Dual immunocytochemical staining of annexin A10 and p53 in low-grade and papillary urothelial carcinoma contributes to improvement of diagnostic accuracy in urine cytology.

BACKGROUND: Urothelial carcinoma (UC) is a common type of human cancer and, although urine cytology is a useful method for identifying high-grade UC (HGUC), its ability to diagnose low-grade UC (LGUC) is limited. The authors previously reported that annexin A10 (ANXA10) expression was strongly linked to both papillary and early stage LGUC and was inversely correlated with p53 expression in upper tract UC (UTUC) and bladder UC. However, it remains largely unknown whether ANXA10 is useful as a diagnostic marker for urine cytology. METHODS: In this study, the authors used 104 biopsy and 314 urine cytology samples to investigate the efficacy of ANXA10 and p53 expression by immunohistochemistry and immunocytochemistry. RESULTS: In immunohistochemistry analysis, expression levels of ANXA10 and p53 were either weak or absent in noncancerous tissues, whereas ANXA10 overexpression was observed patients with LGUC, and strong expression of p53 was identified in patients with HGUC. In immunocytochemistry analysis, sensitivity was not good for the detection of UC, especially UTUC, by cytology alone, but it was improved by combining cytology with ANXA10 and p53 to detect both bladder UC and UTUC. Receiver operating characteristic curve analysis also confirmed the diagnostic superiority of cytology combining ANXA10 and p53 for the detection of all UCs, including both HGUC and LGUC (area under the curve, 0.84). CONCLUSIONS: To the authors' knowledge, this is the first report that the combination of ANXA10 and p53 has potential application as a diagnostic immunomarker for improving the diagnostic accuracy of urine cytology.

Analysis of MicroRNA in Bile Cytologic Samples Is Useful for Detection and Diagnosis of Extrahepatic Cholangiocarcinoma.

OBJECTIVES: This study aimed to develop reliable biomarkers that improve the ability of bile cytology to diagnose cholangiocarcinoma vs benign biliary lesions. METHODS: Many studies indicate that microRNAs (miRNAs) are potential candidates for the early diagnosis of cancer. We analyzed the expression of five tumor-associated miRNAs (miR-31-5p, miR-122-5p, miR-378d, miR-182-5p, and miR-92a-3p) in cytology samples using quantitative reverse transcription polymerase chain reaction. We collected 52 surgically resected tissue samples, 84 cytologic specimens from smears (53 cases of cancer and 31 cases of noncancer), and 40 residual sediments after smearing for routine cytology at Hiroshima University Hospital. RESULTS: The expression of miR-31-5p, miR-378d, and miR-122-5p was significantly higher in cancer tissues than those in normal tissues, while miR-182-5p expression was lower. The expression of miR-31-5p, miR-378d, miR-182-5p, and miR-92a-3p was significantly higher in detached cell samples from smears of cholangiocarcinoma cases than in those from noncancer cases. CONCLUSIONS: These results suggest that the analysis of miRNAs in bile cytologic specimens is a promising auxiliary tool for distinguishing cholangiocarcinoma from benign biliary lesions.

Utility of cytopathological specimens and an automated image analysis for the evaluation of HER2 status and intratumor heterogeneity in breast carcinoma.

OBJECTIVES: Although updated HER2 testing guidelines have been improved by a collaboration between the American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP) in 2013, HER2 evaluation is still problematic because of issues involving CEP17 polysomy, heterogeneity, and HER2 score 2+ cases. The aim of this retrospective study was to evaluate the relationship between HER2 gene heterogeneity, or so called CEP17 polysomy, using breast carcinoma cells sampled by scraping and the IHC score graded by automated image analysis using whole slide image. MATERIAL AND METHODS: We randomly selected 23 breast carcinoma cases with a HER2 score 0, 24 cases with a HER2 score 1+, 24 cases with HER2 score 2+, and 23 cases with HER2 score 3+ from the records of patients with breast cancer at Hiroshima University Hospital. We compared the results of fluorescent in situ hybridization (FISH) using formalin-fixed, paraffin-embedded (FFPE) tissues and cytological samples and compared the HER2 score calculated using an automated image analysis using wholly scanned slide images and visual counting. RESULTS: We successfully performed the FISH assay in 78 of 94 cases (83%) using FFPE tissues and in all 94 (100%) cases using cytological samples. Frequency of both HER2 amplification and CEP17 polysomy was higher when cytological samples were used than when FFPE tissue was used. Frequency of HER2 heterogeneity using cytological samples was higher that than using FFPE tissue, except for the IHC score 3+ cases. CONCLUSIONS: When assessment of HER2 status based on FISH using FFPE tissue cannot be accomplished, FISH using cytological samples should be considered. When intensity of HER2 is heterogeneous in the tumor tissue, particularly in cases regarded as score 2+, they should be evaluated by automated image analysis using the whole slide image.

Discordant HER2 status between primary breast carcinoma and recurrent/metastatic tumors using fluorescence in situ hybridization on cytological samples.

OBJECTIVE: The aim of this study was to show the usefulness of examining HER2 status on fluorescence in situ hybridization using cytological samples taken from recurrent/metastatic tumors. METHODS: One hundred freshly aspirated or scraped cytological samples were obtained from locoregional recurrences or distant metastases. Fluorescence in situ hybridization assay for HER2 amplification was performed on both these samples and the formalin-fixed, paraffin-embedded tissues of the paired primary tumors of breast cancer, and the relationships between various clinico-pathological factors and HER2 amplification of both tumors were examined. RESULTS: A change in HER2 status was observed in nine cases (9%): six cases (6%) underwent a positive-to-negative conversion in HER2 status and three cases (3%) underwent a negative-to-positive conversion in HER2 status. A positive-to-negative conversion of HER2 status was noted in 4 (36%) of 11 'luminal-B' cases. The change in HER2 status in recurrent or metastatic tumor was noted in more cases treated with drug therapy than in those with no drug therapy (P < 0.05; Fisher's exact probability). Although the time to relapse was 3 years or more in three cases showing a negative-to-positive conversion in HER2 status, the time to relapse was less than 3 years in six cases showing a positive-to-negative conversion (P < 0.05; Fisher's exact probability). CONCLUSIONS: HER2 examination on fluorescence in situ hybridization using fine-needle aspiration cytology samples of tumors in recurrent/metastatic sites or disseminated tumor cells in effusion is beneficial, particularly when the primary tumor is not suitable for the testing of HER2 status or negative for HER2 amplification, because aspiration using a needle is technically feasible and not as traumatic as biopsy.

Comparison of evaluations of hormone receptors in breast carcinoma by image-analysis using three automated immunohistochemical stainings.

The aim of this study was to compare the results of immunohistochemistry (IHC) assays evaluated by human examiners with the results evaluated by computerized image analysis, and to compare the computerized image analysis results among three automated IHC assays, namely the BioGenex, Dako and Ventana assays. All slides were semiquantitatively evaluated according to the Allred score and J-score by human examiners. The images were analyzed using MacSCOPE version 2.6 for Macintosh according to the H-score and the percentage of positive-stained nuclei per area of carcinoma cells (PP) irrespective of the intensity of the stained nuclei. The H-score for the estrogen receptor (ER) was significantly correlated with the Allred score (P<0.0001) and the PP for the ER was significantly correlated with the J-score (P<0.0001), suggesting that the image analysis used in the present study is a useful method for the evaluation of ER status. Several discrepancies were identified between the Allred score and H-score and between the PP and J-score due to the positive-stained cytoplasm area of carcinoma cells and/ or the positive-stained nuclei area of non-carcinoma cells, including benign epithelial cells, lymphocytes and stromal cells. Accordingly, advances in the algorithm of the digitized analyzing system is necessary.

Localization of laminin alpha3B chain in vascular and epithelial basement membranes of normal human tissues and its down-regulation in skin cancers.

The basement membrane (BM) proteins laminins, which consist of alpha, beta and gamma chains, play critical roles in the maintenance of tissue structures. One of laminin alpha chains, alpha3 has two isoforms, the truncated form alpha3A and the full-sized form alpha3B. In contrast to alpha3A laminins, little is known about alpha3B laminins. To show the histological distribution of the laminin alpha3B chain, we prepared alpha3B-specific monoclonal antibodies. Immunohistochemical analysis showed that the alpha3B chain was colocalized with the alpha3A, beta3 and gamma2 chains in the epithelial BMs of the skin, esophagus, breast and lung, suggesting the presence of laminin-3B32 (laminin-5B) and laminin-3A32 (laminin-5A). In the lung alveoli, laminin-3B32 was dominant over laminin-3A32, but vice versa in other epithelial BMs. In contrast, the BMs of blood vessels including capillaries were strongly positive for alpha3B, but almost or completely negative for alpha3A, beta3 and gamma2. alpha3B was colocalized with beta1 and gamma1 in these BMs. The alpha3B chain was scarcely detected in the vessels of malignant skin cancers, though the gamma2 and beta3 chains were highly expressed in the cancer cells. These results strongly suggest that the laminin alpha3B chain is widely expressed in vascular BMs of normal tissues, probably as laminin-3B11/3B21 (laminin-6B/7B).