MicroRNAs associated with postoperative outcomes in patients with limited stage neuroendocrine carcinoma of the esophagus.

Esophageal neuroendocrine carcinoma (E-NEC) is an aggressive disease with a poor prognosis. The present study aimed to assess the role of surgery in the treatment of patients with resectable E-NEC, and identify a microRNA (miRNA/miR) signature in association with positive postoperative outcomes. Between February 2017 and August 2019, 36 patients with E-NEC who underwent curative surgery at the Japan Neuroendocrine Tumor Society partner hospitals were enrolled in the study. A total of 16 (44.4%) patients achieved disease-free survival (non-relapse group), whereas 20 (55.6%) patients developed tumor relapse (relapse group) during the median follow-up time of 36.5 months (range, 1-242) after surgery with a 5-year overall survival rate of 100 and 10.8%, respectively (P<0.01). No clinicopathological parameters, such as histological type or TNM staging, were associated with tumor relapse. Microarray analysis of 2,630 miRNAs in 11 patients with sufficient quality RNA revealed 12 miRNAs (miR-1260a, -1260b, -1246, -4284, -612, -1249-3p, -296-5p, -575, -6805-3p, -12136, -6822-5p and -4454) that were differentially expressed between the relapse (n=6) and non-relapse (n=5) groups. Furthermore, the top three miRNAs (miR-1246, -1260a and -1260b) were associated with overall survival (P<0.01). These results demonstrated that surgery-based multidisciplinary treatment is effective in a distinct subpopulation of limited stage E-NEC. A specific miRNA gene set is suggested to be associated with treatment outcome.

Cell Lines of Circulating Tumor Cells: What Is Known and What Needs to Be Resolved.

The importance of circulating tumor cells (CTC) is well recognized. However, the biological characteristics of CTC in the bloodstream have not yet been examined in detail, due to the limited number of CTC cell lines currently available. Thirty-nine CTC cell lines were reported by 2021. For successful cell culturing, these CTC cell lines were reviewed. Previous studies on short-term cultures of CTC also analyzed approaches for establishing the long-term culture of CTC. Negative selection, hypoxic conditions, three-dimensional conditions, and careful management are preferable for the long-term culture of CTC. However, the establishment of CTC cell lines is dependent on the specific characteristics of each cell type. Therefore, a method to establish CTC cell lines has not yet been developed. Further efforts are needed to resolve this issue.

Adverse Effects of Kampo Medicines.

This review summarizes the adverse effects of Kampo medicines. These adverse effects in terms of immunoallergic reactions include interstitial pneumonia, liver injury, allergic cystitis, and drug eruption. Many cases of interstitial pneumonia, liver injury, and allergic cystitis associated with Kampo formulas have been reported to be caused by formulas containing Scutellariae Radix (Scutellaria root, ogon). The known adverse effects linked to overdose of Kampo formulas include pseudoaldosteronism [caused by Glycyrrhizae Radix (licorice, kanzo)], sympathomimetic symptoms [caused by Ephedrae Herba (ephedra, mao)], aconite poisoning [caused by Aconiti Tuber (processed aconite root, bushi and uzu)], and diarrhea [caused by Rhei Rhizoma (rhubarb, daio)]. In recent years, mesenteric phlebosclerosis caused by the long-term administration of Gardeniae Fructus (gardenia fruit, sanshishi) has also been reported. It is necessary to consider these potential adverse effects when prescribing Kampo medicines in clinical practice.

Shikonin induces an anti­tumor effect on murine mammary cancer via p38­dependent apoptosis.

Breast cancer is the most common malignancy in women. Apoptosis is important for tumor suppression and may delay cancer progression. It was found that shikonin induced apoptosis in 4T1 murine mammary cancer cells and MDA­MB­231 human breast cancer cells in vitro. Total p38 and c­Jun N­terminal kinase (JNK) levels were maintained in 4T1 cells, and p38 phosphorylation, but not JNK phosphorylation, was significantly increased. Caspase­3/7 activity was detected, which suggested that the p38 pathway, but not the JNK signaling pathway, induced apoptosis in 4T1 cells. The anti­tumor effects of shikonin on orthotopic mouse models were also examined. On day 7 after inoculation of 4T1 cells into mice, tumor volumes in the shikonin­treated and the control groups began to differ. On day 13, tumors were weighed, and shikonin was revealed to suppress tumor growth in the orthotopic 4T1 model in vivo. In conclusion, shikonin is a potential anti­tumor drug for breast cancer.

Highly efficient capture of cancer cells expressing EGFR by microfluidic methods based on antigen-antibody association.

Epidermal growth factor receptor (EGFR) was evaluated as a target antigen for cancer cell capture by microfluidic methods based on antigen-antibody association. A polymer CTC-chip microfluidic device was surface-functionalized with three different anti-EGFR antibodies and used to capture EGFR-expressing cancer cells. Capture efficacy depended on the type of antibody used, and cetuximab efficiently captured cancer cell lines that had a wide range of EGFR expression. Capture efficiency was analyzed from the viewpoint of antigen-antibody association in a kinetic process, i.e., cell rolling well-known in leukocyte adhesion, and antibodies with a smaller dissociation constant were shown to result in more efficient capture. Moreover, a lower limit of cellular EGFR expression level for the capture was estimated and methods to decrease the limit were discussed based on densities of anti-EGFR antibody on the device surface.

FGFRL1 deficiency reduces motility and tumorigenic potential of cells derived from oesophageal squamous cell carcinomas.

Oesophageal squamous cell carcinoma (ESCC) is an aggressive cancer that resulted in ~400,000 mortalities worldwide in 2012. It was reported previously that fibroblast growth factor receptor-like 1 (FGFRL1) is highly expressed in ESCC patients with lymph node metastasis and poor prognosis accordingly. FGFRL1 is an FGFR that lacks tyrosine kinase activity, whereas the activity is critical for other FGFRs to activate intracellular signalling. The mechanism by which FGFRL1 promotes the aggressiveness of ESCCs is unknown. In the present study, two independent FGFRL1-deficient cell lines were generated from human ESCC KYSE520 cells, in order to investigate the relationship of FGFRL1 with the aggressiveness of ESCCs. FGFRL1-deficiency did not affect proliferation of KYSE520 cells in vitro. However, a xenograft mouse model demonstrated that FGFRL1-deficiency decelerated tumour growth in vivo. The haematoxylin-eosin staining identified that FGFRL1-deficient cells formed well-differentiated squamous cell carcinomas, whereas wild-type cells formed moderately differentiated squamous cell carcinomas. Microarray analysis of mRNA expression revealed that FGFRL1-depletion resulted in decreased expression of proteins associated with motility and invasion of tumour cells, matrix metalloproteinase-1 and fibroblast growth factor binding protein 1. The wound-healing assay indicated that depleting FGFRL1 reduced cell motility. Furthermore, the invasiveness of FGFRL1-deficient cells was lesser than that of wild-type KYSE520 cells. In the FGFRL1-deficient KYSE520 cells, actin filaments around the nucleus were observed sparsely, whereas the filaments along the plasma membranes were observed as frequently as those in the parent KYSE520 cells. These results demonstrate that FGFRL1 may be involved in regulation of protein expression, actin filament assembly and tumorigenic potential of ESCC cells.

The Novel Pathogenesis of Retinopathy Mediated by Multiple RTK Signals is Uncovered in Newly Developed Mouse Model.

Pericyte desorption from retinal blood vessels and subsequent vascular abnormalities are the pathogenesis of diabetic retinopathy (DR). Although the involvement of abnormal signals including platelet-derived growth factor receptor-ß (PDGFRß) and vascular endothelial growth factor-A (VEGF-A) have been hypothesized in DR, the mechanisms that underlie this processes are largely unknown. Here, novel retinopathy mouse model (N-PRß-KO) was developed with conditional Pdgfrb gene deletion by Nestin promoter-driven Cre recombinase (Nestin-Cre) that consistently reproduced through early non-proliferative to late proliferative DR pathologies. Depletion of Nestin-Cre-sensitive PDGFRß+NG2+αSMA- pericytes suppressed pericyte-coverages and induced severe vascular lesion and hemorrhage. Nestin-Cre-insensitive PDGFRß+NG2+αSMA+ pericytes detached from the vascular wall, and subsequently changed into myofibroblasts in proliferative membrane to cause retinal traction. PDGFRα+ astrogliosis was seen in degenerated retina. Expressions of placental growth factor (PlGF), VEGF-A and PDGF-BB were significantly increased in the retina of N-PRß-KO. PDGF-BB may contribute to the pericyte-fibroblast transition and glial scar formation. Since VEGFR1 signal blockade significantly ameliorated the vascular phenotype in N-PRß-KO mice, the augmented VEGFR1 signal by PlGF and VEGF-A was indicated to mediate vascular lesions. In addition to PDGF-BB, PlGF and VEGF-A with their intracellular signals may be the relevant therapeutic targets to protect eyes from DR.

Prostacyclin mimetics afford protection against lipopolysaccharide/d-galactosamine-induced acute liver injury in mice.

Prostacyclin (PGI2) serves as a protective, anti-inflammatory mediator and PGI2 mimetics may be useful as a hepatoprotective agent. We examined whether two PGI2 mimetics, ONO-1301 and beraprost, are beneficial in acute liver injury and attempted to delineate the possible mechanism underlying the hepatoprotective effect. Acute liver injury was induced by lipopolysaccharide/d-galactosamine (LPS/GalN) in mice. Mice were given an intraperitoneal injection of PGI2 mimetics 1h before LPS/GalN challenge. Both ONO-1301 and beraprost significantly declined the LPS/GalN-induced increase in serum aminotransferase activity. ONO-1301 and, to a lesser extent, beraprost inhibited hepatic gene expression levels of pro-inflammatory cytokines, which were sharply elevated by LPS/GalN. The hepatoprotective effects of ONO-1301, to a lesser extent, of beraprost were also supported by liver histopathological examinations. The PGI2 receptor antagonist CAY10441 abrogated their hepatoprotective effects. The mechanisms behind the benefit of PGI2 mimetics in reducing LPS/GalN-induced liver injury involved, in part, their suppressive effects on increased generation of reactive oxygen species (ROS), since their ability to prevent LPS/GalN-induced hepatic apoptosis was mimicked by the antioxidant N-acetyl-l-cysteine. They significantly diminished LPS/GalN-induced activation of signal transducers and activators of transcription 3 (STAT3) in liver tissues, an effect which was highly associated with their hepatoprotective effects. We indicate that IP receptor activation with PGI2 mimetics can rescue the damage in the liver induced by LPS/GalN by undermining activation of STAT3 and leading to a lower production of ROS. Our findings point to PGI2 mimetics, especially ONO-1301, as a potential novel therapeutic modality for the treatment of acute liver injury.

Flow Cytometric Detection of Circulating Tumor Cells Using a Candidate Stem Cell Marker, p75 Neurotrophin Receptor (p75NTR).

The most widely studied detection for circulating tumor cells (CTCs) in peripheral blood of cancer patients has been based on immunomagnetic enrichment using antibodies against epithelial cell adhesion molecule (EpCAM), which is overexpressed in epithelial cells. A neurotrophin receptor p75 (p75NTR) is expressed in a candidate stem cell fraction in esophageal squamous cell carcinoma (ESCC), which shows significantly higher colony formation, enhanced tumor formation in mice, along with strong expression of epithelial mesenchymal transition-related genes. Here, we describe a method to detect CTCs in ESCC based on the combined expression of EpCAM and p75NTR using flow cytometry, demonstrating the feasibility of expression analysis of multiple cell surface markers in viable cells.

Clinical Relevance of a Candidate Stem Cell Marker, p75 Neurotrophin Receptor (p75NTR) Expression in Circulating Tumor Cells.

Despite advances in its diagnosis and multimodal therapies, the prognosis of esophageal squamous cell carcinoma (ESCC) patients remains poor, because of high incidences of metastasis . Recent reports suggested that circulating tumor stem cells (CTSCs), rather than circulating tumor cells (CTCs), were more accurate diagnostic marker for metastasis, because tumor stem cells or cancer stem cells (CSCs) are more responsible for metastasis through processes such as epithelial mesenchymal transition (EMT) and tumor initiation. A neurotrophin receptor p75 (p75NTR) is expressed in a candidate CSC s in ESCC, which possess enhanced tumorigenicity along with strong expression of EMT-related genes. Our recent report using two-color flow cytometry demonstrated that CTC counts based on a combined expression of epithelial cell adhesion molecule (EpCAM) and p75NTR was significantly higher in peripheral blood samples of ESCC patients than healthy controls. In addition, EpCAM + p75NTR+, but not EpCAM + p75NTR- CTC counts, correlated with clinically diagnosed distant metastasis and pathological venous invasion in surgically resected primary ESCC tumors. Malignant cytology of the isolated EpCAM + p75NTR+ cells was microscopically confirmed as well. These results demonstrated that EpCAM + p75NTR+ CTC count was a more accurate diagnostic marker than EpCAM+ CTC count, suggesting the highly metastatic potential of CTCs with p75NTR expression.Investigation using the isolated EpCAM + p75NTR+ CTCs to assess their stem cell properties may shed light on their roles in tumor metastasis in ESCC.Further investigations based on large-scale prospective studies with long term follow up may provide us with evidences for its clinical use.

Enhanced cancer stem cell properties of a mitotically quiescent subpopulation of p75NTR-positive cells in esophageal squamous cell carcinoma.

Mitotically quiescent cancer stem cells (CSCs) possess higher malignant potential than other CSCs, indicating their higher contribution to therapeutic resistance than that of other CSCs. In esophageal squamous cell carcinoma (ESCC), p75 neurotrophin receptor (p75NTR) is expressed in a candidate CSC population showing high tumorigenicity and chemoresistance. In the present study, we isolated and characterized quiescent CSCs population in ESCC based on p75NTR expression and cell cycle status. Expression of p75NTR and Ki-67 in ESCC cell lines (KYSE cells) and surgically resected ESCC specimens was detected by performing immunocytochemical analysis. p75NTR-positive KYSE cells were fractionated into quiescent and proliferating cells by performing flow cytometry with a fluorescent DNA-staining dye to determine their CSC phenotype. Immunocytochemical analysis showed that 21.8 and 36.5% of the p75NTR-positive cells were Ki-67-negative (G0), which accounted for 11.4 and 15.7% of cells in KYSE-30 and KYSE-140 cell lines, respectively. Flow cytometric cell sorting showed that p75NTR-positive cells in the G0-G1 phase (p75NTR-positive/G0-1 cells) but not in the S-G2-M phase (p75NTR-positive/S-G2-M cells) showed strong expression of stem cell-related genes Nanog, BMI-1, and p63; high colony formation ability; high tumorigenicity in a mouse xenograft model; and strong chemoresistance against cisplatin because of the expression of drug resistance genes ABCG2 and ERCC1. Label-retention assay showed that 3.4% p75NTR-positive cells retained fluorescent cell-tracing dye, but p75NTR-negative cells did not. Immunohistochemical analysis of ESCC specimens showed p75NTR expression in 39 of 95 (41.1%) patients, with a median of 13.2% (range, 3.0-80.1%) p75NTR-positive/Ki-67-negative cells, which were found to be associated with poorly differentiated histology. Our results suggest that p75NTR-positive/G0-1 cells represent quiescent CSCs in ESCC and indicate that these cells can be used as targets to investigate molecular processes regulating CSC phenotype and to develop novel therapeutic strategies.

Prognostic significance of KLF4 expression in gastric cancer.

To understand the roles of pluripotent stem cell-inducing genes in gastric cancer, the expression of Krüppel-like factor 4 (KLF4), Nanog, octamer-binding transcription factor 4 (Oct4), avian myelocytomatosis viral oncogene homolog (c-Myc) and sex-determining region Y-box 2 (SOX2) was examined using the newly developed gastric carcinoma tissue microarray. The associations between the immunohistochemical expression levels of the pluripotency-inducing factors and the clinicopathological data of 108 patients with gastric cancer were analyzed. No associations were identified between the expression levels of the five pluripotency-inducing factors and the tumor-node-metastasis (TNM) classification or clinicopathological characteristics of the patients. In addition, multivariate analysis revealed no association of Nanog, Oct4, SOX2 or c-Myc with the prognosis of the gastric cancer patients; however, low expression of KLF4 was determined to be an independent negative prognostic factor (P=0.0331), particularly in patients who underwent R0 resection (TNM stages 2 and 3; P=0.0048). In summary, low KLF4 expression was found to be negatively associated with overall survival, and may therefore be a useful prognostic marker in gastric cancer patients.

KLF4 and NANOG are prognostic biomarkers for triple-negative breast cancer.

BACKGROUND: Prognosis of breast cancer patients has been reported to depend on the expression of induced pluripotent stem (iPS) cell-inducing factors: KLF4 and NANOG. However, the relationship between KLF4 or NANOG expression in each breast cancer subtype and the life prognosis has not been elucidated. METHOD: KLF4 and NANOG expression levels were evaluated in 208 patients using a newly developed tissue microarray (TMA). In vitro, siRNA against klf4 (siKLF4) was transfected in TNBC cell line MDA-MB-231, and the expression of KLF4 was inhibited. RESULTS: Triple-negative breast cancer (TNBC) patients in KLF4 high-expression (upper) group had more favorable overall survival (OS) and disease-free survival (DFS) rates than KLF4 lower group (p = 0.0453 and p = 0.0427). In contrast, patients in the NANOG upper group had significantly poorer prognosis than lower group in TNBC breast cancer subtypes (p < 0.0001). Multivariate analysis showed that KLF4 (p = 0.0313), NANOG (p = 0.0002), and TNM stage (p = 0.0001) are mutually independent prognostic factors. It was also shown that the proliferation and invasion ability of siKLF4-induced TNBC cells were up-regulated significantly. CONCLUSION: Our findings suggested that KLF4 and NANOG expression levels were favorable prognostic factors for TNBC patients. KLF4 also had an ability to inhibit the proliferation and invasion of TNBC.

Carbon ion radiotherapy for desmoid tumor of the abdominal wall: a case report.

BACKGROUND: Desmoid tumors, which are associated with familial adenomatous polyposis (FAP), tend to occur frequently in the abdominal wall and mesentery. Currently, there are no recognized treatments other than surgery, and frequent surgeries result in gastrointestinal obstructions and functional gastrointestinal disorders. CASE PRESENTATION: After surgery that was performed on a 39-year-old patient with FAP, we performed a second tumor excision which was the procedure used for frequently occurring mesenteric desmoid tumors. It was determined that the enlarged tumor would be difficult to operate on through an abdominal incision. Subsequently, the carbon ion radiotherapy of 50 Gy was then performed on the patient. Three years later, the tumor still remains reduced in size. In addition, we have not observed any negative effect on the digestive tract. CONCLUSIONS: This is the first instance that the carbon ion radiotherapy has been effective for the unresected desmoid tumor, and it is believed that this will become the one effective option for the treatment of desmoid tumors.

The expression of microRNA 574-3p as a predictor of postoperative outcome in patients with esophageal squamous cell carcinoma.

BACKGROUND: Despite advances in radical esophagectomies and adjuvant therapy, the postoperative prognosis in esophageal squamous cell carcinoma (ESCC) patients remains poor. The aim of this study was to identify a molecular signature to predict postoperative favorable outcomes in patients with ESCC. METHODS: As a training data set, total RNA was extracted from formalin-fixed paraffin-embedded samples of surgically removed specimens from 19 ESCC patients who underwent curative esophagectomy. The expression of microRNA (miRNA) was detected using a miRNA oligo chip on which 885 genes were mounted. As a validation data set, we obtained frozen samples of surgically resected tumors from 12 independent ESCC patients and the expression of miR-574-3p was detected by quantitative real-time PCR. RESULTS: Our microarray analysis in the training set patients identified three miRNAs (miR-574-3p, miR-106b, and miR-1303) and five miRNAs (miR-1203, miR-1909, miR-204, miR-371-3p, miR-886-3p) which were differentially expressed between the patients with (n = 14) and without (n = 5) postoperative tumor relapse (p < 0.01 and p < 0.05, respectively). Higher expression of miR-574-3p, which showed the most significant association with non-relapse (p = 0.001), was associated with favorable overall survival (p = 0.016). Real-time PCR experiments on the validation set patients confirmed that higher expression of miR-574-3p was associated with non-tumor relapse (p = 0.029) and better overall survival (p = 0.004). CONCLUSIONS: Our results suggest that the aberrant expression of the miRNAs identified in this study plays key roles in the progression of ESCC. miR-574-3p was suggested to have a tumor suppressor effect, and thus, to be a predictor of postoperative outcome in patients with ESCC.

Capture of esophageal and breast cancer cells with polymeric microfluidic devices for CTC isolation.

The present study evaluated the capture efficiency of esophageal and breast cancer cells with a modified 'polymeric circulating tumor cells (CTC)-chip' microfluidic device, which was developed for the isolation of circulating tumor cells. Esophageal cancer cell lines KYSE150, KYSE220 and KYSE510, and breast cancer cell lines MCF7, SKBR3 and MDA-MB-231 were used for evaluation. The capture efficiencies of the esophageal cancer cell lines in phosphate-buffered saline (PBS) were ~0.9, irrespective of epithelial cell adhesion molecule (EpCAM) expression, which was represented as the mean fluorescent intensity from 528 to 76. In the breast cancer cell lines, efficient capture was observed for MCF7 and SKBR3 in PBS; however, a low value of ~0.1 was obtained for MDA-MB-231. Fluorescent imaging of immunolabeled cells revealed marginal EpCAM expression in MDA-MB-231. Using whole blood, no clogging occurred in the microstructure-modified CTC-chip and efficiency of capture was successfully evaluated. Capture efficiencies for KYSE220 and MCF7 in whole blood were >0.7, but were of either equal or lesser efficiency in comparison to PBS. Therefore, the modified CTC-chip appears useful for clinical application due to its cost, practicality of use, and efficient cancer cell capture.

Chrysin Inhibits Lymphangiogenesis in Vitro.

The induction of lymphangiogenesis is an important process to promote cancer growth and cancer metastasis via the lymphatic system. Identifying the compounds that can prevent lymphangiogenesis for cancer therapy is urgently required. Chrysin, 5,7-dihydroxyflavone, a natural flavone extracted from Thai propolis, was used to investigate the effect on the lymphangiogenesis process of TR-LE, rat lymphatic endothelial cells. In this study, maximal nontoxic doses of chrysin on TR-LE cells were selected by performing a proliferation assay. The process of lymphangiogenesis in vitro was determined by cord formation assay, adhesion assay and migration assay. Chrysin at a nontoxic dose (25 µM) significantly inhibited cord formation, cell adhesion and migration of TR-LE cells when compared with the control group. We also found that chrysin significantly induced vascular endothelial growth factor C (VEGF-C) mRNA expression and nitric oxide (NO) production in TR-LE cells which was involved in decreasing the cord formation of TR-LE cells. In conclusion, we report for the first time that chrysin inhibited the process of lymphangiogenesis in an in vitro model. This finding may prove to be a natural compound for anti-lymphangiogenesis that could be developed for use in cancer therapy.

p75 neurotrophin receptor expression is a characteristic of the mitotically quiescent cancer stem cell population present in esophageal squamous cell carcinoma.

Mitotically quiescent cancer stem cells (CSC) are hypothesized to exhibit a more aggressive phenotype involving greater therapeutic resistance and metastasis. The aim of our study was to develop a method for identifying quiescent CSC in esophageal squamous cell carcinoma (ESCC) based on their expression of the p75 neurotrophin receptor (p75NTR) and other proposed CSC markers, such as CD44 and CD90. Double immunostaining of surgical ESCC specimens revealed that the mean Ki-67-labeling index of the p75NTR-positive cells was significantly lower than that of the p75NTR-negative cells. Real-time PCR analysis of sorted fractions of ESCC cell lines (KYSE cells) revealed that stem cell-related genes (Nanog, p63 and Bmi-1) and epithelial-mesenchymal transition (EMT)-related genes (N-cadherin and fibronectin) were expressed at significantly higher levels in the p75NTR-positive fractions than in the CD44-positive or CD90-positive fractions. In addition, the p75NTR-positive fractions exhibited significantly higher colony formation in vitro, significantly enhanced tumor formation in mice, and significantly greater chemoresistance against cisplatin (CDDP) than the CD44­positive or CD90­positive fractions. Furthermore, in both the cultured cells and those from the mouse xenograft tumors, the p75NTR­positive/CD44-negative and p75NTR­positive/CD90-negative KYSE cell fractions contained significantly higher proportions of mitotically quiescent cells. These results suggest that the mitotically quiescent CSC population in ESCC can be identified and isolated based on their p75NTR expression, providing researchers with a novel diagnostic and therapeutic target.

Detection of circulating tumor cells by p75NTR expression in patients with esophageal cancer.

BACKGROUND: The p75 neurotrophin receptor (p75NTR) is a cancer stem cell (CSC) marker in esophageal squamous cell carcinoma (ESCC). This study aimed to assess the use of p75NTR in detecting circulating tumor cells (CTCs) in ESCC. METHODS: Peripheral blood mononuclear cell expression of epithelial cell adhesion molecule (EpCAM) and p75NTR was detected in 23 ESCC patients (13 received chemo- or chemoradiotherapy and 10 received curative surgery) and 10 healthy controls by flow cytometry. RESULTS: EpCAM+p75NTR+ cell counts (average±SD) were significantly higher in patients (n=23, 16.0±18.3) compared to controls (n=10, 0.4±0.9, p=0.013). The sensitivity and specificity to differentiate ESCC patients from controls were 78.3 and 100% (cut-off value 4.0), respectively. EpCAM+p75NTR+, but not EpCAM+p75NTR- cell counts, correlated with clinically diagnosed distant metastasis (n=13, p=0.006) and pathological venous invasion in resected primary tumors (n=10, p=0.016). Malignant cytology was microscopically confirmed in isolated EpCAM+p75NTR+ cells with immunocytochemical double staining. CONCLUSIONS: p75NTR is suggested to be a useful marker for clinically significant CTCs, which exhibit highly metastatic features in ESCC.