Clinical application of the anti-human telomerase reverse transcriptase (hTERT) antibody (SCD-A7) immunocytochemistry in liquid-based urine cytology: A prospective, single institute study.

OBJECTIVES: Urine cytology is the most widely used noninvasive screening tool for urothelial carcinoma diagnosis and surveillance. Although highly specific, urine cytology exhibits suboptimal sensitivity. This study aimed to determine whether hTERT immunocytochemistry (ICC) could be applicable as an ancillary test in routine cytology practice. METHODS: A total of 561 urinary tract samples were initially screened in this study. All of them were prepared using SurePath liquid-based cytology (LBC), while additional LBC slides were made and subsequently used for hTERT (SCD-A7) ICC. RESULTS: From the 561 samples screened, 337 were finally analyzed, all having an adequate cellularity and available follow-up histology. The hTERT ICC-positive rate was 95.9% (n = 208/217), 96% (n = 24/25), and 100% (n = 4/4) in cytology samples with high-grade urothelial carcinoma, carcinoma in situ, and low-grade urothelial carcinoma subsequent histology. Among the 64 atypical cytology cases histologically confirmed as urothelial carcinomas, 92.2% (n = 59/64) were immunoreactive to hTERT, whereas the two histologically benign cases were ICC-negative. 87/90 (96.7%) of the cytology cases confirmed to be benign in follow-up were hTERT-negative. The overall sensitivity and specificity of hTERT ICC were 96.3% and 98.8%, respectively (AUROC = 0.963; 95% CI = 0.960-0.967). CONCLUSIONS: The hTERT ICC test exhibited consistent and intense staining in malignant urothelial cells, suggesting its value as an ancillary test in liquid-based urine cytology.

Identification of TUBB2A by quantitative proteomic analysis as a novel biomarker for the prediction of distant metastatic breast cancer.

BACKGROUND: Metastasis of breast cancer to distal organs is fatal. However, few studies have identified biomarkers that are associated with distant metastatic breast cancer. Furthermore, the inability of current biomarkers, such as HER2, ER, and PR, to differentiate between distant and nondistant metastatic breast cancers accurately has necessitated the development of novel biomarker candidates. METHODS: An integrated proteomics approach that combined filter-aided sample preparation, tandem mass tag labeling (TMT), high pH fractionation, and high-resolution MS was applied to acquire in-depth proteomic data from FFPE distant metastatic breast cancer tissues. A bioinformatics analysis was performed with regard to gene ontology and signaling pathways using differentially expressed proteins (DEPs) to examine the molecular characteristics of distant metastatic breast cancer. In addition, real-time polymerase chain reaction (RT-PCR) and invasion/migration assays were performed to validate the differential regulation and function of our protein targets. RESULTS: A total of 9441 and 8746 proteins were identified from the pooled and individual sample sets, respectively. Based on our criteria, TUBB2A was selected as a novel biomarker candidate. The metastatic activities of TUBB2A were subsequently validated. In our bioinformatics analysis using DEPs, we characterized the overall molecular features of distant metastasis and measured differences in the molecular functions of distant metastatic breast cancer between breast cancer subtypes. CONCLUSIONS: Our report is the first study to examine the distant metastatic breast cancer proteome using FFPE tissues. The depth of our dataset allowed us to discover a novel biomarker candidate and a proteomic characteristics of distant metastatic breast cancer. Distinct molecular features of various breast cancer subtypes were also established. Our proteomic data constitute a valuable resource for research on distant metastatic breast cancer.

Moesin (MSN) as a Novel Proteome-Based Diagnostic Marker for Early Detection of Invasive Bladder Urothelial Carcinoma in Liquid-Based Cytology.

Bladder urothelial carcinoma (BUC) is the most lethal malignancy of the urinary tract. Treatment for the disease highly depends on the invasiveness of cancer cells. Therefore, a predictive biomarker needs to be identified for invasive BUC. In this study, we employed proteomics methods on urine liquid-based cytology (LBC) samples and a BUC cell line library to determine a novel predictive biomarker for invasive BUC. Furthermore, an in vitro three-dimensional (3D) invasion study for biological significance and diagnostic validation through immunocytochemistry (ICC) were also performed. The proteomic analysis suggested moesin (MSN) as a potential biomarker to predict the invasiveness of BUC. The in vitro 3D invasion study showed that inhibition of MSN significantly decreased invasiveness in BUC cell lines. Further validation using ICC ultimately confirmed moesin (MSN) as a potential biomarker to predict the invasiveness of BUC (p = 0.023). In conclusion, we suggest moesin as a potential diagnostic marker for early detection of BUC with invasion in LBC and as a potential therapeutic target.

Downregulation of N-myc and STAT Interactor Protein Predicts Aggressive Tumor Behavior and Poor Prognosis in Invasive Ductal Carcinoma.

PURPOSE: We investigated the expression of the N-myc and STAT interactor (NMI) protein in invasive ductal carcinoma tissue and estimated its clinicopathologic significance as a prognostic factor. The expression levels and prognostic significance of NMI were also analyzed according to the molecular subgroup of breast cancers. METHODS: Human NMI detection by immunohistochemistry was performed using tissue microarrays of 382 invasive ductal carcinomas. The correlation of NMI expression with patient clinicopathological parameters and prognostic significance was analyzed and further assessed according to the molecular subgroup of breast cancers. Moreover, in vitro experiments with 13 breast cancer cell lines were carried out. We also validated NMI expression significance in The Cancer Genome Atlas cohort using the Human Protein Atlas (HPA) database. RESULTS: Low NMI expression was observed in 190 cases (49.7%). Low NMI expression was significantly associated with the "triple-negative" molecular subtype (p < 0.001), high nuclear grade (p < 0.001), high histologic grade (p < 0.001), and advanced anatomic stage (p = 0.041). Patients with low NMI expression had poorer progression-free survival (p = 0.038) than patients with high NMI expression. Low NMI expression was not significantly associated with patient prognosis in the molecular subgroup analysis. In vitro, a reduction of NMI expression was observed in 8 breast cancer cell lines, especially in the estrogen receptor-positive and basal B type of triple-negative breast cancer molecular subgroups. The HPA database showed that low NMI expression levels were associated with a lower survival probability compared with that associated with high NMI expression (p = 0.053). CONCLUSION: NMI expression could be a useful prognostic biomarker and a potential novel therapeutic target in invasive ductal carcinoma.

Comparison of clinical performance of two high-throughput liquid bead microarray assays, GeneFinder and CareGENE, for cervical screening in the general population.

Recently, a clinical need for an improved human papilloma virus (HPV) test that covers a broad range of genotypes has emerged as a valuable primary screening tool for cervical lesions. The liquid bead microarray (LBMA) assay is a recently developed high-throughput platform covering a broad range of genotypes. Here, we compared the clinical performance of two recently developed LBMA assays, GeneFinderTM HPV Liquid Bead Microarray (GeneFinder) and CareGENETM HPV genotyping kit-O (CareGENE), in the Korean general population. A total of 3,148 cervical swabs were tested by the GeneFinder and CareGENE assays. Cases with discrepant results between the two assays were subjected to direct sequencing as a reference method for evaluating the performance of the two LBMA assays. Among all swabs tested, 12.6% showed HPV positivity, and the prevalent HPV genotypes were HPV53, 70, 16, 39, and 51, in that order. The concordance rates between the two assays for the detection of HPV and for genotyping were 96.6% (kappa = 0.836) and 94.5% (kappa = 0.779), respectively. The two LBMA assays showed comparable sensitivity and specificity for HPV detection (GeneFinder: sensitivity 94.4% and specificity 98.7%, CareGENE: sensitivity 89.8% and specificity 99.6%) and for genotyping (GeneFinder: sensitivity 91.0% and specificity 96.6%, CareGENE: sensitivity 90.2% and specificity 99.1%). This is the first demonstration that CareGENE has comparable clinical performance to GeneFinder, which has been established to show excellent performance for screening HPV in previous studies. Both LBMA platforms are thus considered to be valuable tools for HPV detection and genotyping to improve cervical screening in the general population.

High Cytoplasmic CXCR4 Expression Predicts Prolonged Survival in Triple-Negative Breast Cancer Patients Treated with Adjuvant Chemotherapy.

BACKGROUND: Chemokine receptor CXC chemokine receptor type 4 (CXCR4) and its ligand CXC motif chemokine 12 (CXCL12; stromal cell-derived factor-1) are implicated in tumor growth, metastasis, and tumor cell-microenvironment interaction. A number of studies have reported that increased CXCR4 expression is associated with worse prognosis in triple-negative breast cancer (TNBC), but its prognostic significance has not been studied in TNBC patients treated with adjuvant chemotherapy. METHODS: Two hundred eighty-three TNBC patients who received adjuvant chemotherapy were retrospectively analyzed. Tissue microarray was constructed from formalinfixed, paraffin-embedded tumor tissue and immunohistochemistry for CXCR4 and CXCL12 was performed. Expression of each marker was compared with clinicopathologic characteristics and outcome. RESULTS: High cytoplasmic CXCR4 expression was associated with younger age (p = .008), higher histologic grade (p = .007) and lower pathologic stage (p = .045), while high CXCL12 expression was related to larger tumor size (p = .045), positive lymph node metastasis (p = .005), and higher pathologic stage (p = .017). The patients with high cytoplasmic CXCR4 experienced lower distant recurrence (p = .006) and better recurrence-free survival (RFS) (log-rank p = .020) after adjuvant chemotherapy. Cytoplasmic CXCR4 expression remained an independent factor of distant recurrence (p = .019) and RFS (p = .038) after multivariate analysis. CONCLUSIONS: High cytoplasmic CXCR4 expression was associated with lower distant recurrence and better RFS in TNBC patients treated with adjuvant chemotherapy. This is the first study to correlate high CXCR4 expression to better TNBC prognosis, and the underlying mechanism needs to be elucidated in further studies.

Novel cytomorphologic characteristics suggesting human papillomavirus infection in patients diagnosed as negative for intraepithelial lesion or malignancy and a comparison of diagnostic performance of three human papillomavirus tests.

BACKGROUND: Although the Papanicolaou (Pap) test is the first-line screening method for cervical cancer, it has low sensitivity for detection of human papillomavirus (HPV)-infected cervical lesion compared to the HPV test. The aims of this study are to determine novel cytomorphologic parameters for HPV infection in patients previously diagnosed as negative for intraepithelial lesion or malignancy (NILM) and to comparatively analyze the detection performance of 3 HPV tests: nested PCR, the DNA Chip test, and the Liquid Beads Microarray (LBMA) assay. METHODS: In total, 232 patients diagnosed with NILM were enrolled and assessed using 8 cytomorphologic parameters. RESULTS: Six non-classical cytomorphologic features were identified as novel characteristics suggesting HPV infection in patients initially diagnosed with NILM. A combination of these 6 variables showed the best predictive performance for HPV infection (area under the curve, 0.722). In terms of diagnostic ability, the LBMA assay showed better performance in detection of HPV infection (39.7%) in NILM cases compared to the other tests. CONCLUSIONS: Our results suggest that the novel cytomorphologic features used in this study can be used as supportive morphologic parameters to increase the sensitivity of cytological screening tests. The LBMA assay could be used as an advanced method for HPV detection.

Quantitative Proteomic Analysis Identifies AHNAK (Neuroblast Differentiation-associated Protein AHNAK) as a Novel Candidate Biomarker for Bladder Urothelial Carcinoma Diagnosis by Liquid-based Cytology.

Cytological examination of urine is the most widely used noninvasive pathologic screen for bladder urothelial carcinoma (BLCA); however, inadequate diagnostic accuracy remains a major challenge. We performed mass spectrometry-based proteomic analysis of urine samples of ten patients with BLCA and ten paired patients with benign urothelial lesion (BUL) to identify ancillary proteomic markers for use in liquid-based cytology (LBC). A total of 4,839 proteins were identified and 112 proteins were confirmed as expressed at significantly different levels between the two groups. We also performed an independent proteomic profiling of tumor tissue samples where we identified 7,916 proteins of which 758 were differentially expressed. Cross-platform comparisons of these data with comparative mRNA expression profiles from The Cancer Genome Atlas identified four putative candidate proteins, AHNAK, EPPK1, MYH14 and OLFM4. To determine their immunocytochemical expression levels in LBC, we examined protein expression data from The Human Protein Atlas and in-house FFPE samples. We further investigated the expression of the four candidate proteins in urine cytology samples from two independent validation cohorts. These analyses revealed AHNAK as a unique intracellular protein differing in immunohistochemical expression and subcellular localization between tumor and non-tumor cells. In conclusion, this study identified a new biomarker, AHNAK, applicable to discrimination between BLCA and BUL by LBC. To our knowledge, the present study provides the first identification of a clinical biomarker for LBC based on in-depth proteomics.

Cytologic Diagnosis of Metastatic Alveolar Rhabdomyosarcoma in Cerebrospinal Fluid: A Case Report.

Rhabdomyosarcoma is a malignant soft tissue tumor which shows skeletal muscle differentiation. Leptomeningeal metastasis can occur as a late complication, but currently there are no reports that have documented the cytologic features in cerebrospinal fluid (CSF). We report a case of metastatic alveolar rhabdomyosarcoma diagnosed in the CSF of a 28-year-old male who was originally diagnosed with rhabdomyosarcoma on the neck, and that went through systemic therapy. The tumor was positive for anaplastic lymphoma kinase, but progressed despite additional therapy with crizotinib. The CSF specimen revealed small round cells, large atypical cells with abundant cytoplasm and eccentric nuclei, and cells with horseshoe-shaped nuclei. These cytologic findings were in agreement with previous literature and well-correlated with histopathology. This is the first report to document the cytologic feature of rhabdomyosarcoma in CSF. In many cases it is difficult to perform ancillary tests in a CSF specimen and cytopathologists should be aware of the cytomorphologic characteristics to avoid misdiagnosis.

Duodenal Adenocarcinoma of Brunner Gland Origin: A Case Report.

We report a case of adenocarcinoma originating from the duodenal Brunner glands in a 47-year-old female patient. The lesion was 0.8 cm in extent and located at the posterior wall of the first part of the duodenum. Histologically, the tumor showed transition from non-neoplastic Brunner glands through dysplastic epithelium into adenocarcinoma. The carcinoma cells were strongly positive for MUC6 protein, which is an epithelial marker for the Brunner glands. Tumor protein p53 was overexpressed in the carcinoma cells, but not in the non-neoplastic or dysplastic epithelium. Dystrophic calcification was predominant. This is the first case report of duodenal adenocarcinoma of Brunner gland origin in Korea.

SM22alpha inhibits cell proliferation and protects against anticancer drugs and gamma-radiation in HepG2 cells: involvement of metallothioneins.

Smooth muscle protein 22-alpha (SM22alpha) has been postulated to affect the structure and function of the actin filament. In this study, we report on the significant induction of SM22alpha by cytotoxic agents in HepG2 cells. SM22alpha-overexpression inhibited the activation of IGF-1Rbeta/Akt and Erk, consequently suppressing cell proliferation. On the other hand, SM22alpha-overexpressing cells became resistant to apoptotic cell death caused by cytotoxic agents, in which metallothionein (MT) isoforms, especially MT1G, were significantly induced. MT1G-overexpression also conferred cellular resistance, and SM22alpha regulated the expression of MT1G at a transcriptional level. This study provides the first demonstration of SM22alpha-induced blockage of cell proliferation and cellular resistance to overcome the detrimental effects of damaging agents.