Haruhiko Yoshioka

Discriminant Analysis of Malignant Mesothelioma and Reactive Mesothelium Using Three-Dimensional Nuclear Estimation

Objective: The cytological diagnosis of coelomic fluid is essential for examining malignant mesothelioma (MM). However, reactive mesothelium (RM), caused by various factors, is morphologically similar to MM and thus often complicates the differential diagnosis. Here, nuclear luminance and steric alterations were assessed for the discriminant analysis of MM and RM. Study Design: Thirteen epithelial MM and 11 RM cases were included. One hundred alterations in the numbers of nuclear pixels and focus layers and the coefficient of variation of nuclear luminance among layers were determined for each case to conduct discriminant analysis using the Mahalanobis distance. Results: A cutoff value of 0.072 allowed highly accurate discrimination of MM (89.5%) and RM (89.6%). Fifteen cells appeared in 6 agglomerates of indiscriminable MM cases. The 6 agglomerates were individually examined. Malignant cells were dominant in 3 agglomerates (50%), allowing the discrimination of malignant cases. Conclusion: Discrimination using nuclear luminance and steric alterations is useful for morphologically indiscriminable MM cases. Three-dimensional analysis of agglomerates will be further investigated to improve the diagnostic accuracy.

Usefulness of Discriminant Analysis Using Nuclear Three-Dimensional Analysis in Atypical Cells Prepared from Bronchial Brushing Cytology Cases That Are Cytologically Challenging

Objective: Since well-differentiated adenocarcinoma cells of the lung (G1 cancer cells) show mild atypia, their differentiation from benign columnar epithelial cells (benign cells) is often difficult based on morphology. We performed discriminant analysis to distinguish benign from malignant cells by measuring 3-dimensional (3D) changes in nuclear luminance. Study Design: Discriminant analysis of 231 atypical cells prepared by bronchial brushing cytology, which were difficult to morphologically classify as benign or malignant, was performed using 100 G1 cancer cells. One hundred benign cells of bronchial brushing cytology specimens served as controls. The number of pixels of the nucleus, the number of focus layers and the level of change in the coefficient of variation (CV) of nuclear luminance between layers (3D-CV) were used as analytic parameters, and benign cells were discriminated from malignant cells based on the Mahalanobis distance. Results: As a result of discriminant analysis using a cutoff value determined in the control group, about 90% of the atypical cells difficult to classify as benign or malignant could be classified. Conclusion: For cells difficult to morphologically classify as benign or malignant, discriminant analysis based on 3D changes in nuclear luminance may be useful. This method can provide objective parameters for cancer diagnosis.

Usefulness of Evaluation of Nuclear Color by Visible-Microscopic Spectroscopy for Objective Differentiation between Non-Cancer and Cancer Cells Prepared Using Liquid-Based Cytology

Objective: The aims of this study were to investigate the usefulness of color evaluation of the nuclear region using visible-microscopic spectroscopy (Vis-MS) and to clarify whether it can serve as an index to distinguish cancer cells in liquid-based cytology (LBC). Vis-MS is a spectral analysis technique widely used for absorption and fluorometric analyses in the analytical chemistry field. Vis-MS has been applied to histological diagnosis, but only a few studies on its application to cytology have been performed, and no investigations have been performed for LBC, which is expected to become widely used in Japan. Study design: Using culture cell lines of non-cancer cells and cancer cells, transmittance at 530 nm (maximum absorption wavelength of eosin), 580 nm (hematoxylin), and 630 nm (light green), and 530 nm/580 nm and 630 nm/580 nm transmittance ratios were analyzed. Results: Two variances of the transmittance at 580 nm and 630 nm/580 nm transmittance ratio were finally extracted as effective items after applying forward and backward variance selection and investigation of multicollinearity. The odds ratios of 580 nm transmittance and 630 nm/580 nm transmittance ratio were 0.48 and 0.72, respectively. The cancer cell discrimination predictive value determined using the logistic regression equation was 98.0%, being favorable. Conclusion: It was suggested that Vis-MS is useful to evaluate the color of the nuclear region and serves as a cancer cell discrimination index for LBC. We are planning to apply Vis-MS to clinical materials and develop nuclear color evaluation using Vis-MS into an objective index for cases in which cancer cell judgment is difficult.

L-type amino acid transporter 1 expression increases in well-differentiated but decreases in poorly differentiated endometrial endometrioid adenocarcinoma and shows an inverse correlation with p53 expression.

Objectives The aims of this study were to determine whether the altered L-type amino acid transporter 1 (LAT1) expression is related to clinicopathologic factors, expressions of Ki-67, p53, estrogen receptor, and progesterone receptor and clarify the significance of LAT1 as a prognostic factor and the novel possibility of using it to treat endometrial endometrioid adenocarcinoma. Methods The LAT1 expression was analyzed immunohistochemically in atrophic (6 cases), secretory phase (6 cases), proliferative phase endometria (6 cases), atypical hyperplasia (6 cases), and endometrioid adenocarcinoma (26 well-differentiated [G1], 17 moderately differentiated, and 11 poorly differentiated [G3] adenocarcinoma patients). Results The LAT1 expression was observed in the cell membrane. Its expression increased in the atrophic, secretory, and proliferative phases of the endometrium in that order. There was no difference between the proliferative phase endometrium, atypical hyperplasia, and G1 adenocarcinoma. The LAT1 expression in G1 adenocarcinoma was significantly higher than that in G3 adenocarcinoma. The LAT1 expression was inversely correlated with p53 expression but not with those of Ki-67, estrogen receptor, or progesterone receptor. Conclusions It is suggested that the significance of LAT1 as a prognostic factor is low because LAT expression was low in G3 adenocarcinoma, not correlated with the International Federation of Gynecology and Obstetrics stage and proliferative activity and inversely correlated with p53. The LAT1 inhibitors can be used as anticancer drugs for G1 and moderately differentiated adenocarcinoma that express high LAT1.

Significance of binucleated cells with compression in atypical squamous cells of undetermined significance.

Objective: To increase the accuracy of the diagnosis of atypical squamous cells of undetermined significance (ASC-US), ASC-US were divided into high-risk human papillomavirus (HPV HR+) and non-high-risk HPV (HPV HR-) cases to analyze the significance of binucleated cells with compression. Study Design: ThinPrep specimens of ASC-US were examined. This study included 21 CIN and HPV HR+ (CIN+), 19 benign and HPV HR- (B-) and 10 benign and HPV HR+ (B+) cases. The number of cells were examined by defining binucleated cells with their nuclei pressing against each other as positive compression, and their relation to the relative light units (RLUs) of the DNA Hybrid capture 2 (HC2) was determined. Results: 95.2% of CIN+ and 15.8% of B- cases were compression positive, while 4.8% of CIN+ and 84.2% of B- cases were compression negative, which was significantly different. The average number of cells with positive compression was 5.7 ± 5.3 in CIN+, 2.0 ± 0.7 in B- and 5.5 ± 1.5 in B+ cases, with significant differences between CIN+ and B- and between B- and B+ cases. The number of compression-positive cells increased as HPV HC2 RLUs became higher. Conclusion: Positive compression is useful in determining ASC-US with HPV HR+. The identification of positive compression is highly practical because it can be observed morphologically.

Discriminating analysis of atypical squamous cells of undetermined significance of the uterine cervix using nuclear three-dimensional analysis.

Objective: The Bethesda System (TBS) of uterine cervical cytology is a classification method that can improve accuracy in management and it includes descriptions on adequate specimens, human papillomavirus (HPV) involvement and estimated lesions. However, the judgment of atypical squamous cells of undetermined significance (ASC-US) using TBS features complicated diagnostic criteria and poor reproducibility due to the definition of ASC-US. Of patients diagnosed with ASC-US in the initial cytology, cervical intraepithelial neoplasia (CIN)1-2 cases positive for high-risk HPV (CIN+) and benign cases in histology negative for high-risk HPV (B-) were selected for discriminant analysis based on Mahalanobis distance, in order to improve the accuracy of the ASC-US diagnosis. Study Design: ASC-US cases featuring koilocytosis with little nuclear atypia (koilocytosis) and squamous epithelial cells with nuclear atypia (SC with atypia), morphologically diagnosed with liquid-based cytology specimens prepared using ThinPrep were included. The nuclei of koilocytosis cases (CIN+, 8 cases, and B-, 10 cases) and SC with atypia (CIN+, 19 cases, and B-, 15 cases) were three-dimensionally analyzed to conduct a discriminant analysis based on Mahalanobis distance. Results: Discrimination rates were 78.9% for CIN+ and 66.7% for B- in koilocytosis, and 50.7% for CIN+ and 72.1% for B- in SC with atypia. Conclusion: The present method allows the objective analysis of nuclear chromatin, providing effective cytology regarding CIN+ in koilocytosis and B- in SC with atypia of ASC-US cases.

Evaluation of Endometrial Endometrioid Carcinoma Grade Using Ultraviolet- Visible-Light Microscopic Spectroscopy

Objective: The objective of this study was to investigate the possibility of an objective index to evaluate the grades of endometrial endometrioid carcinomas by analyzing the spectral values of ultraviolet(UV) and visible(Vis) transmittances, and the degree of UV- absorbing materials influencing nuclear colors using ultraviolet- visible-light microscopic spectroscopy (UV-Vis MS). Study design: The materials were histological preparations from 4 patients, each with proliferative-phase endometrium (PPE) and grade 1(G1) and grade 3 (G3) of endometrial endometrioid carcinomas. Both spectra were measured in the same 50 nuclei of each specimen. Results: 1) In the ultraviolet spectrum, transmittance of 260, 280, 300, and 320 nm were decreased in G3 compared to those in G1. All of the ultraviolet absorbing materials increased in G3. 2) In the visible spectrum, 520 nm (purplish red), 540 nm (purple), and 560 nm (violet) in G3 were deeper than all colors in G1, and violet in G3 was lighter than purplish red and purple in G1. 3) The following 3 characteristics were clarified with regard to the relationship between UV-absorbing materials and the violet color based group. 1) In G3, the number of the significant canonical correlation coefficient between ultraviolet light-absorbing materials and the violet color based group was only at one. 2) Purplish red and purple of the nuclei in G3 was strongly influenced by ultraviolet lightabsorbing materials (260-320 nm). 3) Purple and violet in G1 was strongly influenced by light-absorbing materials at 300 nm and 320 nm. Conclusion: It was indicated that UV-Vis MS analysis could be an objective index to evaluate the grade of endometrial endometrioid cancerby analyzing the spectral values of transmittances of ultraviolet and visible light, and the degree of ultraviolet light- absorbing materials influencing nuclear colors in the same nuclear region.

New Method to Restore Fading Papanicolaou Specimen Using an Optical Clearing Agentx

Cytological specimens are used only once for staining, and cannot be reused after preservation in paraffin blocks. If a technique is developed allowing restaining of precious/rare specimens or those for education, which have been discolored due to long-term preservation, to restore their former states, it will be extremely useful for the reuse of specimens. We performed this study to determine whether discolored specimens can be restored to their former condition by applying an optical clearing agent. For restaining, 131 Papanicolaou (Pap) specimens prepared from 13 to more than 30 years ago were used. Using an optical clearing agent (SCALEVIEW-A2), Pap restaining, endurance tests of the optical clearing agent by repeated use, and immunostaining were performed. After treatment with the optical clearing agent for5-10 minutes, discolored cytological specimens could be stably restained just as the initial Pap specimens. As a result of the immunostaining of specimens treated using this method, both the nucleus (Ki-67) and cell membrane (MOC31) were positively stained. The optical clearing agent did not deteriorate when used repeatedly ten times. Discolored precious cytological specimens could be restored using a optical clearing agent. Specimens processed using this method could also be immunostained, and so this method is extremely useful

Discriminant Analysis between Malignant Mesothelioma and Reactive Mesothelium Using Nuclear Three-Dimensional Analysis Is Useful for Morphologically Suspicious Cases

Objective: Morphological discrimination between malignant mesothelioma (MM) and reactive mesothelium (RM) is often difficult. Stereological analysis of nuclear luminance using centrifuged smear samples from coelomic fluid and discriminant analysis based on Mahalanobis distance may help to more accurately discriminate between MM and RM. In the present study, discriminant analysis was conducted on cytological specimens using the auto-smear method in a blinded manner with regard to histological results. Study Design: Coelomic fluid samples of 28 cases, cytologically diagnosed using the auto-smear method, were analyzed to determine pixel counts, the number of focus layers, 3-dimensional variation in the coefficient of variation of nuclear luminance between the focus layers as well as roundness in about 30-50 atypical cell nuclei per case. These measurements were employed to determine malignancy based on Mahalanobis distance. Results: Discrimination rates were as high as 91.7% for MM and 82.7% for RM. The discrimination rates of MM with histology were >80% in 8 of 10 suspicious cases with the initial cytology. Conclusion: Our method allowed accurate discrimination between MM and RM and provides a useful alternative for the diagnosis of suspicious cases where morphological diagnosis of malignancy is difficult.