Morphometrical Differences among Endometrial Endometrioid Carcinoma Grade 1, Grade 3, and Serous Carcinoma in Endometrial Liquid-Based Cytology Preparations.

INTRODUCTION: In Japan, the direct smearing preparation (conventional preparation) has been widely used for cytological examination of the endometrium. Problems with the conventional preparation can be dissolved by liquid-based cytology (LBC) preparation. The Yokohama System is a method for reporting endometrial cytology, but the system lumps cancers together and does not distinguish between histological types. The objective of this study was to clarify morphometrical differences among endometrial endometrioid carcinoma grade 1 (G1), grade 3 (G3), and serous carcinoma (Serous) by image analysis of endometrial LBC. METHODS: Using Papanicolaou smears prepared by LBC after sampling with a brush from 32 G1, 16 G3, and 16 Serous patients, image analysis was performed concerning the following 11 items: (1) number of layers of cluster, (2) area of cluster, (3) perimeter of cluster, (4) roundness of cluster, (5) complexity of cluster, (6) area of nucleus, (7) perimeter of nucleus, (8) roundness of nucleus, (9) complexity of nucleus, (10) area of nucleolus, and (11) nucleolus/nucleus (N/N) ratio. The data were statistically compared among G1, G3, and Serous. RESULTS: Significant differences were observed in the number of layers of cluster (G1G3G3, G1>Serous), complexity of cluster (G1Serous), and N/N ratio (G1>G3, G3

Exosomal MicroRNA as Biomarkers for Diagnosing or Monitoring the Progression of Ovarian Clear Cell Carcinoma: A Pilot Study.

Ovarian cancer is the most common cause of gynecological malignancy-related mortality since early-stage disease is difficult to diagnose. Advanced clear cell carcinoma of the ovary (CCCO) has dismal prognosis, and its incidence has been increasing in Japan, emphasizing the need for highly sensitive diagnostic and prognostic CCCO biomarkers. Exosomal microRNAs (miRNAs) secreted by tumor cells are known to play a role in carcinogenesis; however, their involvement in ovarian cancer is unclear. In this study, we performed expression profiling of miRNAs from exosomes released by five cell lines representing different histological types of ovarian cancer. Exosomes isolated from culture media of cancer and normal cells were compared for miRNA composition using human miRNA microarray. We detected 143 exosomal miRNAs, whose expression was ≥1.5-fold higher in ovarian cancer cells than in the control. Among them, 28 miRNAs were upregulated in cells of all histological ovarian cancer types compared to control, and three were upregulated in CCCO cells compared to other types. Functional analyses indicated that miR-21 overexpressed in CCCO cells targeted tumor suppressor genes PTEN, TPM1, PDCD4, and MASP1. The identified miRNAs could represent novel candidate biomarkers to diagnose or monitor progression of ovarian cancer, particularly CCCO.

Usefulness of Fractal Analysis of Kirsch Edge Images for the Tissue Fragment Inner Structure in Breast FNAB.

OBJECTIVE: Recent advances in high-precision mammography and ultrasound screening have led to an increase in the detection of early lesions (ductal carcinoma in situ and small cancers) appearing as microcalcified lesions or microcystic images, and there needs to be an improvement in the accuracy of breast fine-needle aspiration biopsy (FNAB) assessing these lesions. The objective of this study was to investigate whether fractal analysis of Kirsch edge images for the tissue fragment inner structure (FKT) is useful in breast FNAB. FKT measures tissue fragment chromasia of hyperchromatic crowded tissue fragments (HCG), tissue fragment shape unevenness, and tissue fragment inner structure complexity. Study Design Materials: Nineteen epithelial tissue fragments of fibroadenoma (FA) from 7 patients and 52 tissue fragments of invasive breast carcinoma of no special type (IBC-NST) (grade 1-2) from 11 patients were assessed. First, tissue fragments were classified into small (smaller than 60 × 102 µm2), medium, and large (100 × 102 µm2 or larger), and the appearance rate of each size was determined. Second, for FKT, the luminance value of tissue fragment chromasia, the unevenness and fractal value, and the tissue fragment inner structure complexity were determined. In statistical analysis, the Steel-Dwass test, nonlinear discriminant analysis, and receiver operating characteristic analysis were performed, setting the significance level at p < 0.05. RESULTS: "Unevenness of the tissue fragment shape," "fractal value of the tissue fragment shape," and "fractal value of the tissue fragment inner structure" were significantly higher in small and large tissue fragments in IBC-NST compared with those in FA. The specificity and sensitivity were the highest (100%) in small tissue fragments in multivariate analysis using 4 variables ("luminance value of tissue fragment chromasia," "unevenness of tissue fragment shape," "fractal value of the tissue fragment shape," and "fractal value of the tissue fragment inner structure"). CONCLUSION: FKT, which evaluates "tissue fragment darkness," "tissue fragment shape unevenness," and "tissue fragment inner structure complexity" focusing on small tissue fragments of HCG in breast FNAB, is useful as a system that assists cytopathological assessment of breast FNAB.

Morphological Differences between Liquid-Based Cytology and Conventional Preparation in Endometrial Endometrioid Carcinoma Grade 1 and Grade 3, and the Differentiation of Grades in Each Method.

INTRODUCTION: Direct smearing preparation (conventional preparation [CP]) has been widely used for endometrial cytology in Japan. In CP, sampling and screening errors are problematic. In liquid-based cytology preparation (LBC), the problems of CP can be solved. But there is a problem that cytological findings of LBC are different from those of CP. The purpose of this study was to evaluate the differences of morphological findings of endometrial cytology between LBC and CP, and the usefulness of the endometrial LBC to differentiate endometrioid carcinoma grade 1 (G1) from grade 3 (G3). METHODS: Thirteen cases of endometrioid carcinoma G1, and 5 cases of G3 collected by the Softcyte device and prepared by LBC and CP (split specimen) were used. We focused on the following items: (1) the number of clusters per cm2, (2) the number of layers of clusters, (3) area of clusters, (4) perimeter of clusters, (5) roundness of clusters, (6) complexity of clusters, (7) area of nucleus, (8) perimeter of nucleus, (9) roundness of nucleus, (10) complexity of nucleus, (11) area of nucleolus, and (12) nucleolus-nucleus ratio (N/N). RESULTS: Compared with CP, the number of clusters and layers of the clusters in LBC were significantly larger in G1. The area and perimeters of the clusters and the nucleus were significant smaller, and the N/N ratio was greater in LBC than that in CP in both G1 and G3. Regarding morphological differences between G1 and G3 in LBC and CP, the number of layers was significantly larger in G1 than in G3 in LBC and CP. The area of the clusters in LBC was significantly larger in G1 than in G3. The area and perimeters of the nucleus in CP and the area of the nucleolus and N/N ratio in LBC and CP were significantly smaller in G1 than in G3. CONCLUSION: In the endometrial cytology, it became clear that the cell image was different between LBC and CP and between G1 and G3. By microscopic examination understanding the characteristics of the cell image in LBC, endometrial LBC could be useful to diagnose endometrial carcinoma.

Fractal Analysis Method for the Complexity of Cell Cluster Staining on Breast FNAB.

OBJECTIVE: Because of the increased precision of ultrasound breast cancer screening, early cancer cases with no clear mass or extraction of microcysts on imaging have recently increased, and improvement of the accuracy of breast fine-needle aspiration biopsy (FNAB) cytology is needed. The objective of this study was to investigate the usefulness of cluster gray image-fractal analysis evaluating the darkness of clusters, cluster unevenness, and complexity of hyperchromicity (cluster density) of deep-stained cell clusters, known as hyperchromatic crowded cell groups (HCG), on FNAB as a cytology assistance system for breast FNAB. STUDY DESIGN: One hundred clusters collected from 10 patients with fibroadenoma (FA), 90 clusters from 9 patients with ductal carcinoma in situ (DCIS), and 122 clusters from 11 patients with invasive breast carcinoma of no special type (IBC-NST) were used. (1) Cluster size classification: clusters were classified into small, middle, and large clusters (small cluster: smaller than 40 × 102 µm2; large cluster: 100 × 102 µm2 or larger; middle cluster: intermediate), and their frequency was calculated. (2) Cluster gray image-fractal analysis: (a) the darkness of clusters (luminance), (b) cluster unevenness (complexity), and (c) complexity of cluster density (roundness-corrected fractal value) were assessed. For statistical analysis, the multiple comparison Steel-Dwass test was used, with a significance level of p < 0.05. RESULTS: (1) Cluster size classification: in FA, small, middle, and large clusters appeared at a similar frequency, and the frequency (30%) of large clusters was significantly higher than that in other diseases. In IBC-NST, many small clusters (61%) appeared and their frequency was significantly higher than that in other diseases, whereas the frequency of large clusters was significantly lower. (2) Cluster gray image-fractal analysis: in IBC-NST, the luminance of small clusters was low (dark), the cluster unevenness was high, and the complexity of cluster density was high, whereas the luminance of large clusters was high (bright), the cluster unevenness was high, and complexity of cluster density was high compared with those in FA. CONCLUSION: Cluster gray image-fractal analysis evaluating the darkness of clusters, cluster unevenness, and complexity of cluster density in breast FNAB HCG is a useful cytology assistance system for breast FNA.

Outer Cutoff Value for the Box-Counting Method for Fractal Analysis of the Nucleus Using Kirsch Edge Detection.

OBJECTIVE: The complexity of chromatin (i.e., irregular geometry and distribution) is one of the important factors considered in the cytological diagnosis of cancer. Fractal analysis with Kirsch edge detection is a known technique to detect irregular geometry and distribution in an image. We examined the outer cutoff value for the box-counting (BC) method for fractal analysis of the complexity of chromatin using Kirsch edge detection. MATERIALS: The following images were used for the analysis: (1) image of the nucleus for Kirsch edge detection measuring 97 × 122 pix (10.7 × 13.4 µm) with a Feret diameter of chromatin mesh (n = 50) measuring 17.3 ± 1.8 pix (1.9 ± 0.5 µm) and chromatin network distance (n = 50) measuring 4.4 ± 1.6 pix (0.49 ± 0.18 µm), and (2) sample images for Kirsch edge detection with varying diameters (10.4, 15.9, and 18.1 µm) and network width of 0.4 µm. METHODS: Three types of bias that can affect the outcomes of fractal analysis in cytological diagnosis were defined. (1) Nuclear position bias: images of 9 different positions generated by shifting the original position of the nucleus in the middle of a 256 × 256 pix (28.1 µm) square frame in 8 compass directions. (2) Nuclear rotation bias: images of 8 different rotations obtained by rotating the original position of the nucleus in 45° increments (0°, 45°, 90°, 135°, 180°, 225°, 270°, and 315°). (3) Nuclear size bias: images of varying size (diameter: 190 pix [10.4 µm], 290 pix [15.9 µm], and 330 pix [18.1 µm]) with the same mesh pattern (network width: 8 pix [0.4 µm]) within a 512 × 512 pix square. Different outer cutoff values for the BC method (256, 128, 64, 32, 16, and 8 pix) were applied for each bias to assess the fractal dimension and to compare the coefficient of variation (CV). RESULTS: The BC method with the outer cutoff value of 32 pix resulted in the least variation of fractal dimension. Specifically, with the cutoff value of 32 pix, the CV of nuclear position bias, nuclear rotation bias, and nuclear size bias were <1% (0.1, 0.4, and 0.3%, respectively), with no significant difference between the position and rotation bias (p = 0.19). Our study suggests that the BC method with the outer cutoff value of 32 pix is suitable for the analysis of the complexity of chromatin with chromatin mesh.

Bmi-1 Immunohistochemical Expression in Endometrial Carcinoma is Correlated with Prognostic Activity.

Background and objectives: B-lymphoma Mo-MLV insertion region 1 (Bmi-1) is a stem cell factor that is overexpressed in various human cancer tissues. It has been implicated in cancer cell proliferation, cell invasion, distant metastasis, and chemosensitivity, and is associated with patient survival. Several reports have also identified Bmi-1 protein overexpression in endometrial carcinoma; however, the relationship between Bmi-1 expression and its significance as a clinicopathological parameter is still insufficiently understood. Accordingly, the present study aimed to clarify whether immunohistochemical staining for Bmi-1 in human endometrial carcinoma and normal endometrial tissues can be used as a prognostic and cell proliferation marker. Materials and Methods: Bmi-1 expression was assessed in endometrioid carcinoma (grade 1-3) and normal endometrial tissues (in the proliferative and secretory phases) by immunohistochemistry; protein expression was evaluated using the nuclear labeling index (%) in the hot spot. Furthermore, we examined other independent prognostic and proliferation markers, including the protein levels of Ki-67, p53, and cyclin A utilizing semi-serial sections of endometrial carcinoma tissues. Results: The expression of the Bmi-1 protein was significantly higher in all grades of endometrial carcinoma than in the secretory phase of normal tissues. Moreover, Bmi-1 levels tended to be higher in G2 and G3 tissues than in G1 tissue, without reaching significance. Bmi-1 expression showed no notable differences among International Federation of Gynecology and Obstetrics (FIGO) stages in endometrial carcinoma. Furthermore, we observed a significant positive relationship between Bmi-1 and Ki-67, cyclin A, or p53 by Spearman's rank correlation test, implying that high Bmi-1 expression can be an independent prognostic marker in endometrial carcinoma. Conclusions: Our study suggests that Bmi-1 levels in endometrial carcinoma tissues may be useful as a reliable proliferation and prognostic biomarker. Recently, the promise of anti-Bmi-1 strategies for the treatment of endometrial carcinoma has been detected. Our results provide fundamental data regarding this anti-Bmi-1 strategy.

Cyclin A is a reliable proliferation marker in endometrial cancer cell lines.

Cyclin A, a cell cycle regulatory protein, promotes cell proliferation and has been observed to be highly expressed in cancer and to promote tumor growth; however, its value as a marker for endometrial carcinoma has not yet been established. Accordingly, the aim of the present study was to clarify whether cyclin A can be used as a cell proliferation marker using the endometrial carcinoma cell lines Ishikawa and HEC-50B, derived from patients with low-grade and high-grade cancer, respectively. The expression of cyclin A was determined by flow cytometry using double staining with FITC and 7-AAD, and immunocytochemical staining. The results were compared to those of Ki-67, the widely used cell proliferation marker that is considered to be a prognostic marker in endometrial cancer. The flow cytometry results revealed that cyclin A expression was significantly higher in HEC-50B than in Ishikawa cells during the logarithmic growth phase. In addition, cyclin A expression was consistently higher than Ki-67 expression in the examined cell lines. Immunocytochemical staining confirmed cyclin A expression in HEC-50B and Ishikawa cells, demonstrating significantly higher expression during the logarithmic growth phase than during the stationary phase. By contrast, Ki-67 was expressed in almost 90% of the cells, irrespective of their growth state. These results indicate that cyclin A expression is significantly increased in cells with higher proliferative ability and is specifically expressed in cells that have passed the G1-S checkpoint. Therefore, cyclin A may be a reliable proliferation biomarker for endometrioid carcinoma.

Diverse functions of the thrombopoietin receptor agonist romiplostim rescue individuals exposed to lethal radiation.

In cases of radiological accidents, especially victims exposed to high-dose ionizing radiation, the administration of appropriate approved pharmaceutical drugs is the most rapid medical treatment. However, currently, there are no suitable candidates. The thrombopoietin receptor (TPOR) agonist romiplostim (RP) is a therapeutic agent for immune thrombocytopenia and has potential to respond to such victims. Here, we show that RP administration in mice exposed to lethal-dose radiation leads not only to the promotion of haematopoiesis in multiple organs, including the lungs but also a reduction in damage to organs and cells. RP also causes a rapid increase in the number of mesenchymal stem cells in the spleen. In addition, RP suppresses the expression of several miRNAs involved in radiation-induced leukemogenesis, suggesting the presence of targets other than TPOR. Among the currently approved pharmaceutical drugs, RP is the most suitable candidate for victims exposed to high-dose ionizing radiation.

Usefulness of Cytological Scoring Method by Breast Fine Needle Aspiration Cytology on Breast Duct Dilatation and Cystic Lesions.

AIM: The objective of this study was to apply a scoring method to fine needle aspiration cytology on breast duct dilatation and cystic lesions, to set an optimum cut-off value to differentiate between benign and malignant cases, and to identify features useful for cell judgment. MATERIALS AND METHODS: Samples were 23 preparations of specimens (12 benign and 11 malignant cases) suspected with intraductal lesions or cystic change by ultrasonography or mammography and cytology. The scoring system comprised the following 10 items, and each item was scored 1-3, with a total score of 10-30. Three items were concerning structural atypia: 1, scattered epithelial cells; 2, uneven irregular cluster edge; and 3, overlapping nuclei of epithelial cells, and seven items were concerning cellular atypia: 4, irregular nuclear size; 5, irregular nuclear morphology; 6, deep dyeing chromatin; 7, chromatin granularity; 8, chromatin distribution; 9, nucleolus; and 10, absence of myoepithelial cells. RESULTS: (1) Scoring cut-off value: malignancy is to be suspected when the score is 20.75 or higher (diagnostic accuracy: 95.7%). (2) Findings useful for cancer judgment: the sensitivity of the following four findings was high: uneven irregular cluster edge, irregular nuclear overlapping, chromatin granularity, and absence of myoepithelial cells. (3) Correlation among the findings: the findings correlated with malignancy were as follows: scattered epithelial cells versus uneven irregular cluster edge (rs = 0.8). CONCLUSION: Cytological evaluation by scoring lesions accompanied by intraductal dilatation and cystic change was a useful method capable of differentiating between benign and malignant cases at a high accuracy.

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.

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.

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.

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.

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.

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.