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The effects of Estracyt®, HN2 (nitrogen mustard) derivative of estradiol-17β, and free HN2 on the cell kinetics of the estrogen receptor (ER) positive human endometrial cancer cell line HEC-1, were investigated using flow cytometry. HN2 at 1 μg/ml showed a marked increase in the S phase and a decrease in the G0 + Gi phase. However, with equivalent doses of Estracyt® at 10 μg/ml, the accumulation in the G2 + M phase was even more marked. Synchronization in the S phase with methotrexate (MTX) showed no increase in sensitivity to these drugs. The above results suggest that the free HN2 has an effect regardless of the cell cycle phase, whereas the effects of Estracyt® may depend on a fixed population of target cells existing in the G0+G1, S and G2 + M phases. Synchronization in the G1 phase with sodium n-butyrate could increase the target effects of Estracyt® in the S phase.

Objectives: The aim of the present study was to investigate the effectiveness of sorting fetal nucleated red blood cells (FNRBC) from maternal peripheral blood, particularly during early gestation periods, by a combination of specific gravity centrifugation and magnetic cell sorter (MACS). Methods: Without prior knowledge of the gender of the fetus, we determined gender by analyzing a Y-chromosome specific sequence by nested-PCR, using 10 ml of the peripheral blood of healthy primigravida women at different stages of gestation (first trimester: n = 17, second trimester: n = 13, and third trimester: n = 19). The results of this prenatal sex determination were compared to the sex of newborns. Results: The specificity, sensitivity, positive predictive value (PPV) and negative predictive value (NPV) of the present method during the first trimester were 100, 81.8, 100, and 75%, respectively; during the second trimester, 80, 50, 80, and 50%, respectively; and during the third trimester, 25, 63.6, 53.8, and 33.3%, respectively. Conclusion: The results show that this prenatal sex determination method has a highly accurate diagnostic rate during the first trimester, suggesting that it could be developed as a practical, non-invasive prenatal diagnostic technique for use during early gestation periods.

We describe here the case of an 82-year-old woman presenting with a hemorrhagic tumor on the anterior vaginal wall. She was diagnosed with malignant fibrous histiocytoma (MFH) from the findings of cytological analysis of biopsied surface tissue, histopathologic analysis of biopsied tissue, immunohistochemical staining, and electron microscopy. Cytological analysis of the biopsy sample harvested from the tumor surface showed multinucleated giant cells and large atypical cells with rough, granular, chromatin, as well as notable nucleoli. A storiform pattern was observed histopathologically, and immunohistochemical staining confirmed positive reactions to alpha 1-antichymotripsin (alpha 1-ACT), vimentin, and lysosome, but negative reactions to epithelial membrane antigen (EMA), cytokeratin, and alpha-smooth muscle action (alpha-SMA). Electron microscopy showed histiocyte-derived cells with a segmented nucleus with a large groove, pseudopodic cytoplasmic projections, and lysosome-like structures. However, intercellular adhesion factors were not notable, and microvilli were absent. Based on the above findings, a diagnosis of MFH originating from the vaginal wall was made. Because of the patient's advanced age, and, in accordance with her wishes, three courses of cancer chemotherapy, consisting of doxorubicin hydrochloride, fluorouracil, and cisplatin were carried out, without surgery. No reduction in the size of the tumor was seen at follow up, and despite the absence of metastasis and no exacerbation of her general condition, she died suddenly at home 2 years after being discharged.

目的:CAS200画像解析装置およびAutopapシステムを用いて子宮頸癌細胞診自動化スクリーニングの基礎的検討を行った.方法:子宮頸癌集団検診受診者, 1481例より子宮頸部搾過検体を採取し標本を作製した. 作製した標本をCAS200画像解析装置にて測定し, 測定結果を日本臨床細胞学会認定細胞検査士によって判定された細胞診判定結果と比較検討した. 次いで日本臨床細胞学会認定細胞検査士が鏡検し, すでに結果が判明している集団検診検体3232検体に対し, AutopapシステムのPrimary Screening用programを用いて検査対象の75%を要再検 (PS-Review) とする設定により検討を行った.成績:CAS200画像解析装置を用いて子宮頸癌集団検診受診者, 計1481例に対し子宮頸癌細胞診自動化スクリーニングを行った結果, 正診率は87.0%, 偽陽性率は13.7%であり, 偽陰性率は4.4%であった.Autopapにより測定された全検体数は, 3232検体であり, この中で測定可能検体は, 2741検体 (84.8%), 測定不能検体は491検体 (15.2%) であった. 測定可能であった2741検体の内, No Further Reviewと判定された検体は, 540検体 (19.7%) であり, CT Reviewと判定された検体は, 2201検体 (80.3%) であった. CT Review2201検体におけるランキング別の陰性, 陽性検体の割合は, それぞれ陰性2181検体 (99.1%) と陽性22検体 (0.9%) であった. しかし, 22検体中におけるClass VあるいはClass IIIaと判定された2検体は低いランクにとどまった.結論:Autopapシステムによる子宮頸癌細胞診自動化スクリーニングのためには, 1) 測定不能検体を少なくするためのより良い標本作成法の開発, 2) ソフトの改変による『しきい値』の改善 (50%以下), 3) 細胞診の異型度とAutopapのランキングの相関性についての検討が必要であると考えられた.

Ovarian tumors with osteoclast-like multinucleated giant cells are rarely encountered, and the existence of osteoclast-like giant cells in ovarian tumors may have no effect on the patients' prognosis. We report a patient with ovarian serous adenocarcinoma containing osteoclast-like multinucleated giant cells in the anaplastic areas of the tumor, who had a poor prognosis. A recurrent tumor showed the same anaplastic appearance as that of the primary tumor, with osteoclast-like giant cells. Forty-one percent of tumor cells were positive for MIB-1 (Ki-67) antigen, and many cells overexpressed p53 protein. However, the giant cells were negative for both antigens, which meant that they were non-cycling cells. The giant cells were also negative for epithelial and non-epithelial antigens. The multinucleated osteoclast-like giant cells, as well as mononucleated tumor cells around the giant cells, expressed p21 (WAF1/ClP1) protein. These findings suggest that the multinucleated osteoclast-like giant cells in this tumor originated from the p21-expressing mononuclear tumor cells. The coexistence of anaplastic foci with multinucleated giant cells in serous adenocarcinomas of the ovary may indicate a poor prognosis.

The cyclin D1 gene is often amplified in solid tumors. Six human melanoma cell lines derived from tumors at various stages of progression were analyzed by double-target fluorescence in situ hybridization (FISH) and dot blot hybridization to detect cyclin D1 gene amplification and deletion. FISH was performed using a probe for cyclin D1 and an alpha satellite of the chromosome 11 probe, to analyze the cyclin D1 copy number relative to the numbers of chromosome 11 in individual cells. Copies of both the cyclin D1 gene and chromosome 11 were observed in the nuclei of all cell lines. However, the copy numbers of the cyclin D1 gene and of chromosome 11 were closely associated and the copy number ratio of cyclin D1 to chromosome 11 (cyclin D1/chromosome 11) was almost one in all cell lines. This finding indicated that the extra copies of cyclin D1 were always accompanied by extra copies of chromosome 11. At the cellular level, cyclin D1 amplification was detected in fewer than 5% of interphase nuclei in only two cell lines, and the gene was deleted in all cell lines to various degrees. Dot blot hybridization revealed no amplification of cyclin D1 in any of the cell lines. We concluded that amplification of the cyclin D1 gene is rare in these melanoma cell lines.

Dual color fluorescence in situ hybridization (FISH) analysis was applied to 22 squamous cell carcinomas (SCC) of the skin in order to detect deletion of the p53 tumor suppressor gene in interphase nuclei using a cosmid probe for p53 and a centromeric probe for chromosome 17. Fewer p53 signals than 17 centromeric signals and zero or one cosmid signals were observed in 5.6±4.9% (mean±SD) of controls. Based on this, 20% deletion was established as the cutoff line to define deletion of the p53 gene. Twenty of the 22 SCC cases demonstrated p53 gene deletion according to this criterion. The percentage of cells with deletion ranged from 22% to 59% (33.9±10.9%). The ratio between the copy number of chromosome 17 centromeres and p53 in the predominant population of the tumor nuclei with p53 gene deletion was either 2/1 or 1/1. Moreover, the major subfraction was composed of chromosome 17 disomic cells in all cases. This suggests that either p53 gene deletion of one allele in disomic cells or the loss of an entire chromosome 17 in aneusomic cells is the main mechanism of p53 gene deletion in SCC of the skin. Immunohistochemical p53 expression was frequently associated with the p53 gene deletion detected by FISH.

細胞遺伝学的知識の集積に伴って染色体分析の必要性と需要が増し, 簡便で迅速な検査法の確立が望まれている.しかし, 分裂した細胞のみを対象とする従来の染色体分析法では熟練と時間が要求され, また培養に伴う細胞群の選択がおこる.蛍光インサイツーハイブリダイゼーション (FISH法) は間期細胞核においても染色体分析を可能にした (間期細胞遺伝学).ハプテン化染色体特異的プローブを用いたFISH法は, 染色体数の計算と数的異常の検出に汎用されてきたが, 約48時間を要し, ハプテン検出に伴う煩雑な免疫細胞化学的処理過程が必要である.この煩雑さを解消するため, 蛍光色素直接標識セントロメアプローブを用いて, 18トリソミー, 21トリソミーおよびXXYの染色体異常患者の末梢リンパ球にFISH法を適用したところ, おのおのの症例に対応するコピー数が得られた.しかも所要時間を2時間まで短縮できたうえ, 操作段階を半減することが可能であった.このように, 蛍光色素直接標識プローブによるFISH法は, 染色体の数的異常の, 迅速かつ手技的間違いのない簡便な検査法として, 染色体数異常疾患の診断に有用であることを明らかにした.

A case of a right ovarian tumor in a 64-year-old patient showing high blood levels of alpha-fetoprotein (AFP) is reported. Histologically, the tumor resembled hepatocellular carcinoma with hyaline globules. Localization of AFP was detected by the immunoperoxidase method. Electron microscopically, the rough-surfaced endoplasmic reticulum had developed into a meshwork, and the mitochondria were present within this meshwork. Because a transition from adenocarcinoma to a region resembling hepatocellular carcinoma was observed, this tumor was considered to originate as a common epithelial carcinoma. In the blood, 67% of the AFP was bound with concanavalin A (Con A), and the fraction pattern obtained by lentil agglutinin affinity chromatography (LCA) was of the germ cell type. From these results, the current case may be labeled clinicopathologically a hepatoid carcinoma of the ovary as described by Ishikura and Scully.

Sodium butyrate (SB) treatment was previously shown to produce seven-fold increases in estrogen hormone receptor binding sites of human endometrial adenocarcinoma (IK) cells. Flow cytometric analysis and histone gel electrophoresis were used to examine cell cycle, cell metabolism, and nuclear histone fractions in IK cells treated with different concentrations of SB. SB-treated cells stained with fluorochromes specific for DNA, RNA, or general protein were analyzed by flow cytometry (FCM). Changes in accessibility to three DNA stains and gel electrophoresis were used to analyze rearrangements in chromatin structure. SB caused an accumulation of cells in the G1 phase and inhibited DNA synthesis, but not cellular levels of RNA and protein. Hoechst accessibility to A-T rich regions on DNA was dramatically increased after removal of SB. H1 histones were dephosphorylated and core histones were acetylated during SB-treatment. Information obtained in these studies may be useful for correlating cellular and biochemical events with SB-induced increases in nuclear steroid hormone binding sites.

Four cases of ovarian cancer (mucinous cystadenocarcinoma, serous cystadenocarcinoma, endometrioid carcinoma and metastatic cancer of the colon) which revealed diffuse localization of carbohydrate antigen 19-9 (CA 19-9) in their tumor tissues were subjected to immunoelectron microscopy. In all cases CA 19-9 was present in plasma membranes, microvilli and glycocalyx in mucinous and metastatic carcinoma, indicating that the antigenic determinant for CA 19-9 is sialylated Lewis A. Reaction products were seldom seen in perinuclear spaces and endoplasmic reticula. Many pictures revealed positive vesicles apparently released from the Golgi apparatus; this feature seemed to be characteristic of the findings. Thus, the Golgi apparatus is considered to be the place where immunoreactive CA 19-9 is first produced in cancer cells. Since positive images were found not only in organelle but also on collagen fibers directly under the basement membranes, it seemed that an important factor for CA 19-9 increase in the blood is the expansion of CA 19-9 to stroma.It was strongly suggested that CA 19-9 was useful as a tumor marker for ovarian cancer which originated in common epithelial cells.

Immunohistochemical localization of Tumor Antigen 4 (TA-4) in squamous cell carcinoma (SCC) tissues of the uterine cervix was studied by means of the immunoperoxidase methods. Light microscopic detection of TA-4 was carried out by means of the avidin-biotin-peroxidase complex method on formalin-fixed paraffin-embedded sections from 92 cases of invasive cervical cancer of the uterus. These tumors were histologically SCC, consisting of 68 of the large cell non-keratinizing (LNK) type and 24 of the keratinizing (K) type. TA-4 positive cells were detected in 65% of LNK cases and 100% of K cases. Positively stained neoplastic cells were seen frequently around the hyperparakeratotic lesions, and they showed a tendency to keratinization.TA-4 localization was also confirmed in the cells of stratum spinosum of the non-cancerous squamous epithelium.Subcellular localization of TA-4 was studied by the preembedding indirect immunoperoxidase method. TA-4 was localized in the cytosols of the neo-plastic cells, but not in tonofilaments. It was suggested that TA-4 was a structural protein of SCC. Therefore, unlike keratin, TA-4 can be considered as an index of differentiation of SCC cells, and TA-4 should be useful as a tumor marker which expresses the characteristics of the neoplastic tissue during squamous maturation.

It has been reported that the response of target cells to steroid hormone (SH) stimulation may depend on their position in the cell cycle. The DNA and RNA contents of malignant cells of the endometrium cultured in vitro were measured using flow cytometry (FCM). We also measured estrogen receptor (ER) and progesterone receptor (PR) levels of cells at different positions in the cell cycle. The G1 and S phases of the cell cycle were investigated using cells synchronized by sodium n-butyrate (G1 block), methotrexate (S block), and excess thymidine (S block). For DNA measurements, the cells were stained with propidium iodide following RNase treatment. For RNA measurements (double-straded RNA) the cells were treated with DNase. We found that S phase synchronization by methotrexate was 136.2% of control (100%). Using the excess thymidine block and release procedure, the S phase fraction was 185.1% of control. G1 phase synchronization by sodium n-butyrate was 134% of control. The estrogen receptor level in G1 phase synchronized cells increased to 5.94 fmol/μg DNA in the cytosol and 12.35 fmol/μg DNA in the nuclear fraction. These levels represent a sevenfold total increase over that of the control estrogen receptor level. Cells in S phase showed no significant increase in estrogen receptor levels over control cells. Based on this study, the functional increase of the steroid receptor was most significant in the G1 phase.

1) 子宮頸部擦過細胞標本10万403検体中21検体 (0.02%) にいわゆるヘマトイジン結晶を認めた.2) 21例中19例の標本は炎症所見が認められ, 7例では背景に出血を認めた. また21例中7例は, 妊娠または分娩後1年以内の症例であった.3) ヘマトイジン結晶は, 黄褐色または赤褐色で直径2u～100uの放射状の結晶であり・標本中には1～28個の結晶が散在し, その周囲には組織球や好中球がロゼット様に分布していた.4) ヘマトイジン結晶の認められた21例中16例の標本に特殊染色を行った結果・PAS染色陽性, アルシアン青陰性, スタインのヨード法陰性, ベルリン青染色陰性, 酵素抗体染色 (フェリチン) 陰性であった. また, PAS染色陽性であったヘマトイジン結晶は, ジアスターゼ・アミラーゼによっても消化されないことより, この物質は血液中の蛋白物質の結晶であることが示唆された.5) 以上のことよりヘマトイジン結晶という呼称は適当ではなく, 再検討が必要である.

The cytologic findings of a hemangioendotheliomaof the uterine cervix are reported. Vaginal and cervical smears were initially diagnosed as suspiciousfor malignancy, then the atypical cells in these specimen were diagnosed as the tumor cell by a induction from observation of imprint and histologicalsections of the cervical hemangioendothelioma.The cytomorphological findings of the tumor cellin vaginal and cervical smears are as follows:1. Structure of nucleus: Round or oval nucleiwith thin nuclear membranes and small unequalchromatin granules. A few small nucleoli are bluishred.2. Nuclear size: 12.5-10.0μ×10.0-7.5μ.3. N/C ratio: Moderately increased.4. Shape of cell: Polymorphic. Occasionally it ispossible to find a pseudopod-like projection.5. Appearance and staining reaction of cytoplasm;Thin and greensh-blue.

1553 women over 30 years of age were tested for uterine cancer. 72 (4.6%) of these women had had the IUD inserted and had worn the device for an average of 3.1 years. 46 (64%) patients had Class 1 Papanicolaou smears and 2 (2.8%) had Class 3 smears compared with 42% Class 2 smears 6.2% Class 3 smears and .5% Class 4 smears in the non-IUD group. It is concluded that the arguments used by those opposing the use of IUDs on carcinogenic grounds are flimsy.

We investigated the G1 and S phases of synchronization using sodium n-butyrate, MTX and excess thymidine. The flow cytometry system was employed for cell cycle analysis while the receptor assay was adopted dextran coated charcoal (DCC) and the wash method. The results were as follows: S phase synchronization by MTX was 138% (control 100%) and by excess thymidine in the block and release method 210%. G1 phase synchronization by sodium n-butyrate was 140%. The progesterone receptor level, by E2 priming increased to 1.45 fmol/ug DNA being more than five times that of the control PR. The estrogen receptor level increased to 18.29 fmol/ug DNA in the G1 phase synchronization, seven times that of the control ER. From this study, the functional increase of the steroid receptor was most significant in the G1 phase.