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.

World wide web-based cytological analysis of atypical squamous cells cannot exclude high-grade intraepithelial lesions.

OBJECTIVE: It has been reported that the low level of consistency of diagnosis of atypical squamous cells cannot exclude high-grade squamous intraepithelial lesions (ASC-H) in uterine cervical cancer screening using the Bethesda System, indicating the necessity of a large-scale survey. We presented cases cytologically judged as ASC-H on our website and invited our members to give their opinions regarding the diagnosis by voting online. The Web voting results were analyzed and ASC-H was cytologically investigated. STUDY DESIGN: Virtual slides of atypical cells in cytology preparations of 53 cases were prepared and presented on a website. ASC-H cases were divided into 42 cases sampled by brush scraping and 11 cases sampled by cotton swab scraping. Fifty-three cases cytologically judged as ASC-H were classified into benign and CIN2/3, and their patterns of arrangement of atypical cells and 8 cytological parameters were morphologically investigated. RESULTS: The frequency of ASC-H diagnosis in the Web votes was low: 29.2% for brush-scraped and 26.2% for cotton swab-scraped cases. Three-dimensionality, coarse chromatin and irregular nuclei were significantly different between high-grade squamous intraepithelial lesions and benign cases. CONCLUSION: Web-based surveys showed the difference of cytological findings between high-grade squamous intraepithelial lesions and benign cases. To increase interobserver consistency, it may be useful to share information online, which avoids geographical and temporal limitations.

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.

Cytological analysis of atypical squamous epithelial cells of undetermined significance using the world wide web.

OBJECTIVE: The low-level consistency of the cytodiagnosis of uterine cervical atypical squamous epithelial cells of undetermined significance (ASC-US) employing the Bethesda System has been reported, suggesting the necessity of a wide survey. We presented cases judged as ASC-US on the Web and analyzed the voting results to investigate ASC-US cytologically. STUDY DESIGN: Cytology samples from 129 patients diagnosed with ASC-US were used. Images of several atypical cells observed in these cases were presented on the Web. The study, based on the voting results, was presented and opinions were exchanged at the meeting of the Japanese Society of Clinical Cytology. RESULTS: The final diagnosis of ASC-US was benign lesions in 76 cases and low- and high-grade squamous intraepithelial lesions in 44, but no definite diagnosis could be made for the remaining 9. The total number of votes was 17,884 with a 36.5% consistency of cases judged as ASC-US. Benign cases were divided into 6 categories. Four categories not corresponding to the features of koilocytosis and small abnormal keratinized cells were judged as negative for an intraepithelial lesion or malignancy at a high rate. CONCLUSION: A Web-based survey would be useful which could be viewed at any time and thereby facilitate the sharing of cases to increase consistency.

Three-dimensional nuclear luminance analysis in well-differentiated adenocarcinoma of the lung.

OBJECTIVE: Well-differentiated papillary adenocarcinoma of the lung (G1 cancer cells) is difficult to distinguish from benign bronchial columnar epithelial cells with reactive atypia (benign cells) in many cases because nuclear atypia is mild. We focused on the 3-dimensional presence of nuclei in cell smears. Several images focused on the nucleus were acquired, and the nuclear luminance was measured and analyzed. STUDY DESIGN: One hundred G1 cancer cells and benign cells (nuclei), respectively, were selected from those on a bronchial brushing preparation for cytology. Images of 41 layers were acquired at 0.25-µm intervals in each cell, and the nuclear luminance was measured (a total of 8,400 images). RESULTS: There were more focus positions in the G1 cancer cell nuclei, showing a 3-dimensional nucleus, compared to benign cells, and the 3-dimensional variation in the coefficient of variation (CV) of nuclear luminance at the focus position was smaller in G1 cancer than in benign cells, showing a significant difference. CONCLUSION: The G1 cancer cells' nuclear structure was more 3-dimensional, and the chromatin distribution was homogeneous. The three-dimensional variation in the nuclear luminance CV could be numerically presented, which might be an objective index for cancer diagnosis.

Estrogen receptor alpha and beta are prognostic factors in non-small cell lung cancer.

PURPOSE: Estrogen receptor-alpha (ER-alpha) and -beta (ER-beta) play important roles in the carcinogenesis of breast tumors. Similarly, there have been several reports of ER expression in lung cancers, but the results have not been consistent, and the receptors' prognostic value remains unclear. Our goal was to investigate ER expression in non-small cell lung cancer (NSCLC) and to assess whether their expression correlates with prognosis. EXPERIMENTAL DESIGN: ER expression was examined using immunohistochemical methods with sections from 132 resected NSCLC specimens. Kaplan-Meier survival curves were analyzed to determine the significance of ER expression in the prognosis of NSCLC patients. RESULTS: ER-alpha was detected in the cytoplasm of 73% of the specimens analyzed, whereas ER-beta was detected in the nucleus of 51%. ER-alpha expression correlated with poorer overall survival (P < 0.001), as did the absence of ER-beta expression (P = 0.048). Likewise, at histopathologic stage I, ER-alpha expression (P = 0.028) or the absence of ER-beta (P = 0.037) correlated with a poorer prognosis, and ER-alpha(+)ER-beta(-) patients had a significantly worse prognosis than ER-alpha(-)ER-beta(+) patients (P = 0.00007). Multivariate Cox regression analysis revealed the absence of ER-beta to be an independent factor predictive of poor disease outcome (hazard ratio, 1.9; 95% confidence interval, 1.1-3.4; P = 0.0264). CONCLUSIONS: ER-alpha expression and the absence of ER-beta expression are associated with a poorer prognosis among NSCLC patients. In particular, the absence of ER-beta could serve as a marker identifying patients at high risk even at an early clinical stage.

Combined overexpression of EGFR and estrogen receptor alpha correlates with a poor outcome in lung cancer.

BACKGROUND: The aim of the present study was to determine the prognostic significance of the combined overexpression of epidermal growth factor receptor (EGFR) and estrogen receptor a (ER-alpha) in non-small cell lung cancer (NSCLC). PATIENTS AND METHODS: An immunohistochemical assessment of EGFR and ER-alpha expression was carried out in tumor specimens from 122 consecutive NSCLC patients after surgery. RESULTS: Of the 122 tumors examined, 57 (46.7%) overexpressed EGFR, 63 (51.6%) overexpressed ER-alpha, while 33 (27%) overexpressed both EGFR and ER-alpha. Univariate analyses showed that the overexpression of EGFR or ER-alpha correlated with the histological grade (p = 0.01 and p = 0.03, respectively) and a poorer prognosis (p = 0.007), while overexpression of ER-alpha also correlated with smoking (p = 0.02). Multivariate analysis showed the combined overexpression of EGFR and ER-alpha to be an independent indicator of poorer prognosis (p = 0.0006). CONCLUSION: The combined overexpression of EGFR and ER-alpha in NCLSC patients is predictive of poor outcome and, thus, represents a valuable prognostic factor.

Cytological Analysis of Low-grade Squamous Intraepithelial Lesion with a Histodiagnosis of Cervical Intraepithelial Neoplasia 3 (Severe Dysplasia) Prospectively.

OBJECTIVE: To reanalyze the cytological features of the first cytological specimens of cervical intraepithelial neoplasia 3 (CIN3) (severe) cases that were initially cytodiagnosed as LSIL and followed up for up to 5 years. STUDY DESIGN: Among 14,556.patients in 2009, 236 (1.6%) were cytodiagnosed with low-grade squamous intraepithelial lesion (LSIL). Among those, 128 cases could be histologically diagnosed from 2009 to 2013. Among those 128 cases, 16 were diagnosed as CIN3 (severe). The first cytological specimens of those 16 cases were reanalyzed morphologically. RESULTS: The first cytological specimens of the 16 cases diagnosed as LSIL and histodiagnosed as CIN3 (severe) showed a few small atypical cells in 13 and a cluster of small atypical cells in 1 case. Atypical cells indicating CIN1 were predominant in 14 LSIL cases and there were a few small atypical cells in these cases (LSIL, cannot exclude high-grade squamous intraepithelial lesion [LSIL-H]). CONCLUSION: We show the existence of LSIL-H (LSIL, cannot exclude HSIL) cases, for which biopsy with colposcopy should be applied actively.

Cytologic difference between benignity and malignancy in suspicious cases employing urine cytodiagnosis using a liquid-based method.

OBJECTIVE: To follow cases with atypical cells or suspicious cases on first examination by liquid-based cytology (LBC), comparing cases that became negative and those confirmed to be positive (urothelial carcinoma) with regard to the cell morphology. STUDY DESIGN: Urine cytology was performed employing LBC in 323 samples. Of 75 suspicious cases on first examination, 5 and 8 cases were identified with (positive) and without (negative) cancer on reexamination, respectively. Cell morphology was investigated in the first suspicious preparations of these cases. RESULTS: Fewer cells were present in the samples of negative cases, and atypia was generally weak. The nuclei were swollen and pale, and hyperchromatism was weak. In contrast, in positive cases, nuclear swelling, flattening, shrinkage, and pale staining were less marked compared with those in negative cases. The nuclei were hyperchromatic and irregular in many cases. Nuclear cannibalism, multinucleation, anisokaryosis, three-dimensionality, and an irregular arrangement were considered to be features strongly suggesting malignancy. CONCLUSION: The efficiency of diagnosis employing the LBC is high because of the cell collection rate. It was shown that the accuracy of diagnoses made employing the LBC method can be increased by understanding the characteristics of the cell morphology in suspicious cases.

Hepatoid carcinoma of the skin: spontaneous rat skin hepatoid carcinoma with eosinophilic globules and crystals immunoreactive to alpha-1-antitrypsin.

We present a case of hepatoid carcinoma of the abdominal skin in a male Wistar rat. Histopathologically, this carcinoma resembled human hepatocellular carcinoma with respect to trabecular-sinusoidal structures. Carcinoma tissues contain numerous eosinophilic globules and crystals, and in this case, we found the characteristic eosinophilic globules in the hepatoid carcinoma cells and the crystals in the extracellular portions. Vivid carcinoma cells full of eosinophilic globules were present near the necrotic areas in tumor tissue, wherein quadrate crystals unstained with eosin were observed. PAS staining after diastase digestion revealed that the globules were PAS positive and diastase resistant. In addition, we found that the hepatoid carcinoma cells were immunoreactive for alpha-1-antitrypsin (anti-A1AT) antibody with the globules and crystals staining peripherally, and a central unstained region. Ultrastructural study of intracytoplasmic globules and extracellular crystals revealed that the fringe of each globule and crystal had no limiting membrane and showed the same level of electron density. These findings suggest that the characteristic crystals in this tumor may have originated from the globules that were emitted from the carcinoma cells after their death as a result of saturation with intracytoplasmic globules.