CK19 mRNA Expression Research Articles

Novel Screening System for Biliary Excretion of Drugs Using Human Cholangiocyte Organoid Monolayers with Directional Drug Transport.

It has recently been reported that cholangiocyte organoids can be established from primary human hepatocytes. The purpose of this study was to culture the organoids in monolayers on inserts to investigate the biliary excretory capacity of drugs. Cholangiocyte organoids prepared from hepatocytes had significantly higher mRNA expression of CK19, a bile duct epithelial marker, compared to hepatocytes. The organoids also expressed mRNA for efflux transporters involved in biliary excretion of drugs, P-glycoprotein (P-gp), multidrug resistance-associated protein 2 (MRP2), and breast cancer resistance protein (BCRP). The subcellular localization of each protein was observed. These results suggest that the membrane-cultured cholangiocyte organoids are oriented with the upper side being the apical membrane side (A side, bile duct lumen side) and the lower side being the basolateral membrane side (B side, hepatocyte side), and that each efflux transporter is localized to the apical membrane side. Transport studies showed that the permeation rate from the B side to the A side was faster than from the A side to the B side for the substrates of each efflux transporter, but this directionality disappeared in the presence of inhibitor of each transporter. In conclusion, the cholangiocyte organoid monolayer system has the potential to quantitatively evaluate the biliary excretion of drugs. The results of the present study represent an unprecedented system using human cholangiocyte organoids, which may be useful as a screening model to directly quantify the contribution of biliary excretion to the clearance of drugs.

An improved method for the isolation and culture of rat pancreatic ductal epithelial cells.

BackgroundThis aim of this study was to explore a novel method that can be used to isolate and culture rat pancreatic ductal epithelial cells.MethodsRetrograde injection of indigo carmine solution into the bile duct of rats revealed the main pancreatic duct, which was isolated using the naked eye (without using a stereomicroscope). The main pancreatic duct was sequentially digested with three enzymes, and the digested cells were cultured in RPMI-1640 medium containing 10–15% fetal bovine serum. After 72 h, the primary pancreatic ductal epithelial cells started to adhere to the wall. The cells reached 70–80% confluence after approximately 7 days and were subsequently digested with 0.25% trypsin and subcultured. Cells of the second and fourth passages were harvested. Cytokeratin (CK)-7 and CK-19 protein expressions in the cells and pancreatic tissue were detected by Western blot analysis. q-PCR was employed to examine CK-7, CK-19, somatostatin, amylase, insulin, and glucagon mRNA expression in the cells and pancreatic tissue after the main pancreatic duct was removed.ResultsThe rats had one or two main pancreatic ducts meeting the bile ducts at a right or an acute angle. Rat pancreatic ductal epithelial cells isolated by this method grew well and showed a cobblestone-like appearance via microscopy. Western blot analysis showed that both the second and fourth passages of pancreatic ductal epithelial cells expressed CK-7 and CK-19 protein. The q-PCR results showed the expression of CK-7 and CK-19 genes in the second and fourth passages of pancreatic ductal epithelial cells, while the somatostatin, amylase, insulin, and glucagon genes were not expressed. The pancreatic tissue harvested after the removal of the main pancreatic duct did not express CK-7 or CK-19, while the somatostatin, amylase, insulin, and glucagon genes were expressed.ConclusionsThe preliminary results show that this method can be applied to successfully isolate and culture rat pancreatic ductal epithelial cells.

Expression of Cytokeratin in Experimentally Created Inflammatory Cyst in Vivo and in Vitro.

The purpose of this study was to investigate the behavior of epithelial lining derived from Malassez's epithelial rest (MER) cells in experimentally created inflammatory cysts in vivo and in vitro. Porcine MER cells were cultured in vitro with or without interleukin (IL)-1β (1 ng/ml) or IL-6 (1 ng/ml). Cell proliferation was assessed and expression levels of CK19 and CK13 mRNA determined using RT-PCR. In vivo, a cavity was created in the first molar of Sprague-Dawley male rats and tissue repair observed using immunohistochemical methods. In vitro, treatment with IL-1β or IL-6 increased proliferation of MER cells and decreased expression of CK19 mRNA, but increased CK13 mRNA at day 1 (p<0.05). In vivo, at 2 weeks, CK19-positive epithelial cells were observed adjacent to the cementum, in the cystic lesion, and in connective tissue. At 3 weeks, they were only detected in cells adjacent to the connective tissue. Cells positive for CK13 were observed throughout the epithelium, except in cells adjacent to connective tissue at weeks 2 and 3. Exposure to IL-1β and/or IL-6 induced proliferation and differentiation of MER cells.

Quantitative RT-PCR Assay for Detecting Lymph Node Metastasis in Endometrial Cancer: A Preliminary Study

Objective: The detection accuracy of sentinel lymph node (SLN) metastasis by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) for endometrial cancer (EC) remains unclear and was assessed in this preliminary study. Methods: We studied primary cancer tissues and pelvic lymph nodes (PLN) from 105 patients with EC. qRT-PCR assay was performed to determine the copy numbers of CK19 mRNA in EC tissues, and negative and positive LN samples. Further, qRT-PCR results were compared with pathological findings. Results: CK19 mRNA expression was detected in 98% (104/106) of the tumors, with a median copy number of 3.0 × 10⁵/μL. Twelve LN were diagnosed as positive by pathological examination. The median copy number of CK19 mRNA for positive and negative LN was 8.1 × 10⁴/μL and 90.4/µL, respectively. CK19 mRNA expression was higher in pathologically positive LN than in pathologically negative LN (p < 0.01); the pathological and qRT-PCR findings showed no discrepancy. When the cutoff value was set at 4,500 copies/µL, qRT-PCR assay using CK19 mRNA exhibited high sensitivity and specificity. Conclusions: Our results demonstrated that qRT-PCR assay, using CK19 mRNA, exhibits a high accuracy for detecting LN metastasis in EC and represents a useful alternative to conventional pathological diagnosis of EC.

CK19, CK20, EGFR and HER2 status of circulating tumor cells in patients with breast cancer.

The major cause of death in breast cancer patients is metastasis. Various biomarkers have been used for the early detection of circulating tumor cells in the peripheral blood of breast cancer patients. The aims of the current study were to analyze circulating tumor cells in the blood of breast cancer patients by investigating EGFR, CK19, CK20 and HER2 expression profiles and to evaluate their prognostic importance. CK19, CK20 and EGFR gene expression profiles were evaluated in the blood samples of 84 female patients with primary invasive ductal breast cancer and 20 healthy female volunteers using SYBR green-based real-time qPCR assays. HER2 expression analyses were conducted in 46 patients who had an HER2-positive primary tumor and in 30 healthy women to determine the cutoff level of positivity. The positive rates of CK20, EGFR, CK19 and HER2 mRNA expression in the peripheral blood were 28.57% (24/84), 20.23% (17/84), 5.95% (5/84) and 2.17% (1/46), respectively. The high positive ratio of CK20 mRNA expression in the peripheral blood of breast cancer was identified for the first time in the current study. Significant differences were identified in CK20 expression status and several clinical parameters related with aggressiveness of tumors using a binary logistic regression analysis. Higher CK20-positive levels were observed in patients who had lymph node metastasis and advanced-grade primary tumors, which were estrogen receptor-negative. We have demonstrated that CK20 may be a novel biomarker that is useful to identify circulating tumor cells and predict breast cancer progression. The results suggest that the investigation of CK20 mRNA with other biomarkers in the peripheral blood of breast cancer patients may be useful to monitor the presence of disseminated tumor cells in the blood circulation and to predict the prognosis of breast cancer.

CK20 mRNA Expression in Serum as a Biomarker for Colorectal Cancer Diagnosis: A Meta-analysis.

The aim of this study was to evaluate the diagnostic value of serumCK20 mRNA as a biomarker for colorectal cancer diagnosis by meta-analysis.Clinical studies related to serum CK20 mRNA expression for colorectal cancer diagnosis were searched in the databases of Pubmed, Cochrane Library, Embase, ISI Web of Knowledge, CNKI and Wanfang. The number of true positive (tp), false positive (fp), false negative (fn) and true negative (tn) of the original included publications were extracted by two reviewers independently. The diagnostic sensitivity, specificity, positive likely hood ratio (+LR), negative likelyhood ratio (-LR), diagnostic odds ratio (DOR) and area under the symmetric ROC curve (AUC) were pooled by random or fixed effect method according to the statistical heterogeneity among the studies.After screening the databases, nineteen publications met the inclusion criteria and were finally included in this meta-analysis. The diagnostic sensitivity and specificity were pooled by random effect model(I2>50%). The pooled diagnostic sensitivity and specificity of CK20 mRNA in serum as biomarker for colorectal cancer were 0.49 (95% CI:0.46 to 0.51) and 0.94 (95%CI:0.92-0.96) respectively. The pooled +LR and −LR were 10.90 (95%CI:5.78 to 20.55) and 0.51 (95%CI:0.45 to 0.57) respectively by random-effect method. The pooled DOR was 22.31 with the 95% CI of 11.65 to 42.71. The pooled area under the ROC curve (AUC) was 0.72for CK20 mRNA in serum as a biomarker for colorectal cancer diagnosis. Conclusion Serum CK20 mRNA expression was significantly elevated in colorectal cancer patients which could be a promising serum biomarker for colorectal cancer diagnosis with high specificity.

Direct comparison study of DNA methylation markers in EpCAM-positive circulating tumour cells, corresponding circulating tumour DNA, and paired primary tumours in breast cancer.

Circulating Tumour Cells (CTCs) and circulating tumour DNA (ctDNA) represent a non-invasive liquid biopsy approach for the follow-up and therapy management of cancer patients. We evaluated whether DNA methylation status in CTCs and ctDNA is comparable and whether it reflects the status of primary tumours. We compared the methylation status of three genes, SOX17, CST6 and BRMS1 in primary tumours, corresponding CTCs and ctDNA in 153 breast cancer patients and healthy individuals, by using real time methylation specific PCR. We report a clear association between the EpCAM-positive CTC-fraction and ctDNA for SOX17 promoter methylation both for patients with early (P = 0.001) and metastatic breast cancer (P = 0.046) but not for CST6 and BRMS1. In early breast cancer, SOX17 promoter methylation in the EpCAM-positive CTC-fraction was associated with CK-19 mRNA expression (P = 0.006) and worse overall survival (OS) (P = 0.044). In the metastatic setting SOX17 promoter methylation in ctDNA was highly correlated with CK-19 (P = 0.04) and worse OS (Ρ = 0.016). SOX17 methylation status in CTCs and ctDNA was comparable and was associated with CK-19 expression but was not reflecting the status of primary tumours in breast cancer. DNA methylation analysis of SOX17 in CTCs and matched ctDNA provides significant prognostic value.

Notch signaling pathway participates in the differentiation of hepatic progenitor cells into bile duct epithelial cells and progression of hepatic fibrosis in cholestatic liver fibrosis rat

Objective: To investigate differentiation direction of hepatic progenitor cells (HPCs) in cholestatic liver fibrosis (CLF), and the role of Notch signaling pathway in the differentiation of HPCs. Methods: A CLF rat model was established by bile duct ligation (BDL) followed by monitoring changes of Notch signal pathway and the cellular origin of proliferating cholangiocytes. After intraperitoneal injection of DAPT (a Notch signaling inhibitor) after bile duct ligation, the progress of liver fibrosis and the proliferation of cholangiocytes after inhibition of the Notch pathway were analyzed. Results: Data showed that bile duct proliferation gradually increased along with inflammatory cell infiltration and proliferating bile duct cells surrounded by abundant collagen in the BDL group. Immunostaining confirmed markedly increased expression of CK19, OV6, Sox9 and EpCAM. In addition, RT-PCR results showed that Notch signaling pathway was activated significantly. Once the Notch signaling pathway was inhibited by DAPT, bile duct proliferation markedly suppressed along with significantly decreased the mRNA expression of CK19, OV6, Sox9 and EpCAM, compared with BDL group [(10.2±0.7) vs. (22.3±0.8), (7.6±1.5) vs. (18.1±3.7), (1.4±0.4) vs. (4.1±1.1), (1.3±0.3) vs. (5.0±1.4), respectively, P<0.01]. Moreover, liver fibrosis was also reduced significantly. Conclusion: Notch signaling activation is required for HPCs differentiation into cholangiocytes in CLF and inhibition of the Notch signaling pathway may offer a therapeutic option for treating CLF.

Expression of CK19-mRNA and CEA-mRNA biomarkers in pleural fluid of patients with non-small cell lung cancer

Expression of CK19-mRNA and CEA-mRNA biomarkers in pleural fluid of patients with non-small cell lung cancer

One step nucleic acid amplification (OSNA) study in Indonesia

ABSTRACT Sentinel lymph node (SLN) is defined as the first of a few selected lymphatic nodes, into which lymphatic fluid from a primary tumor drains. Streamlined processing of sentinel lymph nodes (SLN) for detection of lymph node metastasis involves the able command over methodical blocks of SLN identification, surgical removal of SLN and SLN analysis. One Step Nucleic Acid Amplification (OSNA) method, which relies on CK19 mRNA expression to detect intraoperatively lymph node metastases in breast cancer cases, emerged as a plausible alternative to the current gold standard that uses histopathological node analysis. Sixty selected axillary sentinel lymph nodes from thirty breast cancer patients. Sentinel lymph nodes were directly bi-halved after collection using customized lymph node cutting device (Sysmex), or scalpel. The first halves were subjected to histopathological examination and were stored in specimen containers containing fresh formaldehyde prior to processing. The adjacent halves were weighed to comply with the required mass by OSNA detection in the range of 50 – 600 mg and wrapped in clean foils for storage in -80°C prior to OSNA analysis. 60 SLNs were same diagnosis using both methods. 25 SLNs were negative and 25 SLNs were positive using both methods. 3 SLNs were positive on OSNA but negative on histology. Other 7 SLNs were negative on OSNA but positive on histology, and these 1 nodes contained only micrometastasis lesion. These results suggest that OSNA is a useful for detecting SLNs metastasis, but a copy number of CK19 might be an indepedent factor from prediction and prognosis of breast cancer.

Flow cytometry as the new ‘gold standard’ for detection of free tumour cells in abdominal lavage fluid in gastric cancer patients: A comparative study of molecular and conventional methods

AimThe ideal method for detection of free tumour cells in abdominal lavage fluid should be rapid, reliable and widely available. Flow cytometry potentially covers these properties, but there are only a few studies directly comparing flow cytometry for detecting gastric cancer cells to other methods. Therefore, we compared free tumour‐cell detection in abdominal lavage fluids using cytology with immunocytochemistry, RT‐qPCR and flow cytometry.MethodsPeritoneal lavage fluid samples were collected from 10 patients. Detection of free tumour cells was performed using cytological and immunocytochemical analysis, and using RT‐qPCR and flow cytometry. Tumour cells were detected according to CEA and CK20 mRNA expression levels using RT‐qPCR, and with the epithelial markers EpCAM/CD326 and CEA using flow cytometry.ResultsThe sensitivity and specificity of cytology were 40% and 100%, respectively, while the RT‐qPCR had a sensitivity of 80% and specificity of 100%. Flow cytometry showed no false‐negative results and only one false‐positive result, giving a sensitivity and specificity of 100% and 80%, respectively. The RT‐qPCR and flow cytometry could detect free tumour cells in each of the three false‐negative cases with cytology. In the samples with detected tumour cells, tumour cell clusters were observed with imaging flow cytometry, providing additional morphological confirmation.ConclusionThe combination of quantitative and qualitative data from flow cytometry and the images of tumour cells in borderline cases provide a rapid, reliable and reproducible method for detection of free tumour cells in abdominal lavage fluid, which is necessary for intraoperative selection of patients for intraperitoneal chemotherapy.

Inhibition of notch signaling pathway prevents cholestatic liver fibrosis by decreasing the differentiation of hepatic progenitor cells into cholangiocytes

Although hepatic progenitor cells (HPCs) are known to contribute to cholestatic liver fibrosis (CLF), how Notch signaling modulates the differentiation of HPCs to cholangiocytes in CLF is unknown. Thus, using a rat model of CLF that is induced by bile duct ligation, we inhibited Notch signaling with DAPT. In vivo, CK19, OV6, Sox9, and EpCAM expression was increased significantly. Notch signaling increased after bile duct ligation, and DAPT treatment reduced the expression of CK19, OV6, Sox9, and EpCAM and blocked cholangiocyte proliferation and CLF. In vitro, treatment of a WB-F344 cell line with sodium butyrate resulted in increased mRNA and protein expression of CK19, Sox9, and EpCAM, but Notch signaling was activated. Both of these processes were inhibited by DAPT. This study reveals that Notch signaling activation is required for HPC differentiation into cholangiocytes in CLF, and inhibition of the Notch signaling pathway may offer a therapeutic approach for treating CLF.

Biliary fibrosis drives liver repopulation and phenotype transition of transplanted hepatocytes

Current research focuses on developing alternative strategies to restore decreased liver mass prior to the onset of end-stage liver disease. Cell engraftment/repopulation requires regeneration in normal liver, but we have shown that severe liver injury stimulates repopulation without partial hepatectomy (PH). We have now investigated whether a less severe injury, secondary biliary fibrosis, would drive engraftment/repopulation of ectopically transplanted mature hepatocytes. Ductular proliferation and progressive fibrosis in dipeptidyl-peptidase IV (DPPIV)(-) F344 rats was induced by common bile duct ligation (BDL). Purified DPPIV(+)/green fluorescent protein (GFP)(+) hepatocytes were infused without PH into the spleen of BDL rats and compared to rats without BDL. Within one week, transplanted hepatocytes were detected in hepatic portal areas and at the periphery of expanding portal regions. DPPIV(+)/GFP(+) repopulating cell clusters of different sizes were observed in BDL rats but not untreated normal recipients. Surprisingly, some engrafted hepatocytes formed CK-19/claudin-7 expressing epithelial cells resembling cholangiocytes within repopulating clusters. In addition, substantial numbers of hepatocytes engrafted at the intrasplenic injection site assembled into multicellular groups. These also showed biliary "transdifferentiation" in the majority of intrasplenic injection sites of rats that received BDL but not in untreated recipients. PCR array analysis showed upregulation of osteopontin (SPP1). Cell culture studies demonstrated increased Itgβ4, HNF1β, HNF6, Sox-9, and CK-19 mRNA expression in hepatocytes incubated with osteopontin, suggesting that this secreted protein promotes dedifferentiation of hepatocytes. Our studies show that biliary fibrosis stimulates liver repopulation by ectopically transplanted hepatocytes and also stimulates hepatocyte transition towards a biliary epithelial phenotype.

Correlation of Cyfra 21-1 levels in saliva and serum with CK19 mRNA expression in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) accounts for 90% of malignant lesions of oral cavity. The study assessed the potential of Cyfra 21-1 as a tumor marker in OSCC. The study included 50 patients of OSCC to evaluate levels of Cyfra 21-1 in serum and saliva by electrochemiluminescent immunoassay (ECLIA) and CK19 messenger RNA (mRNA) expression in tissue by florescent quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) along with healthy individuals as control. The salivary and serum Cyfra 21-1 levels in patients of OSCC were significantly higher compared to controls (p value < 0.01). There was a 2.75-fold increase in CK19 mRNA expression in OSCC cases compared to controls. A significant positive correlation was found between serum and salivary Cyfra 21-1, serum Cyfra 21-1, and CK19 mRNA expression and between salivary Cyfra 21-1 and CK19 mRNA expression. Among these, correlation between serum and salivary Cyfra 21-1 was highly significant. Salivary and serum Cyfra 21-1 showed significantly elevated levels in grade II OSCC compared to grade I histopathologically. Elevated levels of salivary Cyfra 21-1 were associated with recurrence in OSCC patients. Reverse operating curve constructed using 3ng/ml as a cutoff for serum Cyfra 21-1 revealed the sensitivity and specificity to be 88 and 78.2%, respectively. Using a cutoff value of 8.5ng/ml for salivary Cyfra 21-1, the sensitivity was found to be 93.8% and specificity 84.3%. We advocate salivary Cyfra 21-1 as a better diagnostic marker over serum Cyfra 21-1 as well as a potential marker in the prognosis of OSCC.

Can widely used cell type markers predict the suitability of immortalized or primary mammary epithelial cell models?

BackgroundMammary cell cultures are convenient tools for in vitro studies of mammary gland biology. However, the heterogeneity of mammary cell types, e.g., glandular milk secretory epithelial or myoepithelial cells, often complicates the interpretation of cell-based data. The present study was undertaken to determine the relevance of bovine primary mammary epithelial cells isolated from American Holstein (bMECUS) or Swiss Holstein–Friesian (bMECCH) cows, and of primary bovine mammary alveolar epithelial cells stably transfected with simian virus-40 (SV-40) large T-antigen (MAC-T) for in vitro analyses. This was evaluated by testing their expression pattern of cytokeratin (CK) 7, 18, 19, vimentin, and α-smooth muscle actin (α-SMA).ResultsThe expression of the listed markers was assessed using real-time quantitative PCR, flow cytometry and immunofluorescence microscopy. Characteristic markers of the mesenchymal (vimentin), myoepithelial (α-SMA) and glandular secretory cells (CKs) showed differential expression among the studied cell cultures, partly depending on the analytical method used. The relative mRNA expression of vimentin, CK7 and CK19, respectively, was lower (P < 0.05) in immortalized than in primary mammary cell cultures. The stain index (based on flow cytometry) of CK7 and CK19 protein was lower (P < 0.05) in MAC-T than in bMECs, while the expression of α-SMA and CK18 showed an inverse pattern. Immunofluorescence microscopy analysis mostly confirmed the mRNA data, while partly disagreed with flow cytometry data (e.g., vimentin level in MAC-T). The differential expression of CK7 and CK19 allowed discriminating between immortal and primary mammary cultures.ConclusionsThe expression of the selected widely used cell type markers in primary and immortalized MEC cells did not allow a clear preference between these two cell models for in vitro analyses studying aspects of milk composition. All tested cell models exhibited to a variable degree epithelial and mesenchymal features. Thus, based on their characterization with widely used cell markers, none of these cultures represent an unequivocal alveolar mammary epithelial cell model. For choosing the appropriate in vitro model additional properties such as the expression profile of specific proteins of interest (e.g., transporter proteins) should equally be taken into account.

Label-Free Isolation and mRNA Detection of Circulating Tumor Cells from Patients with Metastatic Lung Cancer for Disease Diagnosis and Monitoring Therapeutic Efficacy.

We develop an inertial-based microfluidic cell sorter combined with an integrated membrane filter, allowing for size-based, label-free, and high-efficiency separation and enrichment of circulating tumor cells (CTCs) in whole blood. The cell sorter is composed of a double spiral microchannel that hydrodynamically focuses and separates large CTCs from small blood cells. The focused CTCs with the equilibrium position around the midline of microchannel are further captured and enriched by a membrane filter (pore size of 8 μm) attached at the middle outlet. This integrated microfluidic device can process 1 mL of whole blood containing spiked tumor cells (A549, human lung adenocarcinoma epithelial cell line) within 15 min, with the capture efficiency of 74.4% at the concentration as low as tens of A549 cells per mL of whole blood. This microfluidic cell sorter is further adopted for isolation of CTCs from peripheral blood samples of patients with metastatic lung cancer. The immunostaining and CK-19 mRNA detection are applied for identification of captured CTCs, showing that our method can detect 90% of metastatic lung cancer patients before therapy, whereas the commercially used system can only detect 40% of the same patients. We also use the expression of CK-19 mRNA from captured CTCs as an indicator for monitoring the therapeutic efficiency, which correlates well with X-ray computed tomography (CT) assessment of the disease.

Upregulation of circulating cytokeratin 20, urokinase plasminogen activator and C-reactive protein is associated with poor prognosis in gastric cancer.

Gastric cancer is one of the most common types of cancer, with a high mortality rate. The aim of this study was to investigate the role of several key molecules, including cytokeratin (CK) 19 and CK20, urokinase plasminogen activator (uPA), C-reactive protein (CRP) and matrix metalloproteinase (MMP)-9, which are involved in cancer invasion and metastasis, in order to determine whether they may be considered as novel prognostic factors for gastric cancer. Peripheral blood was collected from 165 patients with gastric adenocarcinoma who underwent curative surgical resection at Zhejiang Cancer Hospital (Hangzhou, China) between 2010 and 2011. The mRNA levels of CK19, CK20, uPA and MMP-9 were detected by reverse transcription-quantitative polymerase chain reaction. The protein expression of CRP was measured by immunoturbidimetry. The Students t-test was used in the univariate analyses and the Kaplan-Meier method was used to analyze the survival curves. The relative mRNA expression of CK19 and MMP-9 was not found to be significantly associated with gender, age or cancer stage, whereas that of CK20 and uPA was associated with gastric cancer stage: The low-expression group was associated with early-stage and the high-expression group with more advanced-stage disease (P<0.05). The CRP protein level was associated with gender and cancer stage: The low-expression group was predominantly associated with male gender and early-stage disease, whereas the high-expression group was associated with female gender and advanced-stage disease (P<0.05). The expression of CK19, CK20, uPA and CRP, but not MMP-9, was negatively associated with overall survival (OS): The OS rate in the high-expression groups was significantly lower compared with that in the low-expression groups (P<0.05). In conclusion, the upregulation of CK20, uPA and CRP was found to be a negative prognostic factor for gastric cancer.

One-Step Nucleic Acid Amplification Assay for Detection of Axillary Lymph Node Metastases in Breast Cancer Patients Treated with Neoadjuvant Chemotherapy.

The accuracy of one-step nucleic acid amplification (OSNA) for the detection of lymph node (LN) metastasis in breast cancer patients has been well established. This study aimed to evaluate its accuracy for patients treated with neoadjuvant chemotherapy (NAC). For this study, 300 LNs, 115 sentinel lymph nodes (SLNs), and 185 non-SLNs from 88 breast cancer patients treated with NAC were examined by means of histology (hematoxylin and eosin staining and pancytokeratin immunostaining) and OSNA. The diagnostic accuracy, sensitivity, and specificity of OSNA were respectively 92.3, 88.5, and 93.3 % for all LNs, and the corresponding values were 87.8, 75.0, and 91.2 % for SLNs and 95.1, 97.3, and 94.6 % for non-SLNs. The diagnostic accuracy of OSNA was significantly lower for SLNs than for non-SLNs (P = 0.021), which was attributable to the low sensitivity for detection of micrometastases (micromets) due to lower CK19 mRNA expression detected by in situ hybridization (ISH) in SLN micromets than in non-SLN micromets. For primary breast tumors, CK19 mRNA expression showed a significant reduction after NAC (P = 0.040). The diagnostic accuracy of OSNA for NAC-treated patients is similar to that for NAC-nontreated patients, but its accuracy is significantly lower for SLNs than for non-SLNs. The findings obtained with CK19 mRNA ISH suggest that most SLN micromets cannot be detected by OSNA due to the reduced expression of CK19 mRNA induced by NAC.

Effects of Herceptin on circulating tumor cells in HER2 positive early breast cancer.

The objective of this study was to determine the changes in peripheral blood circulating tumor cells in HER2-positive early breast cancer before and after Herceptin therapy, and to explore the effects of the HER2 gene and Herceptin on circulating tumor cells. CK19 mRNA expression in peripheral blood was evaluated by qRT-PCR as an index of circulating tumor cells in 15 cases of HER-2-positive breast cancer and 18 cases of HER2-negative breast cancer before, and after chemotherapy as well. Ten cases of HER2-positive breast cancer continued on Herceptin therapy for 3 months after chemotherapy, and their peripheral blood was again drawn and assayed for CK-19 mRNA expression. Preoperatively, all cases of HER2-positive cancer were positive for CK19 mRNA in peripheral blood, but 6 cases of HER2-negative breast cancer were positive (33.3%), where there was a substantial difference between the two groups. After 6 cycles of adjuvant chemotherapy, CK19 positive rates in cases of HER2-positive and -negative breast cancer reduced by 93.3 and 11.1%, respectively, with a significant difference still existing. After 3 months of Herceptin therapy, expression of CK19 mRNA declined considerably in 10 cases of HER2 positive breast cancer (113.66 ± 88.65 vs 63.35 ± 49.27, P = 0.025). HER-2 gene expression closely correlated with circulating tumor cells in peripheral blood of early breast cancer patients. Moreover, Herceptin, a monoclonal antibody for HER2, can reduce the number of circulating tumor cells, which can be an early predictive factor for Herceptin therapy effectiveness against breast cancer.

Abstract 4813: DNA methylation of tumor suppressor and metastasis suppressor genes in primary tumors, circulating tumor cells and cell free DNA in the same breast cancer patients

Abstract Introduction: Blood based specimens such as Circulating Tumor Cells (CTCs) and cell free DNA (cfDNA) provide a very promising and non-invasive liquid biopsy approach. In our study, we first compared the promoter methylation status of BRMS1 and SOX17 genes in DNA samples isolated from primary tumors, matched CTCs and cfDNA isolated from the same breast cancer patients. Methods: We first developed and validated real time Methylation Specific PCR (MSP) assays for studying BRMS1, CST6 and SOX17 promoter methylation. We further analysed 165 paired DNA samples isolated from: a) the EpCAM positive CTC fraction from 92 operable, 61 metastatic breast cancer patients and 12 healthy individuals, b) the corresponding plasma from the same individuals. All CTC were also checked for CK-19 mRNA expression by RT-qPCR. Additionally, we investigated the promoter methylation status of BRMS1 and SOX17 in corresponding primary tumors (n=75). Results: In operable breast cancer, comparison of SOX17 promoter methylation status in CTCs and corresponding cfDNA revealed a direct association (P = 0.001), while it was also highly correlated with CK-19 mRNA expression (P=0.006). In patients with verified metastasis, there was a direct connection between SOX17 promoter methylation in CTCs and cfDNA (P=0.046), while SOX17 promoter methylation in cfDNA was highly correlated with CK-19 mRNA expression (P=0.04). Our results are shown below Conclusion: Our findings indicate a direct connection between SOX17 promoter methylation in CTCs and cfDNA both in patients with operable breast cancer, and verified metastasis. resultsBreast CancerGenesCTC fractioncfDNAFFPEsOperable (N=92)CST624/92(26.1%)33/92(35.9%)not analyzedSOX1719/92(20.7%)24/92(26.1%)26/43(60.5%)BRMS120/92(21.7%)22/92(23.9%)19/43(44.1%)Verified metastasis (N=61)CST621/61(34.4%)26/61(42.6%)not analyzedSOX1726/61(42.6%)22/61(36.1%)21/32(65.6%)BRMS126/61(42.6%)4/61(6.6%)5/32(15.6%) Citation Format: Maria Chimonidou, Areti Strati, Nikos Malamos, Vassilis Georgoulias, Evi Lianidou. DNA methylation of tumor suppressor and metastasis suppressor genes in primary tumors, circulating tumor cells and cell free DNA in the same breast cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4813. doi:10.1158/1538-7445.AM2014-4813