Characterization of Mesothelin Glycosylation in Pancreatic Cancer: Decreased Core Fucosylated Glycoforms in Pancreatic Cancer Patients' Sera.

Currently, there are no reliable biomarkers for the diagnosis of pancreatic cancer (PaC). Glycoproteomic approaches that analyze the glycan determinants on specific glycoproteins have proven useful to develop more specific cancer biomarkers than the corresponding protein levels. In PaC, mesothelin (MSLN) is a neo-expressed glycoprotein. MSLN glycosylation has not been described and could be altered in PaC. In this work, we aimed to characterize MSLN glycans from PaC cells and serum samples to assess their potential usefulness as PaC biomarkers. First, we analyzed MSLN glycans from PaC cell lines and then we developed an enzyme-linked lectin assay to measure core fucosylated-MSLN (Cf-MSLN) glycoforms. MSLN glycans from PaC cells were analyzed by glycan sequencing and through Western blotting with lectins. All of the cell lines secreted MSLN, with its three N-glycosylation sites occupied by complex-type N-glycans, which were mainly α2,3-sialylated, core fucosylated and highly branched. The Cf-MSLN glycoforms were quantified on PaC serum samples, and compared with MSLN protein levels. The Cf-MSLN was significantly decreased in PaC patients compared to control sera, while no differences were detected by using MSLN protein levels. In conclusion, Cf-MSLN glycoforms were differently expressed in PaC, which opens the way to further investigate their usefulness as PaC biomarkers.

Characterisation of the main PSA glycoforms in aggressive prostate cancer.

Serum levels of prostate specific antigen (PSA) are commonly used for prostate cancer (PCa) detection. However, their lack of specificity to distinguish benign prostate pathologies from PCa, or indolent from aggressive PCa have prompted the study of new non-invasive PCa biomarkers. Aberrant glycosylation is involved in neoplastic progression and specific changes in PSA glycosylation pattern, as the reduction in the percentage of α2,6-sialic acid (SA) are associated with PCa aggressiveness. In this study, we have characterised the main sialylated PSA glycoforms from blood serum of aggressive PCa patients and have compared with those of standard PSA from healthy individuals' seminal plasma. PSA was immunoprecipitated and α2,6-SA were separated from α2,3-SA glycoforms using SNA affinity chromatography. PSA N-glycans were released, labelled and analysed by hydrophilic interaction liquid chromatography combined with exoglycosidase digestions. The results showed that blood serum PSA sialylated glycoforms containing GalNAc residues were largely increased in aggressive PCa patients, whereas the disialylated core fucosylated biantennary structures with α2,6-SA, which are the major PSA glycoforms in standard PSA from healthy individuals, were markedly reduced in aggressive PCa. The identification of these main PSA glycoforms altered in aggressive PCa opens the way to design specific strategies to target them, which will be useful to improve PCa risk stratification.

Knockdown of α2,3-Sialyltransferases Impairs Pancreatic Cancer Cell Migration, Invasion and E-selectin-Dependent Adhesion.

Aberrant sialylation is frequently found in pancreatic ductal adenocarcinoma (PDA). α2,3-Sialyltransferases (α2,3-STs) ST3GAL3 and ST3GAL4 are overexpressed in PDA tissues and are responsible for increased biosynthesis of sialyl-Lewis (sLe) antigens, which play an important role in metastasis. This study addresses the effect of α2,3-STs knockdown on the migratory and invasive phenotype of PDA cells, and on E-selectin-dependent adhesion. Characterization of the cell sialome, the α2,3-STs and fucosyltransferases involved in the biosynthesis of sLe antigens, using a panel of human PDA cells showed differences in the levels of sialylated determinants and α2,3-STs expression, reflecting their phenotypic heterogeneity. Knockdown of ST3GAL3 and ST3GAL4 in BxPC-3 and Capan-1 cells, which expressed moderate to high levels of sLe antigens and α2,3-STs, led to a significant reduction in sLex and in most cases in sLea, with slight increases in the α2,6-sialic acid content. Moreover, ST3GAL3 and ST3GAL4 downregulation resulted in a significant decrease in cell migration and invasion. Binding and rolling to E-selectin, which represent key steps in metastasis, were also markedly impaired in the α2,3-STs knockdown cells. Our results indicate that inhibition of ST3GAL3 and ST3GAL4 may be a novel strategy to block PDA metastasis, which is one of the reasons for its dismal prognosis.

A nucleus-directed bombesin derivative for targeted delivery of metallodrugs to cancer cells.

We have synthesized a set of bombesin derivatives with the aim of exploring their tumor targeting properties to deliver metal-based chemotherapeutics into cancer cells. Peptide QRLGNQWAVGHLL-NH2 (BN3) was selected based on its high internalization in gastrin-releasing peptide receptor (GRPR)-overexpressing PC-3 cells. Three metallopeptides were prepared by incorporating the terpyridine Pt(II) complex [PtCl(cptpy)]Cl (1) (cptpy = 4'-(4-carboxyphenyl)-2,2':6,2″-terpyridine) at the N-terminus of BN3 or at the NƐ- or Nα-amino group of an additional Lys residue (1-BN3, Lys-1-BN3 and 1-Lys-BN3, respectively). 1-Lys-BN3 displayed the best cytotoxic activity (IC50: 19.2 ±â€¯1.7 µM) and similar ability to intercalate into DNA than complex 1. Moreover, the polypyridine Ru(II) complex [Ru(bpy)2)(cmbpy)](PF6)2 (2) (bpy = 2,2'-bipyridine; cmbpy = 4-methyl-2,2'-bipyridine-4'-carboxylic acid), with proven activity as photosensitizer, was coupled to BN3 leading to metallopeptide 2-Lys-BN3. Upon photoactivation, 2-Lys-BN3 displayed 2.5-fold higher cytotoxicity against PC-3 cells (IC50: 7.6 ±â€¯1.0 µM) than complex 2. To enhance the accumulation of the drugs into the cell nucleus, the nuclear localization signal (NLS) PKKKRKV was incorporated at the N-terminus of BN3. NLS-BN3 displayed higher cellular internalization along with nuclear biodistribution. Accordingly, metallopeptides 1-NLS-BN3 and 2-NLS-BN3 showed increased cytotoxicity (IC50: 12.0 ±â€¯1.1 µM and 2.3 ±â€¯1.1 µM). Interestingly, the phototoxic index of 2-NLS-BN3 was 8-fold higher than that of complex 2. Next, the selectivity towards cancer cells was explored using 1BR3.G fibroblasts. Higher selectivity indexes were obtained for 1-NLS-BN3 and 2-NLS-BN3 than for the unconjugated complexes. These results prove NLS-BN3 effective for targeted delivery of metallodrugs to GRPR-overexpressing cells and for enhancing the cytotoxic efficacy of metal-based photosensitizers.

Glycoprotein biomarkers for the detection of pancreatic ductal adenocarcinoma.

Pancreatic cancer (PaC) shows a clear tendency to increase in the next years and therefore represents an important health and social challenge. Currently, there is an important need to find biomarkers for PaC early detection because the existing ones are not useful for that purpose. Recent studies have indicated that there is a large window of time for PaC early detection, which opens the possibility to find early biomarkers that could greatly improve the dismal prognosis of this tumor. The present manuscript reviews the state of the art of the existing PaC biomarkers. It focuses on the anomalous glycosylation process and its role in PaC. Glycan structures of glycoconjugates such as glycoproteins are modified in tumors and these modifications can be detected in biological fluids of the cancer patients. Several studies have found serum glycoproteins with altered glycan chains in PaC patients, but they have not shown enough specificity for PaC. To find more specific cancer glycoproteins we propose to analyze the glycan moieties of a battery of glycoproteins that have been reported to increase in PaC tissues and that can also be found in serum. The combination of these new candidate glycoproteins with their aberrant glycosylation together with the existing biomarkers could result in a panel, which would expect to give better results as a new tool for early diagnosis of PaC and to monitor the disease.

Erratum: Improvement of Prostate Cancer Diagnosis by Detecting PSA Glycosylation-Specific Changes: Erratum.

[This corrects the article DOI: 10.7150/thno.15226.].

Synthesis and Biological Evaluation of Ru(II) and Pt(II) Complexes Bearing Carboxyl Groups as Potential Anticancer Targeted Drugs.

The synthesis and characterization of Pt(II) (1 and 2) and Ru(II) arene (3 and 4) or polypyridine (5 and 6) complexes is described. With the aim of having a functional group to form bioconjugates, one uncoordinated carboxyl group has been introduced in all complexes. Some of the complexes were selected for their potential in photodynamic therapy (PDT). The molecular structures of complexes 2 and 5, as well as that of the sodium salt of the 4'-(4-carboxyphenyl)-2,2':6',2″-terpyridine ligand (cptpy), were determined by X-ray diffraction. Different techniques were used to evaluate the binding capacity to model DNA molecules, and MTT cytotoxicity assays were performed against four cell lines. Compounds 3, 4, and 5 showed little tendency to bind to DNA and exhibited poor biological activity. Compound 2 behaves as bonded to DNA probably through a covalent interaction, although its cytotoxicity was very low. Compound 1 and possibly 6, both of which contain a cptpy ligand, were able to intercalate with DNA, but toxicity was not observed for 6. However, compound 1 was active in all cell lines tested. Clonogenic assays and apoptosis induction studies were also performed on the PC-3 line for 1. The photodynamic behavior for complexes 1, 5, and 6 indicated that their nuclease activity was enhanced after irradiation at λ = 447 nm. The cell viability was significantly reduced only in the case of 5. The different behavior in the absence or presence of light makes complex 5 a potential prodrug of interest in PDT. Molecular docking studies followed by molecular dynamics simulations for 1 and the counterpart without the carboxyl group confirmed the experimental data that pointed to an intercalation mechanism. The cytotoxicity of 1 and the potential of 5 in PDT make them good candidates for subsequent conjugation, through the carboxyl group, to "selected peptides" which could facilitate the selective vectorization of the complex toward receptors that are overexpressed in neoplastic cell lines.

Analysis of urinary PSA glycosylation is not indicative of high-risk prostate cancer.

The levels of core fucosylation and α2,3-linked sialic acid in serum Prostate Specific Antigen (PSA), using the lectins Pholiota squarrosa lectin (PhoSL) and Sambucus nigra agglutinin (SNA), can discriminate between Benign Prostatic Hyperplasia (BPH) and indolent prostate cancer (PCa) from aggressive PCa. In the present work we evaluated whether these glycosylation determinants could also be altered in urinary PSA obtained after digital rectal examination (DRE) and could also be useful for diagnosis determinations. For this purpose, α2,6-sialic acid and α1,6-fucose levels of urinary PSA from 53 patients, 18 biopsy-negative and 35 PCa patients of different aggressiveness degree, were analyzed by sandwich ELLA (Enzyme Linked Lectin Assay) using PhoSL and SNA. Changes in the levels of specific glycosylation determinants, that in serum PSA samples were indicative of PCa aggressiveness, were not found in PSA from DRE urine samples. Although urine is a simpler matrix for analyzing PSA glycosylation compared to serum, an immunopurification step was necessary to specifically detect the glycans on the PSA molecule. Those specific glycosylation determinants on urinary PSA were however not useful to improve PCa diagnosis. This could be probably due to the low proportion of PSA from the tumor in urine samples, which precludes the identification of aberrantly glycosylated PSA.

Comparative Study of Blood-Based Biomarkers, α2,3-Sialic Acid PSA and PHI, for High-Risk Prostate Cancer Detection.

Prostate Specific Antigen (PSA) is the most commonly used serum marker for prostate cancer (PCa), although it is not specific and sensitive enough to allow the differential diagnosis of the more aggressive tumors. For that, new diagnostic methods are being developed, such as PCA-3, PSA isoforms that have resulted in the 4K score or the Prostate Health Index (PHI), and PSA glycoforms. In the present study, we have compared the PHI with our recently developed PSA glycoform assay, based on the determination of the α2,3-sialic acid percentage of serum PSA (% α2,3-SA), in a cohort of 79 patients, which include 50 PCa of different grades and 29 benign prostate hyperplasia (BPH) patients. The % α2,3-SA could distinguish high-risk PCa patients from the rest of patients better than the PHI (area under the curve (AUC) of 0.971 vs. 0.840), although the PHI correlated better with the Gleason score than the % α2,3-SA. The combination of both markers increased the AUC up to 0.985 resulting in 100% sensitivity and 94.7% specificity to differentiate high-risk PCa from the other low and intermediate-risk PCa and BPH patients. These results suggest that both serum markers complement each other and offer an improved diagnostic tool to identify high-risk PCa, which is an important requirement for guiding treatment decisions.

Otitis externa maligna en España / Malignant external otitis in Spain

Introducción. La otitis externa maligna, una infección necrosante, se extiende desde el epitelio escamoso del conducto auditivo externo hasta tejidos adyacentes. Se estimó la incidencia y otros datos epidemiológicos de esta enfermedad en España con la serie más amplia de casos descrita. Métodos. Se realizó un estudio retrospectivo de la población ingresada en los hospitales españoles con los datos del conjunto mínimo básico de datos (CMBD) durante el periodo 2008-2013. Se consideró «caso» a los pacientes cuyo diagnóstico (principal o secundario) al alta hospitalaria se hubiera codificado, de acuerdo al CIE 9-MC, como 380.14 (otitis externa maligna). Se calculó la tasa de incidencia por sexo, por grupos etarios, además de la estacionalidad y la mortalidad. Resultados. Se diagnosticaron 355 pacientes (302 como diagnóstico principal y 53 como secundario). La tasa de incidencia global por 1.000.000 de habitantes y año en España fue de 1,30 (IC 95%: 1,17 a 1,44), aunque varió de unas zonas geográficas a otras. La mediana de edad de los casos con diagnostico principal de OEM fue de 74 años (rango: 10 a 95 años). La mayor incidencia se situó por encima de los 84 años (19,3 casos por 106 de habitantes y año). La incidencia fue mayor en hombres y el riesgo relativo hombre-mujer de 2,4. El 74,6% de los pacientes fue diagnosticado de diabetes. El diagnóstico predominó en el último cuatrimestre del año. La tasa de mortalidad bruta intrahospitalaria fue del 3,7%. Conclusiones. La otitis externa maligna, con una incidencia y mortalidad baja, afecta principalmente a varones diabéticos de mayor edad (AU)

Introduction. Malignant external otitis is a necrotizing infection, which extends from the squamous epithelium of the ear canal to the adjacent tissue. The objective of the study was to investigate its incidence and other epidemiological data in Spain, reporting the largest case series to date. Methods. A descriptive, retrospective study of the Spanish population was carried out using the minimum basic data set (MBDS) based on data of patients admitted to hospitals in the 2008-2013 period. Patients whose diagnosis (principal or secondary) at discharge was encoded as 380.14 (malignant external otitis), according to ICD-9-CM, were included as cases. The Spanish incidence rate was calculated for all its communities and provinces, as well as by season and mortality. Results. A total of 355 patients (302 as principal diagnosis and 53 as secondary) were diagnosed. The incidence rate was 1.30 (95% CI, 1.17 to 1.44) per 106 inhabitants and year, although there were variations among geographical areas. The median age of cases with main diagnosis was 74 years (range 10-95 years). The predominant age group was in patients over 84 years old (19.3 cases per 106 inhabitants and year). The incidence was higher in men and the male-female relative risk was 2.4. Diabetes was present in 74.6% of patients. The diagnosis was predominant in the last quarter of the year. The gross in-hospital mortality rate was 3.7%. Conclusions. Malignant external otitis is seen mostly among male elderly and diabetic patients. The incidence and mortality rate are low in Spain (AU)

Early Experience with the Latest-Generation Biological Prosthesis, the Trifecta™ GT.

BACKGROUND: The study aim was to assess the hemodynamic results and implantation technique for the latest-generation St. Jude Medical aortic valve bioprosthesis, the Trifecta™ GT, which was first marketed in 2016. METHODS: The first 100 patients (mean age 74.59 ± 7.41 years) undergoing aortic valve replacement (AVR) with the Trifecta GT, whether associated or not with other procedures, were included and assessed. All patients underwent a baseline ultrasound scan prior to hospital discharge to monitor postoperative gradients and the presence of periprosthetic leakage. RESULTS: The predominant valvular heart disease was aortic stenosis (85%). An isolated AVR was required in 43% of patients. The prosthesis sizes used were 19, 21, 23, 25, and 27 mm. The overall hospital mortality was 5%; all deaths occurred in patients with associated surgeries. Peak gradients measured prior to hospital discharge ranged from 17.95 mmHg to 10.95 mmHg for 19 mm and 27 mm prostheses, respectively; mean gradients were 9.94 and 6.18 mmHg for 19 mm and 27 mm prostheses, respectively. Neither implant-related complications nor significant periprosthetic leakages were recorded. CONCLUSIONS: Based on experience with patients, the Trifecta GT demonstrated an excellent hemodynamic performance after implantation, which involved a simple and safe technique. Further long-term studies to determine the durability of the prosthesis are required.

Comparative analysis of prostate-specific antigen by two-dimensional gel electrophoresis and capillary electrophoresis.

Serum levels of Prostate-Specific Antigen (PSA) are not fully specific for prostate cancer (PCa) diagnosis and several efforts are focused on searching to improve PCa markers through the study of PSA subforms that could be cancer associated. We have previously reported by 2DE a decrease in the sialic acid content of PSA from PCa compared to benign prostatic hyperplasia patients based on the different proportion of the PSA spots. However, faster and more quantitative techniques, easier to automate than 2DE, are desirable. In this study, we examined the potential of CE for resolving PSA subforms in different samples and compared the results with those obtained by 2DE. We first fractionated by OFFGEL the subforms of PSA from seminal plasma according to their pIs and analyzed each separated fraction by 2DE and CE. We also analyzed PSA and high pI PSA, both from seminal plasma, and PSA from urine of a PCa patient. These samples with different PSA spots proportions by 2DE, due to different posttranslational modifications, also presented different CE profiles. This study shows that CE is a useful and complementary technique to 2DE for analyzing samples with different PSA subforms, which is of high clinical interest.

Malignant external otitis in Spain. / Otitis externa maligna en España.

INTRODUCTION: Malignant external otitis is a necrotizing infection, which extends from the squamous epithelium of the ear canal to the adjacent tissue. The objective of the study was to investigate its incidence and other epidemiological data in Spain, reporting the largest case series to date. METHODS: A descriptive, retrospective study of the Spanish population was carried out using the minimum basic data set (MBDS) based on data of patients admitted to hospitals in the 2008-2013 period. Patients whose diagnosis (principal or secondary) at discharge was encoded as 380.14 (malignant external otitis), according to ICD-9-CM, were included as cases. The Spanish incidence rate was calculated for all its communities and provinces, as well as by season and mortality. RESULTS: A total of 355 patients (302 as principal diagnosis and 53 as secondary) were diagnosed. The incidence rate was 1.30 (95% CI, 1.17 to 1.44) per 106 inhabitants and year, although there were variations among geographical areas. The median age of cases with main diagnosis was 74 years (range 10-95 years). The predominant age group was in patients over 84 years old (19.3 cases per 106 inhabitants and year). The incidence was higher in men and the male-female relative risk was 2.4. Diabetes was present in 74.6% of patients. The diagnosis was predominant in the last quarter of the year. The gross in-hospital mortality rate was 3.7%. CONCLUSIONS: Malignant external otitis is seen mostly among male elderly and diabetic patients. The incidence and mortality rate are low in Spain.

Synthetic lethal interaction of cetuximab with MEK1/2 inhibition in NRAS-mutant metastatic colorectal cancer.

KRAS mutations are an established predictor of lack of response to EGFR-targeted therapies in patients with metastatic colorectal cancer (mCRC). However, little is known about the role of the rarer NRAS mutations as a mechanism of primary resistance to the anti-EGFR monoclonal antibody cetuximab in wild-type KRAS mCRC. Using isogenic mCRC cells with a heterozygous knock-in of the NRAS activating mutation Q61K, we aimed to elucidate the mechanism(s) by which mutant NRAS blocks cetuximab from inhibiting mCRC growth. NRASQ61K/+ cells were refractory to cetuximab-induced growth inhibition. Pathway-oriented proteome profiling revealed that cetuximab-unresponsive ERK1/2 phosphorylation was the sole biomarker distinguishing cetuximab-refractory NRASQ61K/+ from cetuximab-sensitive NRAS+/+ cells. We therefore employed four representative MEK1/2 inhibitors (binimetinib, trametinib, selumetinib, and pimasertib) to evaluate the therapeutic value of MEK/ERK signaling in cetuximab-refractory NRAS mutation-induced mCRC. Co-treatment with an ineffective dose of cetuximab augmented, up to more than 1,300-fold, the cytotoxic effects of pimasertib against NRASQ61K/+ cells. Simultaneous combination of MEK1/2 inhibitors with cetuximab resulted in extremely high and dose-dependent synthetic lethal effects, which were executed, at least in part, by exacerbated apoptotic cell death. Dynamic monitoring of real-time cell growth rates confirmed that cetuximab synergistically sensitized NRASQ61K/+ cellsto MEK1/2 inhibition. Our discovery of a synthetic lethal interaction of cetuximab in combination with MEK1/2 inhibition for the NRAS mutant subgroup of mCRC underscores the importance of therapeutic intervention both in the MEK-ERK and EGFR pathways to achieve maximal therapeutic efficacy against NRAS-mutant mCRC tumors.

Improvement of Prostate Cancer Diagnosis by Detecting PSA Glycosylation-Specific Changes.

New markers based on PSA isoforms have recently been developed to improve prostate cancer (PCa) diagnosis. However, novel approaches are still required to differentiate aggressive from non-aggressive PCa to improve decision making for patients. PSA glycoforms have been shown to be differentially expressed in PCa. In particular, changes in the extent of core fucosylation and sialylation of PSA N-glycans in PCa patients compared to healthy controls or BPH patients have been reported. The objective of this study was to determine these specific glycan structures in serum PSA to analyze their potential value as markers for discriminating between BPH and PCa of different aggressiveness. In the present work, we have established two methodologies to analyze the core fucosylation and the sialic acid linkage of PSA N-glycans in serum samples from BPH (29) and PCa (44) patients with different degrees of aggressiveness. We detected a significant decrease in the core fucose and an increase in the α2,3-sialic acid percentage of PSA in high-risk PCa that differentiated BPH and low-risk PCa from high-risk PCa patients. In particular, a cut-off value of 0.86 of the PSA core fucose ratio, could distinguish high-risk PCa patients from BPH with 90% sensitivity and 95% specificity, with an AUC of 0.94. In the case of the α2,3-sialic acid percentage of PSA, the cut-off value of 30% discriminated between high-risk PCa and the group of BPH, low-, and intermediate-risk PCa with a sensitivity and specificity of 85.7% and 95.5%, respectively, with an AUC of 0.97. The latter marker exhibited high performance in differentiating between aggressive and non-aggressive PCa and has the potential for translational application in the clinic.

Improved Pancreatic Adenocarcinoma Diagnosis in Jaundiced and Non-Jaundiced Pancreatic Adenocarcinoma Patients through the Combination of Routine Clinical Markers Associated to Pancreatic Adenocarcinoma Pathophysiology.

BACKGROUND: There is still no reliable biomarker for the diagnosis of pancreatic adenocarcinoma. Carbohydrate antigen 19-9 (CA 19-9) is a tumor marker only recommended for pancreatic adenocarcinoma follow-up. One of the clinical problems lies in distinguishing between this cancer and other benign pancreatic diseases such as chronic pancreatitis. In this study we will assess the value of panels of serum molecules related to pancreatic cancer physiopathology to determine whether alone or in combination could help to discriminate between these two pathologies. METHODS: CA 19-9, carcinoembryonic antigen (CEA), C-reactive protein, albumin, insulin growth factor-1 (IGF-1) and IGF binding protein-3 were measured using routine clinical analyzers in a cohort of 47 pancreatic adenocarcinoma, 20 chronic pancreatitis and 15 healthy controls. RESULTS: The combination of CA 19-9, IGF-1 and albumin resulted in a combined area under the curve (AUC) of 0.959 with 93.6% sensitivity and 95% specificity, much higher than CA 19-9 alone. An algorithm was defined to classify the patients as chronic pancreatitis or pancreatic cancer with the above specificity and sensitivity. In an independent validation group of 20 pancreatic adenocarcinoma and 13 chronic pancreatitis patients, the combination of the four molecules classified correctly all pancreatic adenocarcinoma and 12 out of 13 chronic pancreatitis patients. CONCLUSIONS: Although this panel of markers should be validated in larger cohorts, the high sensitivity and specificity values and the convenience to measure these parameters in clinical laboratories shows great promise for improving pancreatic adenocarcinoma diagnosis.

A comparison of non-biologically active truncated EGF (EGFt) and full-length hEGF for delivery of Auger electron-emitting 111In to EGFR-positive breast cancer cells and tumor xenografts in athymic mice.

INTRODUCTION: EGFt is a truncated form of human epidermal growth factor (hEGF) that is non-biologically active but retains binding and internalization into EGFR-positive cells. Our aim was to compare EGFt and hEGF for delivery of (111)In to human breast cancer (BC) cells and tumors and evaluate its cytotoxicity against EGFR-positive BC cells, mediated by the Auger electron emissions of (111)In. METHODS: The binding, internalization and nuclear localization of EGFt and hEGF in MDA-MB-468 human BC cells were first assessed by confocal fluorescence microscopy. Subcellular fractionation was then used to quantify the cellular and nuclear uptake of (111)In-EGFt and (111)In-hEGF in MDA-MB-468 cells. The effect of exposure in vitro to (111)In-EGFt or (111)In-hEGF on the clonogenic survival of MDA-MB-468 (10(6) EGFR/cell) or MCF-7 cells (10(4) EGFR/cell) was determined. The pharmacokinetics and tumor and normal tissue biodistribution of (111)In-EGFt was compared to (111)In-hEGF in CD-1 athymic mice with s.c. MDA-MB-468 and MCF-7 tumors. Nuclear importation in MDA-MB-468 tumors was determined ex vivo by subcellular fractionation. RESULTS: Fluorescently-labeled EGFt and hEGF were bound, internalized and localized in the nucleus of MDA-MB-468 cells. Binding of (111)In-EGFt to MDA-MB-468 cells was 8-fold lower than (111)In-hEGF, but nuclear importation as a proportion of cell-bound (111)In was 3.6-fold greater than (111)In-hEGF. Nuclear uptake of (111)In-EGFt was lower than (111)In-hEGF when differences in cell binding were taken into account. The cytotoxicity of (111)In-EGFt (1.0MBq/mL; 10 nmols/L) against MDA-MB-468 cells was 9-fold lower than (111)In-hEGF but only 2-fold lower at a higher concentration (1.85 MBq/mL; 40 nmols/L). (111)In-EGFt and (111)In-hEGF exhibited greater cytotoxicity against MDA-MB-468 cells than MCF-7 cells. (111)In-EGFt was eliminated more slowly from the blood of tumor-bearing mice and exhibited lower liver uptake but higher kidney accumulation. Uptake of (111)In-EGFt in MDA-MB-468 tumors was 2.2-fold lower than (111)In-hEGF, and was blocked by anti-EGFR monoclonal antibody, nimotuzumab. Nuclear uptake into MDA-MB-468 tumor cells was higher for (111)In-EGFt than (111)In-hEGF, but when the lower tumor uptake of (111)In-EGFt was considered, there were no overall differences. CONCLUSION: We conclude that the absence of biological activity of EGFt makes it attractive for delivery of Auger electron-emitting (111)In to EGFR-overexpressing BC, but its lower cellular and tumor uptake would limit its effectiveness compared to (111)In-hEGF. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: (111)In-EGFt may reduce the adverse effects previously observed in patients administered (111)In-hEGF since it is not biologically active, but its lower uptake by BC cells and tumors would limit its effectiveness for treatment of breast cancer.

Oncometabolic mutation IDH1 R132H confers a metformin-hypersensitive phenotype.

Metabolic flexibility might be particularly constrained in tumors bearing mutations in isocitrate dehydrogenase 1 (IDH1) leading to the production of the oncometabolite 2-hydroxygluratate (2HG). To test the hypothesis that IDH1 mutations could generate metabolic vulnerabilities for therapeutic intervention, we utilized an MCF10A cell line engineered with an arginine-to-histidine conversion at position 132 (R132H) in the catalytic site of IDH1, which equips the enzyme with a neomorphic α-ketoglutarate to 2HG reducing activity in an otherwise isogenic background. IDH1 R132H/+ and isogenic IDH1 +/+ parental cells were screened for their ability to generate energy-rich NADH when cultured in a standardized high-throughput Phenotype MicroArrayplatform comprising >300 nutrients. A radical remodeling of the metabotype occurred in cells carrying the R132H mutation since they presented a markedly altered ability to utilize numerous carbon catabolic fuels. A mitochondria toxicity-screening modality confirmed a severe inability of IDH1-mutated cells to use various carbon substrates that are fed into the electron transport chain at different points. The mitochondrial biguanide poisons, metformin and phenformin, further impaired the intrinsic weakness of IDH1-mutant cells to use certain carbon-energy sources. Additionally, metabolic reprogramming of IDH1-mutant cells increased their sensitivity to metformin in assays of cell proliferation, clonogenic potential, and mammosphere formation. Targeted metabolomics studies revealed that the ability of metformin to interfere with the anaplerotic entry of glutamine into the tricarboxylic acid cycle could explain the hypersensitivity of IDH1-mutant cells to biguanides. Moreover, synergistic interactions occurred when metformin treatment was combined with the selective R132H-IDH1 inhibitor AGI-5198. Together, these results suggest that therapy involving the simultaneous targeting of metabolic vulnerabilities with metformin, and 2HG overproduction with mutant-selective inhibitors (AGI-5198-related AG-120 [Agios]), might represent a worthwhile avenue of exploration in the treatment of IDH1-mutated tumors.

Identification of potential pancreatic cancer serum markers: Increased sialyl-Lewis X on ceruloplasmin.

Pancreatic adenocarcinoma (PDAC) usually shows an enhanced expression of sialyl-Lewis X (sLe(x)) and related epitopes. PDAC may secrete some of the proteins carrying such increased sLe(x) determinant into serum, so they could be used as PDAC markers. Previously, we identified acute-phase proteins with increased sLe(x) in both PDAC and in chronic pancreatitis patients. In this study, depleted sera from the main acute-phase proteins has been analysed for the search of proteins with increased sLe(x) levels in PDAC. Sera from healthy controls, chronic pancreatitis and PDAC patients were depleted, electrophoresed and subjected to sLe(x) immunodetection. Proteins that differentially expressed sLe(x) in PDAC were trypsin digested and identified by LC-ESI-QTOF mass spectrometry. Five protein bands that differentially expressed sLe(x) in PDAC were identified and corresponded to seven different acute-phase proteins. Among them, ceruloplasmin (CP) was selected for further analysis. N-glycan sequencing of CP confirmed the increase of sLe(x) levels in CP in PDAC patients. Healthy controls, chronic pancreatitis and PDAC patients' sera were immunoprecipitated with anti-CP antibodies, and their sLe(x) and CP levels were analysed by western blot. The sLe(x)/CP ratio tended to be higher for the PDAC group, which altogether suggests that the sLe(x)/CP ratio could be a useful biomarker for PDAC.

The 3f Enable sutureless bioprosthesis: Early results, safeguards, and pitfalls.

OBJECTIVE: The aim of the study was to evaluate the clinical and hemodynamic performance of the 3f Enable (Medtronic Inc, Minneapolis, Minn) sutureless bioprosthesis in the early postoperative period. Its implantation technique was also evaluated highlighting the modifications in regard to the original technique and mistakes made throughout the learning curve. METHODS: This is a prospective, nonrandomized study. From February 2011 to March 2014, a total of 60 patients underwent aortic valve replacement with the 3f Enable valve at the Hospiten Rambla. All intraoperative and postoperative data were prospectively collected. RESULTS: The mean age was 81.3 ± 3.78 years. Mean logistic European System for Cardiac Operative Risk Evaluation I was 13.78%. An associated concomitant procedure was performed in 23 patients (38.3%). The extracorporeal circulation and crossclamping times were 49.8 ± 15.7 minutes and 35.4 ± 8.9 minutes, respectively. The average stay in the intensive care unit was 34.7 hours. Mean systolic pressure gradients ranged from 8.01 mm Hg (size 19 valve) to 7.2 mm Hg (size 25 valve). Mean effective orifice area ranged from 1.9 cm(2) (size 19 valve) to 2.5 cm(2) (size 25 valve). Severe mismatch (<0.65 cm(2)/m(2)) did not occur in any patient. Only 1.66% of patients had more than mild aortic insufficiency at discharge. The early (30-day) mortality was 6.6% (n = 4). CONCLUSIONS: The 3f Enable bioprosthesis is an important alternative to conventional prostheses, with reduction of surgery times and good hemodynamic results on discharge. It is especially useful for high-risk patients and octogenarians. Studies on a greater number of patients and long-term follow-ups are necessary.