Yoshiaki Norimatsu

New Terminology for Intrauterine Endometrial Samples: A Group Study by the Japanese Society of Clinical Cytology

Objective: To evaluate the sensitivity and specificity of endometrial cytology obtained by intrauterine sample using a descriptive reporting format for endometrial cytological diagnosis. Study Design: 10,152 consecutive endometrial scrapings obtained in 13 different Japanese hospitals were analyzed. Cytological results were classified as ‘negative for malignancy’, ‘atypical endometrial cells’ (ATEC), ‘endometrial hyperplasia’, ‘atypical endometrial hyperplasia’ or ‘malignant tumor’. ATEC was subclassified as ‘ATEC, of undetermined significance’ (ATEC-US) and ‘ATEC, cannot exclude atypical endometrial hyperplasia or more’ (ATEC-A). Cytological results were compared with the histological diagnosis as a gold standard. When the cytological result was ‘negative for malignancy’ and there was no subsequent histological examination, the case was considered a true negative when the endometrium was assessed as normal on transvaginal ultrasonography and there was no abnormal uterine bleeding. Results: 1,083 cases in which histology was not performed, 557 cases of ‘unsatisfactory specimen’ and 76 cases of ATEC-US were excluded. In the remaining 8,436 cases, the sensitivity and specificity, positive predictive value and negative predictive value for detecting atypical endometrial hyperplasia or malignant tumors were 79.0 and 99.7, 92.9 and 98.9%, respectively. Conclusion: The current diagnostic standards for endometrial cytology in Japan were established. Specificity is satisfactory for excluding cancer or precancerous diseases.

Cytologic features of the endometrial adenocarcinoma: Comparison of ThinPrep and BD surepath preparations

We compared the cytoarchitectural features used for the cytologic diagnosis of endometrial adenocarcinoma (EC) using ThinPrep® (TPS = ThinPrep Sample) and BD SurePath™ (SPS = SurePath Sample) preparations. In 20 patients, a direct endometrial sample using the Uterobrush was obtained. Nineteen cases of EA and one case of carcinosarcoma were studied. TPS and SPS were performed according to the manufacturers recommendations. Moreover, after the TPS preparation, the residual material was also used to prepare an SPS sample (TP–SPS = ThinPrep–Surepath sample). The following points were investigated in both preparations: (1) number of cell clumps; SPS had a significantly higher (20.9) than TPS (1.7) and TP–SPS (10.3); (2) long axis of clumps; SPS had a significantly higher (215.4) than TPS (146.0); (3) rate of cell clumps with longer axes than 200 μm; SPS had a significantly higher (36.7) than TPS (15.2) and TP–SPS (24.2). TP–SPS showed higher values than TPS; (4) nuclear area; TPS had a significant higher (61.2) than SPS (40.8) and TP–SPS (38.6); (5) degree of overlapping nuclei; SPS (3.4) had a significantly higher number of overlapping nuclei than TPS (0.7) and TP–SPS (2.1); (6) nuclear chromatin pattern; no significant differences for the nuclear chromatin pattern were found in the three different methods. The poor performance of TPS versus SPS and TP–SPS was explained with the heavy blood contamination of the samples, and the absence of adhesive coating in the slides is used for TPS. Further investigation of technical differences in liquid‐based cytology methodologies is needed. Diagn. Cytopathol. 2013;41:673–681.

Evaluation of Endometrial Cytology Prepared with the Becton Dickinson SurePath™ Method: A Pilot Study by the Osaki Study Group

Objective: To evaluate the sensitivity and specificity of the BD SurePath™ liquid-based Papanicolaou test for assessing the cytology of intrauterine endometrial samples according to newly devised cytological diagnostic criteria and a novel descriptive reporting format. Materials and Methods: One hundred and twenty-two endometrial samples were analyzed. All samples were obtained directly from the intrauterine cavity using the Uterobrush or Honest Super Brush. The samples used for the histological examination and cytological tests were collected simultaneously. Our study group devised new cytological diagnostic criteria for examining endometrial samples: the Osaki Study Group method. In this study, histological diagnosis was considered to be the gold standard for cytological diagnosis. A novel descriptive reporting format was also used. Results: Satisfactory cytological specimens were obtained in all cases. The sensitivity and specificity of the SurePath endometrial cytological examination method were 96.4 and 100%, respectively. Conclusion: These results indicate that the SurePath method is acceptable for clinical use. Since the SurePath method seems to be easier and allows greater preparation standardization than the conventional method, coupling it with our newly devised cytological diagnostic criteria and descriptive reporting format might represent a reliable diagnostic method for assessing endometrial specimens.

The Role of Liquid-Based Preparation in the Evaluation of Endometrial Cytology

Objective: Liquid-based preparation (LBP) of the endometrial lesions is an important diagnostic tool for a variety of endometrial abnormalities because of its simplicity and high quali-quantitative diagnostic yield. We aimed to investigate the LBP method for endometrial cytology to evaluate both benign and abnormal endometrial lesions. Study Design: LBP is a semiautomated methodology that has recently become widely available and has gained popularity as a method of collecting and processing both gynecologic and nongynecologic cellular specimens. Results: Some peculiar endometrial cytoarchitectural features were described using LBPs. These were advantageous to screen as compared to conventional slides due to a smaller screening area and an excellent quality of cell preparations. Conclusions: LBP is a useful tool in the cellular diagnosis and follow-up of endometrial abnormalities, which remains complementary to the emerging molecular diagnostic cytopathology. The study of LBPs from endometrial cytology could be challenging since it is affected by numerous look-alikes and diagnostic pitfalls. This review discusses these various entities and takes into consideration the ancillary techniques that may be useful in the diagnostic procedure.

Use of liquid‐based preparations in urine cytology: An evaluation of liqui‐PREP™ and BD SurePath™

Dear Dr. Bedrossian: Conventional smears are tedious and time-consuming to screen due to nonuniform slide preparation and fixation. Features usually associated with conventional smears, such as, thick, overlapping cellular areas, obscuring inflammation, and blood and air-drying artifact result in poor cellular and nuclear preservation. Liquid-based preparations (LBP) are increasingly being used both for gynecologic and nongynecologic cytology including urine and fine-needle aspirations. Urine cytology comprises a large proportion of nongynecological specimens processed in most routine cytology laboratories. A recent paper reported on a larger series comparing conventional urine preparations with LBP. Liqui-PREP (LPR: LGM International, Fort Lauderdale, FL) and BD SurePath (BSP: BD Diagnostics, Franklin, NJ) are two commercially available LBP methods. In this study, the differences between LPR and BSP were evaluated for a variety of parameters including cellularity, cytomorphology, background features, etc. The material consists of 51 fresh voided urine samples from 25 patients with known urothelial carcinoma, which were obtained from January 2008 to June 2009 at the Mihara Medical Associations Hospital and the Shigei Medical Research Hospital. All patients provided informed consent. To equate cell density of LPR and BSP sample, urine samples were split equally. Each half of the urine samples was centrifuged at 3000 rpm for 5 minutes, and the supernatant fluid was discarded. Using a micropipette, 50 lL cell pellets were obtained and poured into a 15 mL centrifuge tube. Then, 1 mL of LPR preservation fluid and BSP CytoRich Red preservative fluid were added into centrifuge tube, respectively. After 30 minutes fixation time, LPR and BSP sample tubes were centrifuged at 3000 rpm for 10 minutes, and the supernatant fluid was discarded. As for the LPR sample, 0.2 mL of LPR Cellular Base Reagent was added in LPR sample tubes, and the cell pellet was resuspended using a vortex for 10 second. Following this step, 100 lL of the suspension were pipetted onto the slide to form a 2.0 cm diameter circle. Two LPR specimens were prepared for each case and were then dried and stained by the Papanicolaou method (Fig. C-1). As for the BSP sample, 0.2 mL of distilled water was added in BSP sample tubes, and the cell pellet was resuspended using a vortex for 10 second. After resuspension, 100 lL of the suspension was transferred into small plastic chambers, mounted on microscope slides, and fixed with 95% ethanol. Two BSP specimens were prepared for each case and were immediately stained by the Papanicolaou method (Fig. C-1). Four of the authors (Y. N., N. K., Y. S., and T. K.) microscopically analyzed all the preparations.

Ketoprofen in topical formulation decreases the matrix metalloproteinase-2 expression and pulmonary metastatic incidence in nude mice with osteosarcoma

The aim of this study was to investigate whether ketoprofen (KP) in topical formulation affected the tumor growth and pulmonary metastasis of LM8 cells, which were inoculated subcutaneously into the back space of male nude mice. At 7 days after inoculation, the tumor was treated topically for 3 weeks with either a KP‐containing patch (KP group) or a placebo‐containing patch (placebo group). The pulmonary metastatic incidence was 100% in the placebo group and 60% in the KP group. The tumor mass of the KP group without pulmonary metastasis, termed the KP/metastasis(−) group, was smaller than that of the placebo group. Immunohistochemical staining for proliferating cell nuclear antigen (PCNA), terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labeling (TUNEL), matrix metalloproteinase‐2 (MMP‐2), and vascular endothelial growth factor (VEGF) was performed. The tumors of the KP/metastasis(−) group contained fewer PCNA‐positive cells and many more TUNEL‐positive cells in comparison to the placebo group. In the placebo group, MMP‐2 and VEGF were extensively expressed within the tumor, whereas in the KP/metastasis(−) group the expression of these two proteins was very low. In conclusion, the topical treatment of osteosarcoma with KP decreased the expression of MMP‐2 and VEGF, thus resulting in the suppression of tumor growth and pulmonary metastasis.

Nuclear characteristics of the endometrial cytology: Liquid‐based versus conventional preparation

The aim of this study was to assess the utility of liquid‐based cytologic preparation (LP) compared with conventional preparation (CP) for the assessment of nuclear findings in endometrial glandular and stromal breakdown (EGBD) which may be misdiagnosed as carcinoma in EGBD cases. The material consists of cytologic smears including 20 cases of proliferative endometrium (PE), 20 cases of EGBD, and 20 cases of endometrioid adenocarcinoma grade1 (G1) for which histopathological diagnosis was obtained by endometrial curettage at the JA Suzuka General Hospital. Nuclear findings were examined in PE cells, EGBD‐stromal cells, EGBD‐metaplastic cells, and G1 cells, respectively. It was examined about the following items; (1) nuclear shape; (2) A long/minor axis ratio in cell nuclei; (3) an area of cell nuclei; (4) overlapping nuclei. Results are as follows: (1) nuclear shape; as for the reniform shape of EGBD‐stromal cells and spindle shape of EGBD‐metaplastic cells, the ratio of the LP method was a higher value than the CP method. (2) The long axis and area of cell nuclei; LP in all groups was a recognizable tendency for nuclear shrinkage. (3) The long/minor axis ratio in cell nuclei; only EGBD‐metaplastic cells recognize a significant difference between CP and LP. (4) Overlapping nuclei; LP was a higher value in comparison with CP in the other groups except PE cells, and the degree of overlapping nuclei was enhanced about three times. Therefore, although a cell of LP has a shrinking tendency, (1) it is excellent that LP preserves a characteristic of nuclear shape than CP; (2) a cellular characteristic becomes clearer, because three‐dimensional architecture of LP is preserved of than CP. As for the standard preparation method for endometrial cytology samples, we considered that a concrete introduction of the LP method poses no problems. Diagn. Cytopathol. 2013.

Endometrial glandular and stromal breakdown, part 3: cytomorphology of "condensed cluster of stromal cells".

The aim of this study was undertaken to clarify the cytological characteristic of the “condensed clusters of stromal cells,” which may be recognized in endometrial glandular and stromal breakdown (EGBD) cases. The material consists of 60 cases of cytologic smears for which histopathological diagnosis was obtained by endometrial curettage; they comprised 30 cases of EGBD and 30 cases of endometrioid adenocarcinoma grade 1 (G1).

Genistein inhibits cell invasion and motility by inducing cell differentiation in murine osteosarcoma cell line LM8

BackgroundOne of the problems associated with osteosarcoma is the frequent formation of micrometastases in the lung prior to diagnosis because the development of metastatic lesions often causes a fatal outcome. Therefore, the prevention of pulmonary metastases during the early stage of tumor development is critical for the improvement of the prognosis of osteosarcoma patients. In Japan, soy is consumed in a wide variety of forms, such as miso soup and soy sauce. The purpose of this study is to investigate the effect of genistein, an isoflavone found in soy, on the invasive and motile potential of osteosarcoma cells.MethodsLM8 cells were treated for 3 days with various concentrations of genistein. The effect of genistein on cell proliferation was determined by DNA measurement in the cultures and 5-bromo-2’-deoxyuridine (BrdU) incorporation study. The assays of cell invasion and motility were performed using the cell culture inserts with either matrigel-coated membranes or uncoated membranes in the invasion chambers. The expression and secretion of MMP-2 were determined by immunohistochemistry and gelatin zymography. The subcellular localization and cellular level of β-catenin were determined by immunofluorescence and Western blot. For examining cell morphology, the ethanol-fixed cells were stained with hematoxylin-eosin (H&E). The expression of osteocalcin mRNA was determined by reverse transcription-polymerase chain reaction (RT-PCR).ResultsGenistein dose-dependently inhibits cell proliferation. Genistein-treated cells were less invasive and less motile than untreated cells. The expression and secretion of MMP-2 were lower in the genistein-treated cultures than in the untreated cultures. β-Catenin in untreated cells was located in the cytoplasm and/or nucleus, while in genistein-treated cells it was translocated near to the plasma membrane. The level of β-catenin was higher in genistein-treated cells than in untreated cells. Treatment of LM8 cells with genistein induced morphological changes, markedly decreased the formation of multilayer masses of cells, and markedly increased the expression of osteocalcin mRNA.ConclusionsGenistein decreased invasive and motile potential by inducing cell differentiation in LM8 cells. Genistein may be useful as an anti-metastatic drug for osteosarcoma through its differentiation-inducing effects.

Expression of immunoreactivity and genetic mutation in eosinophilic and ciliated metaplastic changes of endometrial glandular and stromal breakdown: cytodiagnostic implications

Various metaplastic changes may be present in endometrium, in which also cellular atypias may often be observed. Particularly, eosinophilic and ciliated changes (ECCs) occur in both nonneoplastic and neoplastic endometrium. This may cause confusion in the cytodiagnosis. This study was enterprised to investigate the possible help of immunocytochemical and cytogenetic study in the diagnostic and biologic assessment of ECC cells. In immunocytochemistry for p53 protein, Ki-67, and cyclin A, the material consists of 40 cases of cytologic smears examined by direct sampling of the endometrial cavity comprising 30 cases of ECC in endometrial glandular and stromal breakdown (EGBD) and 10 cases of well-differentiated adenocarcinoma (G1). After cytodiagnosis, immunostaining for p53 protein, Ki-67, and cyclin A was performed on multiple wet-fixed slides from each single case to evaluate the immunoreactivity, intensity of nuclear staining, and nuclear labeling index (N-LI). The intensity of nuclear staining was scored as negative (0), weak (1), moderate (2), or strong (3), and the N-LI was scored as less than 10% (0), from 10% to 25% (1), from 26% to 50% (2), or more than 50% (3), and the final score was calculated by adding both partial scores. A statistical significance test was performed by using Mann-Whitney U test, and the result was judged as significant when the P value was less than .05. For genetic mutation analysis of p53, the material comprised 6 cases of EGBD in which a high score was measured with immunocytochemistry for p53 protein, and the presence of ECC was confirmed on the hematoxylin and eosin. The ECC cells on paraffin-embedded specimens were captured using laser capture microdissection technology. Mutations in p53 gene (exons 5-8) were examined using DNA sequencing analysis. In immunocytochemistry for p53 protein, Ki-67, and cyclin A, the proportions of immunoreactive cells for p53 were statistically higher in ECC compared with those of G1 (P < .05). The proportions of the immunoreactive cells for Ki-67 and cyclin A were statistically higher in G1 compared with those of ECC (P < .05). (2) In genetic mutation analysis of p53, DNA sequencing of p53 in 6 cases revealed no mutations. The percentage of immunoreactive cells for p53 protein were higher in ECC than in G1, but the mutation point was not confirmed in genetic mutation analysis. The differential expression of these biologic parameters in ECC cells could be considered of possible relevance to the cytopathologic diagnosis in the future.