PANoptosis subtypes predict prognosis and immune efficacy in gastric cancer.

PANoptosis is a form of inflammatory programmed cell death that is regulated by the PANoptosome. This PANoptosis possesses key characteristics of pyroptosis, apoptosis, and necroptosis, yet cannot be fully explained by any of these cell death modes. The unique nature of this cell death mechanism has garnered significant interest. However, the specific role of PANoptosis-associated features in gastric cancer (GC) is still uncertain. Patients were categorized into different PAN subtypes based on the expression of genes related to the PANoptosome. We conducted a systematic analysis to investigate the variations in prognosis and tumor microenvironment (TME) among these subtypes. Furthermore, we developed a risk score, called PANoptosis-related risk score (PANS), which is constructed from genes associated with the PANoptosis. We comprehensively analyzed the correlation between PANS and GC prognosis, TME, immunotherapy efficacy and chemotherapeutic drug sensitivity. Additionally, we performed in vitro experiments to validate the impact of Keratin 7 (KRT7) on GC. We identified two PAN subtypes (PANcluster A and B). PANoptosome genes were highly expressed in PANcluster A. PANcluster A has the characteristics of favorable prognosis, abundant infiltration of anti-tumor lymphocytes, and sensitivity to immunotherapy, thus it was categorized as an immune-inflammatory type. Meanwhile, our constructed PANS can effectively predict the prognosis and immune efficacy of GC. Patients with low PANS have a good prognosis, and have the characteristics of high tumor mutation load (TMB), high microsatellite instability (MSI), low tumor purity and sensitivity to immunotherapy. In addition, PANS can also identify suitable populations for different chemotherapy drugs. Finally, we confirmed that KRT7 is highly expressed in GC. Knocking down the expression of KRT7 significantly weakens the proliferation and migration abilities of GC cells. The models based on PANoptosis signature help to identify the TME features of GC and can effectively predict the prognosis and immune efficacy of GC. Furthermore, the experimental verification results of KRT7 provide theoretical support for anti-tumor treatment.

Role of the KRT7 Biomarker in Immune Infiltration and Paclitaxel Resistance in Ovarian.

Context: Paclitaxel (PTX) resistance is often associated with poor outcomes for patients with ovarian cancer (OC), but its mechanism is unknown. Clinicians are increasingly using immunotherapy in the management of OC, and the ability to assess tumor-immune interactions and identify effective, predictive, prognostic molecular biomarkers for OC is an urgent need. Objective: The study intended to explore the potential tumorigenesis mechanisms to identify promising biomarkers and improve survival in OC patients. Design: The research team performed a genetic analysis. Setting: The study took place at First Affiliated Hospital of Jinan University, Guangzhou, Guangdong, China. Outcome Measures: The research team: (1) obtained GSE66957 and GSE81778 gene expression profiles from the Gene Expression Omnibus (GEO) database and identified 468 differentially expressed genes (DEGs); (2) conducted functional enrichment analysis and constructed a protein-to-protein interaction (PPI) network; (3) identified the OC survival-related genes using the Gene Expression Profiling Interactive Analysis 2 (GEPIA2) webserver and compared those genes with upregulated DEGs to identify the core genes; (4) used GEPIA2 and the Kaplan-Meier plotter to explore the expression profiles and the prognostic values of the core genes in OC; (5) used the LinkOmics, Oncomine, and GEPIA2 web servers to perform co-expression analysis and explore functional networks correlated with keratin 7 (KRT7); (6) performed correlation analyses between KRT7, the six main types of tumor-infiltrating lymphocytes (TILs), and immune signatures, using the TIMER tool; and (7) subsequently detected the KRT7 expression in the cell lines IOSE80, A2780, A2780/PTX, ho8910, skov3, and ovcar3 using quantitative reverse transcription-polymerase chain reaction (RT-qPCR) technology. Results: High expression levels of KRT7 were significantly correlated with progression-free survival (PFS) and poor overall survival (OS) for OC patients, with logrank P = .0074 and logrank P = .014, respectively. The expression levels of KRT7 were also significantly correlated with the infiltrated neutrophil levels (r = 0.169, P = .0077). The study identified neutrophils as potential predictors of survival in OC. Moreover, the expression levels of KRT7 in OC were positively correlated with 51 (31.68%) of the 161 immune gene markers. The RT-qPCR analyses revealed a high expression of KRT7 in the paclitaxel-resistant OC cell line. Conclusions: KRT7 is correlated with immune infiltration and paclitaxel resistance in OC patients. Therefore, clinicians could use KRT7 as a prognostic marker and a target in the development of new drugs.

Construction of a novel model based on cell-in-cell-related genes and validation of KRT7 as a biomarker for predicting survival and immune microenvironment in pancreatic cancer.

BACKGROUND: Pancreatic cancer (PC) is a highly malignant tumor featured with high intra-tumoral heterogeneity and poor prognosis. Cell-in-cell (CIC) structures have been reported in multiple cancers, and their presence is associated with disease progression. Nonetheless, the prognostic values and biological functions of CIC-related genes in PC remain poorly understood. METHODS: The sequencing data, as well as corresponding clinicopathological information of PC were collected from public databases. Random forest screening, least absolute shrinkage, and selection operator (LASSO) regression and multivariate Cox regression analysis were performed to construct a prognostic model. The effectiveness and robustness of the model were evaluated using receiver operating characteristic (ROC) curves, survival analysis and establishing the nomogram model. Functional enrichment analyses were conducted to annotate the biological functions. The immune infiltration levels were evaluated by ESTIMATE and CIBERSORT algorithms. The expression of KRT7 (Keratin 7) was validated by quantitative real-time PCR (qRT-PCR), western blotting and immunohistochemistry (IHC) staining. The CIC formation, cell clusters, cell proliferation, migration and invasion assays were applied to investigate the effects of silencing the expression of KRT7. RESULTS: A prognostic model based on four CIC-related genes was constructed to stratify the patients into the low- and high-risk subgroups. The high-risk group had a poorer prognosis, higher tumor mutation burden and lower immune cell infiltration than the low-risk group. Functional enrichment analyses showed that numerous terms and pathways associated with invasion and metastasis were enriched in the high-risk group. KRT7, as the most paramount risk gene in the prognostic model, was significantly associated with a worse prognosis of PC in TCGA dataset and our own cohort. High expression of KRT7 might be responsible for the immunosuppression in the PC microenvironment. KRT7 knockdown was significantly suppressed the abilities of CIC formation, cell cluster, cell proliferation, migration, and invasion in PC cell lines. CONCLUSIONS: Our prognostic model based on four CIC-related genes has a significant potential in predicting the prognosis and immune microenvironment of PC, which indicates that targeting CIC processes could be a therapeutic option with great interests. Further studies are needed to reveal the underlying molecular mechanisms and biological implications of CIC phenomenon and related genes in PC progression.

Cytokeratin 7 and cytokeratin 20 expression in cancer: A tissue microarray study on 15,424 cancers.

Combined analysis of cytokeratin 7 (CK7) and cytokeratin 20 (CK20) is often used for assessing the origin of metastatic cancer. To evaluate the diagnostic utility of CK7 and CK20, tissue microarrays containing 15,424 samples from 120 different tumor types and subtypes and 608 samples of 76 different normal tissue types were analyzed by immunohistochemistry. CK7 positivity was seen in 52% (8.7% weak, 5.9% moderate, 37% strong) and CK20 positivity in 23% (5.1% weak, 3.4% moderate, 15% strong) of interpretable tumors. Of 8390 positive tumors, 1181 (14%) showed positivity for CK7 and CK20, 5380 (64%) showed positivity for CK7 alone, and 1829 (22%) showed positivity for CK20 alone. CK20 predominated in gastrointestinal tract, urothelial and Merkel cell carcinomas. CK7 was usually negative in prostate cancer and colorectal cancer. Combined evaluation of CK7/CK20 revealed the best diagnostic utility in CK20 positive tumors, where CK7 negativity is often linked to colorectal origin while CK7 positivity argues for urothelial origin or mucinous ovarian cancer. Associations with unfavorable tumor features were found for cytokeratin 7 loss in breast cancer of no special type, urothelial and renal cell carcinomas, for CK7 overexpression in high-grade serous ovarian and gastric cancer, and for CK20 overexpression in urothelial carcinoma. CK20 loss was linked to MSI in gastric (p = 0.0291) and colorectal adenocarcinoma (p < 0.0001). These analyses provide comprehensive data on the frequency of CK7 and CK20 immunostaining - alone or in combination - in human cancers. These data facilitate interpretation of CK7/CK20 immunostaining in cancers.

Determining the relationship between cytokeratin expression and prognostic factors in human gastric cancer.

Determining the prognosis of gastric cancer is the most crucial step in the treatment process. Cytokeratins are intermediate filaments found in the intracellular structure of epithelial tissues. Recent researches have focused on determining the relationship between the expression of cytokeratins and the degree and prognosis of tumors. This study aimed to investigate the relationship between the incidence of cytokeratin-20 and cytokeratin-7 in patients with gastric carcinoma with factors influencing the prognosis. In this regard, the study was conducted cross-sectional. The expression of cytokeratin-20 and cytokeratin-7 was evaluated on 50 gastric adenocarcinoma specimens with different degrees of differentiation by the immunohistochemical method. We determined the relationship between the incidence of cytokeratin-20 and cytokeratin-7 with factors affecting the prognosis of patients, including the degree of differentiation of gastric cancer tissue, lymph node involvement, and the depth of tumor invasion. Data were statistically analyzed by Chi-square and Spearman tests. The results showed a statistically inverse relationship between the incidence of cytokeratin-20 and cytokeratin-7 with the degree of tissue differentiation and lymph node involvement in gastric cancer. Although there was a statistically significant relationship between the incidence of tissue invasion in gastric cancer and the incidence of cytokeratin-7, there was no association between the incidence of cytokeratin-20 and tissue invasion. In general, decreased cytokeratin-20 and cytokeratin-7 are associated with decreased tissue differentiation and increased lymph node involvement.

Expression Level of Keratin 7 in Epithelial Ovarian Cancer and Malignant Metastasis of Benign Epithelial Ovarian Tumors.

It was to investigate the diagnostic value of keratin 7 (KRT7) in malignant metastasis of epithelial ovarian cancer and benign epithelial ovarian tumors. From January 2018 to January 2019, 30 fresh tissues of benign epithelial ovarian tumors, 30 fresh tissues of borderline tumors, 30 fresh tissues of metastatic ovarian were collected in The First Affiliated Hospital of Fujian Medical University, and 30 fresh tissues of normal ovarian tissues were collected as the control group. Federation of gynecology and obstetrics (FIGO) staging criteria: 25 cases of stage I, 26 cases of stage II, 16 cases of stage III, and 23 cases of stage IV. The relative expression of KRT7 was detected by real-time fluorescence quantitative PCR, and the relationship between KRT7 expression and epithelial ovarian cancer grading was analyzed. The results showed that the positive expression rate of KRT7 was 12.1% in normal ovarian tissues, 28.4% in benign epithelial ovarian tumors, 53.5% in borderline tumors, and 24.2% in metastatic ovarian cancer. With the increase of tumor stage malignancy, the relative expression of KRT7 decreased significantly, but there was no significant difference between stage I and stage II, stage III and stage IV (P > 0.05). The difference between stage I and stage III, and stage IV was significant (P < 0.05). Patients with epithelial ovarian cancer had a significant difference compared with the control group (P < 0.05). In summary, compared with the control group, the expression of KRT7 in patients with benign epithelial ovarian tumors and borderline tumors was significantly decreased. The expression level of KRT7 in benign epithelial ovarian tumors was lower than that in borderline tumors. The expression of KRT7 was related to the occurrence, development, and deterioration of ovarian cancer, which provided a basis for targeted therapy of tumors.

MicroRNA-216a targets WT1 expression and regulates KRT7 transcription to mediate the progression of pancreatic cancer-A transcriptome analysis.

Gene expression profiling has been broadly performed in the field of cancer research. This study aims to explore the key gene regulatory network and focuses on the functions of microRNA (miR)-216a in pancreatic cancer (PC). PC datasets GSE15471, GSE16515, and GSE32676 were used to screen the differentially expressed genes (DEGs) in PC. A miRNA microarray analysis and gene oncology analysis suggested miR-216a as an important differentially expressed miRNA in PC. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis suggested that miR-216a and the DEGs are largely enriched on the phosphatidyl inositol 3-kinase/protein kinase B (PI3K/AKT) signaling pathway. miR-216a targeted Wilms Tumor 1 (WT1), while WT1 promoted transcription activity of keratin 7 (KRT7). Upregulation of miR-216a reduced proliferation and invasiveness of PC cells, while further upregulation of WT1 blocked the functions of miR-216a. Silencing of KRT7 diminished the oncogenic role of WT1. The in vitro results were reproduced in vivo. High expression of miR-216a while poor expression of WT1 indicated better prognosis of PC patients. The miR-216a/WT1/KRT7 axis influenced the activity of the PI3K/AKT pathway. To conclude, this study evidenced that miR-216a suppressed WT1 expression and blocked KRT7 transcription, which inactivated the PI3K/AKT signaling and reduced PC progression.

Keratin 7 Is a Constituent of the Keratin Network in Mouse Pancreatic Islets and Is Upregulated in Experimental Diabetes.

Keratin (K) 7 is an intermediate filament protein expressed in ducts and glands of simple epithelial organs and in urothelial tissues. In the pancreas, K7 is expressed in exocrine ducts, and apico-laterally in acinar cells. Here, we report K7 expression with K8 and K18 in the endocrine islets of Langerhans in mice. K7 filament formation in islet and MIN6 ß-cells is dependent on the presence and levels of K18. K18-knockout (K18‒/‒) mice have undetectable islet K7 and K8 proteins, while K7 and K18 are downregulated in K8‒/‒ islets. K7, akin to F-actin, is concentrated at the apical vertex of ß-cells in wild-type mice and along the lateral membrane, in addition to forming a fine cytoplasmic network. In K8‒/‒ ß-cells, apical K7 remains, but lateral keratin bundles are displaced and cytoplasmic filaments are scarce. Islet K7, rather than K8, is increased in K18 over-expressing mice and the K18-R90C mutation disrupts K7 filaments in mouse ß-cells and in MIN6 cells. Notably, islet K7 filament networks significantly increase and expand in the perinuclear regions when examined in the streptozotocin diabetes model. Hence, K7 represents a significant component of the murine islet keratin network and becomes markedly upregulated during experimental diabetes.

Functional and genomic characterization of three novel cell lines derived from a metastatic gallbladder cancer tumor.

BACKGROUND: Gallbladder cancer (GBC) is the most common tumor of the biliary tract. The incidence of GBC shows a large geographic variability, being particularly frequent in Native American populations. In Chile, GBC represents the second cause of cancer-related death among women. We describe here the establishment of three novel cell lines derived from the ascitic fluid of a Chilean GBC patient, who presented 46% European, 36% Mapuche, 12% Aymara and 6% African ancestry. RESULTS: After immunocytochemical staining of the primary cell culture, we isolated and comprehensively characterized three independent clones (PUC-GBC1, PUC-GBC2 and PUC-GBC3) by short tandem repeat DNA profiling and RNA sequencing as well as karyotype, doubling time, chemosensitivity, in vitro migration capability and in vivo tumorigenicity assay. Primary culture cells showed high expression of CK7, CK19, CA 19-9, MUC1 and MUC16, and negative expression of mesothelial markers. The three isolated clones displayed an epithelial phenotype and an abnormal structure and number of chromosomes. RNA sequencing confirmed the increased expression of cytokeratin and mucin genes, and also of TP53 and ERBB2 with some differences among the three cells lines, and revealed a novel exonic mutation in NF1. The PUC-GBC3 clone was the most aggressive according to histopathological features and the tumorigenic capacity in NSG mice. CONCLUSIONS: The first cell lines established from a Chilean GBC patient represent a new model for studying GBC in patients of Native American descent.

Clonal expansion of hepatic progenitor cells and differentiation into hepatocyte-like cells.

Hepatic progenitor cells (HPCs) in adult liver are promising for treatment of liver diseases. A biliary-derived HPC population in adult mice has been characterized by co-expression of stem cell marker Sry (sex determining region Y)-box 9 (SOX9) and biliary marker cytokeratin 7 (CK7). However, isolation of these HPCs in adult healthy liver without any selection procedures remains a big challenge in this field. Here, by establishing a simple and efficient method to isolate and expand the CK7+ SOX9+ HPCs in vitro as clones, we acquired a stable and largely scalable cell source. The CK7+ SOX9+ progenitor cells were then further induced to differentiate into hepatocyte-like cells with expression of mature hepatocyte markers albumin (Alb) and hepatocyte nuclear factor 4 alpha (HNF4α), both in vitro and in vivo in the presence of hepatocyte growth factor (HGF) and fibroblast growth factor 9 (FGF9). Furthermore, we found that the HPCs are highly responsive to transforming growth factor-beta (TGF-ß) signals. Collectively, we identified and harvested a CK7+ SOX9+ progenitor cell population from adult mouse liver by a simple and efficient approach. The exploration of this HPC population offers an alternative strategy of generating hepatocyte-like cells for cell-based therapies of acute and chronic liver disorders.

Cytokeratin 7-negative and GATA binding protein 3-negative breast cancers: Clinicopathological features and prognostic significance.

BACKGROUND: Cytokeratin 7 (CK7) and GATA binding protein 3 (GATA3) are considered as immunohistochemical hallmarks of breast cancers; however, there are breast tumors lacking these markers. Clinicopathological characterization of CK7 negative breast cancer has not been addressed previously and similar studies on GATA3 negative tumors are limited. METHODS: This study included 196 consecutive cases of Nottingham Grade 3 breast cancers with 159 cases of Grade 1 and Grade 2 tumors for comparison. CK7 and GATA3 expression was correlated with patient's age, histological type, pathological grade and stage, hormone receptor status, molecular subtype and overall survival. RESULTS: CK7 negativity was seen in 13% of Grade 3, 9% of Grade 2, and 2% of Grade 1 cases (P = 0.0457). Similarly, 28% of Grade 3, 5% of Grade 2 and 2% of Grade 1 cases were GATA3 negative (P < 0.0001). CK7 negative tumors did not show association with other clinicopathological parameters. GATA3 negative tumors were enriched in the basal-like molecular subgroup and were associated with negative estrogen receptor (ER) and negative progesterone receptor (PR) statuses. Both CK7 and GATA3 expression showed no association with overall survival in patients with Grade 3 tumor. CONCLUSIONS: This is the first study to characterize CK7 negative breast tumors in the context of clinicopathology. Profiling the CK7 negative and GATA3 negative breast cancers helps to understand the biology of these specific tumor subgroups and may aid in their diagnosis.

Safety of ovarian tissue transplantation in patients with borderline ovarian tumors.

STUDY QUESTION: Is transplantation of cryopreserved ovarian tissue from patients with borderline ovarian tumors (BOTs) a safe procedure? SUMMARY ANSWER: BOT cells were found in frozen-thawed and xenografted ovarian tissue in 1 of 11 BOT patients. WHAT IS KNOWN ALREADY: The risk of reintroducing malignant cells upon ovarian tissue transplantation has been subject of debate for many years. Reimplantation of cryopreserved ovarian tissue from leukemia patients is unsafe, while results from studies of cryopreserved ovarian tissue from other forms of cancer, such as Hodgkin's lymphoma, are reassuring. STUDY DESIGN, SIZE, DURATION: Prospective experimental study conducted in an academic research unit using ovarian tissue from 11 patients undergoing cryopreservation for BOTs. PARTICIPANTS/MATERIALS, SETTING, METHODS: Histology, immunohistochemistry (IHC) for mucin 1 (MUC1) and cytokeratin 7 (CK7) and molecular analysis by reverse transcription quantitative polymerase chain reaction (RT-qPCR) for CK7 and MUC1 were performed on frozen-thawed ovarian tissue from 11 patients. Long-term (5 months) xenografting of ovarian tissue in immunodeficient mice was performed. The xenografts were analyzed by histology, IHC and RT-qPCR, furthermore IHC for CD10, a marker of endometriosis, was performed on a selected sample. MAIN RESULTS AND THE ROLE OF CHANCE: Analysis by histology, IHC and RT-qPCR indicated 10 of the ovarian tissue samples were negative. Analysis of the xenograft samples indicated nine were negative for malignant cells but in two xenografts glandular lesions were detected by histology. In these two xenografts, CK7 and MUC1 markers were demonstrated by IHC and CK7 expression also by RT-qPCR. A BOT was confirmed in the xenograft in which the original ovarian tissue was positive, while in the other case IHC demonstrated expression of endometriosis marker CD10. LIMITATIONS, REASONS FOR CAUTION: Cryopreserved ovarian fragments cannot be tested before transplantation, therefore the preimplantation analysis cannot guarantee that all cryopreserved fragments will be free of BOT cells. WIDER IMPLICATIONS OF THE FINDINGS: BOT cells can be found in cryopreserved ovarian tissue from BOT patients, therefore preimplantation analysis is an absolute prerequisite. Endometriosis can also be detected in cryopreserved ovarian tissue and caution should also be exercised in this scenario. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS-PDR Convention T.0077.14, Télévie Grant 7.4590.16 awarded to Rossella Masciangelo, and Grant 5/4/150/5 awarded to Marie-Madeleine Dolmans), the Fonds Speciaux de Recherche, and the Foundation Against Cancer. None of the authors have any conflicting interests to declare.

Characteristics of Trophoblasts in Long-Term Culture.

We analyzed more than 40 cytotrophoblast cultures derived from cell islets that grew from trypsinized tissue fragments of placental microvilli. Phenotypic variability of trophoblasts was demonstrated. Changes in trophoblast morphology from epithelium-like or oval cells to bipolar and spindle-shaped or twisted and then to mesenchymal-like cells as well as intensive expression of cytokeratin-7 and vimentin attested to epithelial-mesenchymal transition of trophoblasts during in vitro culturing. Analysis of the expression of specific markers in long-term trophoblast culture (≥7 passages) revealed the possibility of culture contamination with other non-trophoblast cells including fibroblasts. High risk of trophoblast culture contamination with rapidly growing cells necessitates regular control of the cultures used in fundamental studies. Our experiments confirmed the possibility of long-term culturing of cells maintaining trophoblast properties. The identity and purity of 4 trophoblast cultures free from contamination and retaining the properties of pure culture during long-term (>10 passages) culturing in vitro were confirmed.

Morphological Analysis of Biocompatibility of Autologous Bone Marrow Mononuclear Cells with Synthetic Polyethylene Terephthalate Scaffold.

We studied the properties of a tissue-engineered trachea consisting of a polyethylene terephthalate scaffold populated with autologous bone marrow mononuclear cells. The tissue-engineered constructs were obtained before surgery, during the postoperative period, and during autopsy. Cytomorphological analysis during the postoperative period showed the presence of mesenchymal stem cells on the inner surface of the implant on day 3 after surgery and cells of the respiratory epithelium on day 10-14. In autopsy samples, single epithelial cells, endothelial cells, and basal cells were found. Biocompatibility of the tissue-engineered trachea with autologous mononuclear cells of the patient was demonstrated.

Usefulness of immunocytochemistry using a Breast Marker antibody cocktail targeting P63/cytokeratin7/18/cytokeratin5/14 for fine needle aspiration of the breast: a retrospective cohort study of 139 cases.

OBJECTIVE: The Breast Marker Cocktail from Biocare Medical comprises five antibodies recognising p63, and cytokeratins (CKs) 7, 18, 5 and 14. Immunohistochemistry using this cocktail is useful for diagnosing proliferative intraductal breast lesions. However, cytology using the cocktail has not been reported. METHODS: We report 139 cases of mammary samples collected by fine needle aspiration (FNA) for which histological diagnoses were available. After cell transfer, immunocytochemistry was performed using the cocktail, and clusters of cells were classified. A cluster with no or limited CK5/14 expression (<20% of cells) was classified as a monotonous cluster. One with more than 20% of cells showing CK5/14 expression was defined as a mosaic cluster. When at least one p63-positive cell was present, we defined it as a cluster with p63. We also evaluated background p63-positive myoepithelial cell densities. RESULTS: The diagnostic sensitivity and specificity for carcinomas were 97.8% (89/91) and 91.7% (11/12), respectively, using the criterion of two or more monotonous clusters lacking p63. Two false-negative cases were triple-negative cancers; one false-positive was an apocrine papilloma. The numbers of monotonous clusters with p63 differed significantly between benign lesions, ductal carcinoma in situ (DCIS)/lobular carcinoma in situ (LCIS) and invasive carcinomas (P < 0.001). The background myoepithelial cell density was significantly higher in fibroepithelial tumours than in other lesions (P < 0.001). CONCLUSIONS: Immunocytochemistry using this antibody cocktail showed good sensitivity and specificity for diagnosing breast cancers. Thus, this method is useful for mammary cytology using FNA.

Cell-Free RNA Content in Peripheral Blood as Potential Biomarkers for Detecting Circulating Tumor Cells in Non-Small Cell Lung Carcinoma.

Circulating tumor cells (CTCs) have been implicated in tumor progression and prognosis. Techniques detecting CTCs in the peripheral blood of patients with non-small cell lung carcinoma (NSCLC) may help to identify individuals likely to benefit from early systemic treatment. However, the detection of CTCs with a single marker is challenging, owing to low specificity and sensitivity and due to the heterogeneity and rareness of CTCs. Herein, the probability of cell-free RNA content in the peripheral blood as a potential biomarker for detecting CTCs in cancer patients was investigated. An immunomagnetic enrichment of real-time reverse-transcription PCR (RT-PCR) technology for analysis of CTCs in NSCLC patients was also developed. The mRNA levels of four candidate genes, cytokeratin 7 (CK7), E74-like factor 3 (ELF3), epidermal growth factor receptor (EGFR), and erythropoietin-producing hepatocellular carcinoma receptor B4 (EphB4) that were significantly elevated in tumor tissues and peripheral blood mononuclear cells (PBMCs) were determined. The expression of CK7 and ELF3 in tumor tissues and EGFR in PBMCs was associated with lymph node metastasis (all p < 0.05). The expression of CK7 in PBMCs was correlated with age and EphB4 in PBMCs correlated with histopathological type, respectively (all p < 0.05). The expression of all four genes in tumor tissues and PBMCs was significantly correlated with the clinical stage (all p < 0.01). Survival analysis showed that the patients with enhanced expression of CK7, ELF3, EGFR, and EphB4 mRNA in PBMCs had poorer disease-free survival (DFS) and overall survival (OS) than those without (all p < 0.0001). The present study showed that this alteration of cell-free RNA content in peripheral blood might have clinical ramifications in the diagnosis and treatment of NSCLC patients.

An immunohistochemical study on the expression of sex steroid receptors, Ki-67 and cytokeratins 7 and 20 in feline endometrial adenocarcinomas.

BACKGROUND: Endometrial adenocarcinomas are a rare type of tumour in cats. Though different morphologies have been reported, the most frequent histological type of feline endometrial adenocarcinoma (FEA) is the papillary serous. Characterization of molecular markers expression in FEA may contribute to clarify the pathogenesis of these tumours and to assess the differences between normal endometrium and FEA regarding the expression pattern of several proteins. Therefore, this study aimed to evaluate the immunohistochemical profile of a wide panel of antibodies (specific for ER-α, PR, Ki-67, CK7 and CK20) in twenty-four cases of FEA. Comparisons were made between FEA and feline normal cyclic endometrium in follicular (n = 13) and luteal (n = 10) stages. Except for Ki-67, all other molecular markers were assessed independently for the intensity of immunolabeling and for the percentage of cells expressing the protein. RESULTS: This study showed that in FEA a loss of expression occurs for ER-α (P ≤ 0.0001) and less markedly also for PR. The lost in sex steroid receptors concerns a decrease in both the proportion of labelled cells and the intensity of immunolabelling (P = 0.002 and P = 0.024, respectively). Proliferative activity, estimated via Ki-67 immunoreaction, significantly increased in FEA as compared to normal endometrium (P ≤ 0.0001). Feline endometrial adenocarcinomas maintained the CK7+/CK20+ status of normal endometrium. However, FEA showed decreased CK7 intensity of labelling compared to normal endometria (P ≤ 0.0001) and loss of CK20 expression, both in intensity (P ≤ 0.0001) and in percentage of positive cells (P = 0.01), compared to normal tissues. CONCLUSIONS: Data gathered in this study suggest that proliferation in FEA accompanies ER-α down-regulation, possibly following activation of pathways mediated by local growth factors. Moreover, FEA retains combined expression of CK7 and CK20, as evidenced in normal endometrial epithelia, although a decrease in CK7 expression was observed.

Inductive differentiation of conjunctival goblet cells by γ-secretase inhibitor and construction of recombinant conjunctival epithelium.

γ-secretase inhibitor has been shown to promote intestinal goblet cell differentiation. We now demonstrated that the in vitro addition of γ-secretase inhibitor in the culture of human conjunctival epithelial cells significantly promoted the differentiation of conjunctival goblet cells with typical droplet-like phenotype, positive periodic acid-Schiff and goblet cell-specific Muc5Ac, cytokeratin 7 and Helix pomatia agglutinin lectin staining. Moreover, topical application of γ-secretase inhibitor promoted the differentiation of mouse conjunctival goblet cells in vivo. Furthermore, the expression of Notch target gene HES-1 was down-regulated during the differentiation of conjunctival goblet cells. In addition, we found that the recombinant conjunctival epithelium on amniotic membrane showed less goblet cell density and abnormal location when compared with normal conjunctival epithelium, which were improved by the addition of γ-secretase inhibitor in the final induction.

Newly identified biomarkers for detecting circulating tumor cells in lung adenocarcinoma.

Circulating tumor cells (CTCs) have been implicated in cancer prognosis and follow up. Detection of CTCs was considered significant in cancer evaluation. However, due to the heterogeneity and rareness of CTCs, detecting them with a single maker is usually challenged with low specificity and sensitivity. Previous studies concerning CTCs detection in lung cancer mainly focused on non-small cell lung carcinoma. Currently, there is no report yet describing the CTC detection with multiple markers in lung adenocarcinoma. In this study, by employing quantitative real-time PCR, we identified four candidate genes (mRNA) that were significantly elevated in peripheral blood mononuclear cells and biopsy tissue samples from patients with lung adenocarcinoma: cytokeratin 7 (CK7), Ca(2+)-activated chloride channel-2 (CLCA2), hyaluronan-mediated motility receptor (HMMR), and human telomerase catalytic subunit (hTERT). Then, the four markers were used for CTC detection; namely, positive detection was defined if at least one of the four markers was elevated. The positive CTC detection rate was 74.0% in patients with lung adenocarcinoma while 2.2% for healthy controls, 6.3% for benign lung disease, and 48.0% for non-adenocarcinoma non-small cell lung carcinoma. Furthermore, in a three-year follow-up study, patients with an increase in the detection markers of CTCs (CK7, CLCA2, HMMR or hTERT) on day 90 after first detection had shorter survival time compared to those with a decrease. These results demonstrate that the combination of the four markers with specificity and sensitivity is of great value in lung adenocarcinoma prognosis and follow up.

Diagnosis of lymph node micrometastasis at the pN0 stage of lung adenocarcinoma using a combination of markers.

This study aimed to detect micrometastatic tumor cells in the lymph nodes of patients with pN0 lung adenocarcinoma using a combination of thyroid transcription factor-1 (TTF-1) expression and cytokeratin 7 (CK7) expression and to investigate the association of lymph node micrometastasis with the clinicopathological characteristics of patients with lung adenocarcinoma. A total of 54 patients with pN0 lung adenocarcinoma and whose primary tumors were positive for both TTF-1 and CK7 expression were included in this study. In total, 893 lymph nodes were obtained from these 54 patients and were analyzed for micrometastasis by immunohistochemical staining with anti-CK7 and anti-TTF-1 antibodies. CK7- and TTF-1-positive cells were found in the lymph nodes of 9 (16.7%) of 54 patients, and 21 (2.4%) of 893 lymph nodes exhibited positivity for these factors. No cells positive for both CK7 and TTF-1 were detected in the 5 lymph nodes obtained from patients with benign lung tumors. Lymph node micrometastasis was found to be associated with the differentiation grade and primary tumor position (P < 0.05). The detection of lymph node micrometastasis by a combination of CK7 and TTF-1 immunohistochemical staining provides a more accurate assessment of tumor staging for pN0 lung adenocarcinoma.