical tissue from 9 cases, 8 showing heterozygosity at the HUMARA locus and being, therefore, informative for clonality analysis. Uterine cervices were cut into 12 blocks, fixed with formalin and embedded in paraffin, and DNA was extracted from targeted lesions of each block. A total of 30 samples of cervical intra‐epithelial neoplasia 3 (CIN3) (14 samples of carcinoma in situ and 16 samples of severe dysplasia) and 1 sample of CIN2 (moderate dysplasia) were analyzed. Monoclonal composition of the lesions was demonstrated in 30/30 cases of CIN3. Polyclonal composition was seen in the single case of CIN2. In 6 uterine cervices, in which dysplastic lesions were present in more than 3 blocks, the pattern of X‐chromosome inactivation was the same in all lesions, suggesting that these individual lesions were derived from a single cell, with intra‐epithelial extension within the cervical mucosa. By contrast, one uterus contained 2 discontinuous dysplastic foci with different patterns of X‐chromosome inactivation, indicating that the 2 lesions developed independently from each other. Our results demonstrate that (i) lesions of CIN3 (severe dysplasia and carcinoma in situ) are composed of a clonal neoplastic population of cells and (ii) most cases of HSIL are unifocal in origin. Int. J. Cancer 73:339–344, 1997.

Hepatocellular carcinoma: Efficacy of transcatheter oily chemoembolization in relation to macroscopic and microscopic patterns of tumor growth among 100 patients with partial hepatectomy

AbstractPurpose: To evaluate the efficacy of transcatheter oily chemoembolization (TOCE) for hepatoceliular carcinoma (HCC) on the basis of microscopic and macroscopic findings postembolization. Methods: HCCs ranging in size from 0.5 to 13 cm (mean 3.6 cm) were obtained from partial hepatectomies of 100 consecutive patients who had undergone TOCE between 20 and 246 days (mean 59.5 days) prior to surgery. The efficacy of TOCE was assessed on the basis of the necrotic to live cell ratio of the tumors. The microscopic pattern of tumor growth was grouped into expanding type (complete capsule formation) and replacing type (incomplete or no capsule). There were five types of macroscopic groupings: single nodule, single nodule with extranodular growth (SNE), contiguous and noncontiguous multinodular, and massive growth type. Results: Among 79 cases with the expanding type, 29 (37%) had 100% HCC necrosis, but none with 100% necrosis were in the replacing type. By macroscopic grouping, the efficacy of TOCE decreased from the single nodule type (50% of patients had 100% necrosis) to the SNE type (21%), and the other types (9%).

The role of CD8+ and CD4+ cells in islet allograft rejection

The requirements of CDS+ and CD4+ cells for islet graft rejection in combinations with different histoin-compatibilities were investigated by in vivo administration of anti-Lyt-2.2 (CD8) mAb, anti-L3T4 (CD4) mAb, or both to recipient mice. In B10.AQR×B10.A (H-2K-incompatible) and B10.A(5R)×B10.A (H-2K- and IA-incompatible) combinations, administration of either anti-Lyt-2.2 (CD8) or anti-L3T4 (CD4) mAb completely blocked islet graft rejection, indicating that neither CD8+ cells nor CD4+ cells alone were capable of mediating rejection, and that collaboration of CD8+ cells and CD4+ cells was necessary. On the other hand, in the BALB/c×B6 (H-2− and non-H-2-incompatible) combination, administration of anti-Lyt-2.2 (CD8) or anti-L3T4 (CD4) mAb resulted in rejection of most of the grafts, although survival was prolonged significantly, and administration of both anti-Lyt-2.2 (CD8) and anti-L3T4 (CD4) mAb together completely blocked rejection. These results suggested that either CD8+ or CD4+ cells were capable of mediating rejection, but that rejection was maximal in the presence of both T cell subsets. Immunohistochemical analyses showed marked depletion of CD8+ cells and CD4+ cells in grafted islets as well as spleens when anti-Lyt-2.2 (CD8) and anti-L3T4 (CD4) mAb, respectively, were injected.

Sinusoidal Capillarization and Arterial Blood Supply Continuously Proceed with the Advance of the Stages of Hepatocarcinogenesis in the Rat

Hepatocellular carcinoma (HCC) is supplied only with arterial blood and has capillaries instead of sinusoids, unlike the normal hepatic tissue. In order to reveal the sequential changes of sinusoidal structure and blood supply during hepatocarcinogenesis, we examined hyperplastic nodules (HPN), atypical hyperplastic nodules (AHPN) and HCC in F344 rats fed with 2‐acetylaminofluorene for 25–35 weeks. The extent of arterial blood supply and the degree of sinusoidal capillarization were assessed by the staining of hepatic nodules with arterially infused ink and by immunohistochemical and ultrastructural analyses of the sinusoids, respectively. The proportion of arterialized nodules was 47% in HPN, 57% in AHPN and 85% in HCC, which indicates that arterialization begins at the stage of HPN and a few HCC still receive portal venous blood. The proportion of Factor‐VIII‐related antigen (F‐VIII)‐positive nodules was 25% in HPN, 40% in AHPN and 100% in HCC. Non‐arterialized nodules did not express F‐VIII, while some F‐VIII‐negative nodules were already arterialized. Electron microscopically, non‐arterialized HPN exhibited normal features of sinusoids, while arterialized HPN showed disappearance of Kupffer cells and partial defenestration of sinusoidal endothelial cells. In non‐arterialized and arterialized AHPN, basement membrane became continuous and lipid droplet‐containing stellate cells were no longer seen. In HCC, endothelial fenestrae were diminished and basement membrane became thick. The present study has demonstrated that sinusoids are altered in the following order as hepatocarcinogenesis advances from HPN to HCC; onset of arterialization and decrease of endothelial fenestrae, expression of F‐VIII by endothelial cells, disappearance of Kupffer cells, diminution of lipid droplets in stellate cells and thickening of basement membrane.

Immunohistochemical investigation of pulmonary surfactant in perinatal fatalities

In order to verify forensic pathological significance of immunohistochemical investigation of pulmonary surfactant, 11 forensic and 16 clinico-pathological cases of perinatal death were comparatively examined. Surfactant appeared in some infants of 31-32 weeks gestation and was usually positive thereafter, indicating maturity of fetal lungs, although it may not have fully developed until about the 36th week of gestation. It was negative in all cases of the hyaline membrane disease except for a full-term infant (secondary respiratory distress syndrome). In usual cases, surfactant coating the expanded alveolar epithelia with its diffuse deposit in the intra-alveolar spaces was considered to indicate duration of hypoxia under persistent respiration (agonal state). Such finding was most intensely observed in asphyxia and in severe respiratory failure from intrinsic causes in the infants over ca. 36 weeks of gestation. With reference to pulmonary micromorphology, the amount of intra-alveolar surfactant seemed to be most closely related to the alveolar septal (interstitial) edema.

Altered expression of sialyl-Tn, Lewis antigens and carcinoembryonic antigen between primary and metastatic lesions of uterine cervical cancers

SummaryImmunohistochemical examination was performed of serial sections of 24 normal human adult cervical tissues and 53 human cervical carcinomas including 36 cases with lymph node metastasis. For this investigation, monoclonal antibodies directed to Lewis-X, Lewis-Y, sialyl-dimeric Lewis-X (SDLX), sialyl-Tn (STn) and carcinoembryonic antigen (CEA) were used. STn and CEA antigens were expressed very weakly in the normal cervical epithelium but strongly in the cancer cells, indicating the antigens to be oncogenic antigens of cervical squamous cell carcinoma. No significant difference in immunoreactivity was observed between primary and metastatic lesions of carcinoma or between primary lesions with and without metastasis. However, the expression patterns of STn and Lewis-Y antigens were quite different between primary lesions and metastatic lesions. In primary lesions the cancer cell nests tended to be stained centrally, but in metastatic lesions the cancer cell nests tended to be stained peripherally. This finding may reflect an important role of these carbohydrate chains in the process of metastasis of cervical squamous cell carcinoma to regional lymph nodes.

Histochemistry of tartrate-resistant acid phosphatase and carbonic anhydrase isoenzyme II in osteoclast-like giant cells in bone tumours

Using routinely processed, paraffin-embedded tissue specimens, osteoclast-like giant cells in giant cell tumour of bone (GCT), chondroblastoma, osteoblastoma and osteoblastic osteosarcoma were examined histochemically for osteoclast-specific enzymes tartrateresistant acid phosphatase (TRAP) and carbonic anhydrase isoenzyme II (CA-II). Osteoclast-like giant cells and some mononuclear cells possessed TRAP activity. These were further classified with respect to CA-II immunoreactivity, i.e. cells with CA-II were seen in GCT and chondroblastoma, while those in osteoblastoma and osteoblastic osteosarcoma were negative for CA-II. All the cellular components in malignant fibrous histiocytoma and various extraosseous inflammatory lesions including malignant giant cells and macrophage polykaryons were negative for both TRAP and CA-II. These results indicate that osteoclast-like giant cells in GCT, chondroblastoma, osteoblastoma and osteoblastic osteosarcoma are all osteoclasts and generated by fusion of mononuclear cells with the same histochemical characteristics as osteoclast-like giant cells. The difference in CA-II immunoreactivity suggests the functional or maturational difference between osteoclast-like giant cells in GCT and chondroblastoma and those in osteoblastoma and osteosarcoma.

Suppressive effect of the angiogenesis inhibitor TNP-470 on the development of carcinogen-induced hepatic nodules in rats

Tumor metastasis can be prevented by inhibiting angiogenesis. In the present study, we have demonstrated that the angiogenesis inhibitor TNP‐470 also suppresses the development of primary hepatic nodules. Hepatocarcinogenesis was performed by the feeding of 2‐acetylaminofluorene to hepatectomized rats during 8–14 weeks of age. Predominantly arterial‐to‐portal circulation and sinusoidal capillarization were determined by the staining of nodules with arterially infused ink and immunostaining for factor VIII‐related antigen, respectively. Intraperitoneal administration of 30 mg/kg b.w. of TNP‐470 twice a week significantly reduced the number of hepatic nodules. Among the nodules, hyperplastic nodules stained with ink, atypical hyperplastic nodules and hepatocellular carcinoma, all of which possess structurally altered sinusoidal endothelial cells or capillary‐type endothelial cells, were dramatically decreased in number. Suppression was observed equally in nodules of all sizes. TNP‐470 was more effective when administered during 8–20 weeks than during 14–26 weeks. In contrast, ink‐non‐stained hyperplastic nodules, which have normal sinusoidal endothelial cells, were not affected at all. The present results indicate that TNP‐470 suppresses the development of primary hepatic nodules whose microvessels are capillaries or transitional forms from sinusoids to capillaries.

Diagnostic Potential and Pitfalls of Ultrasound-guided Fine-Needle Aspiration Cytology for Breast Lesions

Ultrasound (US)-guided fine-needle aspiration cytology (FNAC) is now widely accepted as a diagnostic procedure for breast lesions. Along with its advantages, US-guided FNAC also has some pitfalls. The recognition of these pitfalls for this procedure is extremely important for the strict management of the disease. We retrospectively investigated the diagnostic potential and pitfalls of US-guided FNAC in the diagnosis of breast lesions. This study consisted of 348 aspirated samples from 274 breast tumors. The rate of sufficient aspirates was 74% after a single aspiration, while sufficient materials were finally obtained from 93% of the tumors by repeated aspirations. The rate was lower in tumors measuring less than 10 mm in diameter (62%), and in sclerosing adenosis (25%). The sensitivity of FNAC was 65%, the specificity was 75%, the border diagnosis rate was 18%, and the positive predictive value was 92%. The false-negative rate was higher in noninvasive carcinoma (45%). The border diagnosis rate was also higher in scirrhous carcinoma (29%). There were also five false-positive cases. Limited to nonpalpable lesions, the sufficient aspirates rate was 70% and the accuracy was 67%.

Characteristic Histologic Features of Human Hepatocellular Carcinoma with Mutant p53 Protein

Abstract. To characterize hepatocellular carcinoma (HCC) cells with mutant (m) p53 protein histologically, we examined 68 main nodules and 20 accessory lesions of 72 patients with HCC who underwent hepatic resection between October 1990 and September 1993. Some sections were fixed in periodate-lysine-paraformaldehyde, embedded in OCT compound, and stained with the mouse monoclonal antibody PAb1801 to m-p53 protein by the immunoperoxidase technique with avidin-biotin complexes. Other sections were fixed in 20% buffered formalin, embedded in paraffin, and stained with the mouse monoclonal antibody DO-1 to m-p53 protein in the same way. Lesions in which cells had nuclei stained for m-p53 protein were defined as being positive for the protein; 25 of the 68 main nodules and 14 of the 20 accessory lesions were positive. Large main nodules were more likely to be positive than small ones. Microscopic examination showed that a larger proportion of poorly differentiated main nodules than well differentiated nodules were positive. Larger proportions of main nodules with extracapsular invasion, septa, portal thrombi, or intrahepatic metastases were positive than main nodules without these features. Accessory lesions that seemed to be metastatic were almost all positive, but few accessory lesions that seemed to be of multicentric occurrence were positive. Our results suggest that lesions with m-p53 protein had a high grade of malignancy and metastasized readily.