Detection and Isolation of Circulating Tumor Cells from Breast Cancer Patients Using CUB Domain-Containing Protein 1.

In cancer metastasis, single circulating tumor cells (CTCs) in the blood and disseminated tumor cells (DTCs) in the bone marrow mediate cancer metastasis. Because suitable biomarker proteins are lacking, CTCs and DTCs with mesenchymal attributes are difficult to isolate from the bulk of normal blood cells. To establish a procedure allowing the isolation of such cells, we analyzed the cell line BC-M1 established from DTCs in the bone marrow of a breast cancer patient by stable isotope labeling by amino acids in cell culture (SILAC) and mass spectrometry. We found high levels of the transmembrane protein CUB domain-containing protein 1 (CDCP1) in breast cancer cell lines with mesenchymal attributes. Peripheral blood mononuclear cells were virtually negative for CDCP1. Confirmation in vivo by CellSearch revealed CDCP1-positive CTCs in 8 of 30 analyzed breast cancer patients. Only EpCam-positive CTCs were enriched by CellSearch. Using the extracellular domain of CDCP1, we established a magnetic-activated cell sorting (MACS) approach enabling also the enrichment of EpCam-negative CTCs. Thus, our approach is particularly suited for the isolation of mesenchymal CTCs with downregulated epithelial cancer that occur, for example, in triple-negative breast cancer patients who are prone to therapy failure.

Cyclin D1 gene amplification is highly homogeneous in breast cancer.

BACKGROUND: Cyclin D1 (CCND1) gene amplification is a molecular key alteration in breast cancer and was suggested to predict resistance to antihormonal therapy. As tissue heterogeneity may affect diagnostic accuracy of predictive biomarkers, CCND1 genetic heterogeneity was assessed in this study. A novel tissue microarray (TMA) platform was manufactured for this purpose. METHODS: Primary breast carcinomas from 147 patients were sampled in a "heterogeneity-TMA" by taking eight different tissue cores from 4 to 8 tumor-containing blocks per case. Additional tissue samples were taken from 1 to 4 corresponding nodal metastases in 35 of these patients. CCND1 amplification was assessed by fluorescence in situ hybridization (FISH). RESULTS: CCND1 amplification was seen in 28 of 133 (21.05 %) informative patients. Amplification was significantly associated with high tumor grade (p = 0.042), but unrelated to tumor type (p = 0.307), stage (p = 0.540) and ER (p = 0.061) or PR (p = 0.871) expression. A discordant Cyclin D1 amplification status was detected in 6 out of 28 (21.43 %) amplified tumors by heterogeneity-TMA analysis. Re-testing on large sections revealed three patients with true heterogeneity of high-level CCND1 amplification and another three patients with variable interpretation of borderline FISH ratios ranging between 1.7 and 2.3. No discrepancies were detected between 22 primary tumors and their matched lymph node metastases. CONCLUSIONS: The high degree of homogeneity seen for CCND1 amplification suggests that this alteration is an early event in the development of a small subset of breast cancers.

A multidisciplinary approach for the diagnosis of hypocalcified amelogenesis imperfecta in two Chilean families.

OBJECTIVE: The purpose of this study was to conduct a multidisciplinary analysis of a specific type of tooth enamel disturbance (amelogenesis imperfecta) affecting two Chilean families to obtain a precise diagnosis and to investigate possible underlying mutations. MATERIALS AND METHODS: Two non-related families affected with amelogenesis imperfecta were evaluated with clinical, radiographic and histopathological methods. Furthermore, pedigrees of both families were constructed and the presence of eight mutations in the enamelin gene (ENAM) and three mutations in the enamelysin gene (MMP-20) were investigated by PCR and direct sequencing. RESULTS: In the two affected patients, the dental malformation presented as soft and easily disintegrated enamel and exposed dark dentin. Neither of the affected individuals presented with a dental and skeletal open bite. Histologically, a high level of an organic matrix with prismatic organization was found. Genetic analysis indicated that the condition is autosomal recessive in one family and either autosomal recessive or due to a new mutation in the other family. Molecular mutational analysis revealed that none of the eight mutations previously described in the ENAM gene or the three mutations in the MMP-20 gene were present in the probands. CONCLUSION: A multidisciplinary analysis allowed for a diagnosis of hypocalcified amelogenesis imperfecta, Witkop type III, which was unrelated to previously described mutations in the ENAM or MMP-20 genes.

Circumferential pulmonary vein isolation with the cryoballoon technique results from a prospective 3-center study.

OBJECTIVES: The purpose of this study was to investigate the efficacy safety of the novel cryoballoon device (Arctic Front, Cryocath, Quebec, Canada). BACKGROUND: Antral pulmonary vein (PV) ablation with radiofrequency energy is widely used as a strategy for catheter ablation of paroxysmal atrial fibrillation (PAF). A novel double lumen cryoballoon catheter was designed for circumferential pulmonary vein isolation (PVI) with the cryoablation technique. METHODS: We consecutively enrolled 346 patients with symptomatic, drug refractory paroxysmal (n = 293) or persistent (n = 53) atrial fibrillation (AF). In all patients, PVI of all targeted PVs was the therapeutic aim. The primary end points of this nonrandomized study were: 1) acute isolation rate of targeted PV; and 2) first electrocardiogram-documented recurrence of AF. The secondary end point was occurrence of PV stenosis or atrio-esophageal fistula. RESULTS: The 1,360 of 1,403 PVs (97%) were targeted with balloons or balloons in combination with the use of Freezor Max (Cryocath). We found that ablation with the cryoballoon resulted in maintenance of sinus rhythm in 74% of patients with PAF and 42% of patients with persistent AF. No PV narrowing occurred. The most frequent complication was right phrenic nerve palsy observed during cryoballoon ablation at the right superior PV. CONCLUSIONS: Pulmonary vein isolation with a new cryoballoon technique is feasible. Sinus rhythm can be maintained in the majority of patients with PAF by circumferential PVI using a cryoballoon ablation system. Cryoablation was less effective in patients with persistent AF than in patients with PAF.

Long-term endothelial changes in phakic eyes with posterior chamber intraocular lenses.

PURPOSE: To evaluate the long-term endothelial cell changes in phakic eyes after implantation of a posterior chamber phakic intraocular lens to correct high ametropia. SETTING: Department of Ophthalmology, University Hospital of Vienna, Vienna, Austria. METHODS: Thirty-four eyes of 21 patients having implantation of an implantable contact lens (ICL, Staar Surgical) for high myopia or hyperopia were prospectively examined. The minimum follow-up was 2 to 4 years. Preoperative and serial postoperative specular microscopy (Noncon Robo SP 8000, Konan) was performed to evaluate the long-term endothelial cell changes. RESULTS: The mean preoperative endothelial cell density was 2854 cells/mm(2). The mean endothelial cell loss from preoperatively was 1.8% at 3 months, 4.2% at 6 months, 5.5% at 12 months, 7.9% at 2 years (n = 34), 12.9% at 3 years (n = 13), and 12.3% at 4 years (n = 11). All other endothelial cell characteristics remained stable during the 4-year follow-up. CONCLUSIONS: Continuous endothelial cell loss was observed after ICL implantation during a 4-year follow-up. There was rapid cell loss until 1 year postoperatively, after which the rate of loss was no longer statistically significant. The percentage of hexagonal cells (polymorphism) and the coefficient of variation (polymegethism) remained stable during the 4-year follow-up.