KRAS Early Testing: Consensus Initiative and Cost-Effectiveness Evaluation for Metastatic Colorectal Patients in an Italian Setting

KRAS testing is relevant for the choice of the most appropriate first-line therapy of metastatic colorectal cancer (CRC). Strategies for preventing unequal access to the test should be implemented, but their relevance in the practice is related to economic sustainability. The study adopted the Delphi technique to reach a consensus on several topics. Issues related to execution of KRAS testing were identified by an expert’s board and proposed to 108 Italian oncologists and pathologists through two subsequent questionnaires. The emerging proposal was evaluated by decision analyses models employed by technology assessment agencies in order to assess cost-effectiveness. Alternative therapeutic strategies included most commonly used chemotherapy regimens alone or in combination with cetuximab or bevacizumab. The survey indicated that time interval for obtaining KRAS test should not exceed 15 days, 10 days being an optimal interval. To assure the access to proper treatment, a useful strategy should be to anticipate the test after radical resection in patients at high risk of relapse. Early KRAS testing in high risk CRC patients generates incremental cost-effectiveness ratios between 6,000 and 13,000 Euro per quality adjusted life year (QALY) gained. In extensive sensitivity analyses ICER’s were always below 15,000 Euro per QALY gained, far within the threshold of 60,000 Euro/QALY gained accepted by regulatory institutions in Italy. In metastatic CRC a time interval higher than 15 days for result of KRAS testing limits access to therapeutic choices. Anticipating KRAS testing before the onset of metastatic disease in patients at high risk does not affect the sustainability and cost-effectiveness profile of cetuximab in first-line mCRC. Early KRAS testing may prevent this inequality in high-risk patients, whether they develop metastases, and is a cost-effective strategy. Based on these results, present joined recommendations of Italian societies of Oncology and Pathology should be updated including early KRAS testing.     

New Stenella and Parastenella species from the Brazilian cerrado

Five new Stenella species were found on native cerrado plants. Stenella erythroxyli-campestris, S. erythroxyli-suberosi and S. erythroxylicola were associated with plant species belonging in the family Erythroxylaceae; S. cyrtopodii was found infecting the rare Cyrtopodium eugenii (Orchidaceae), and S. ocoteae occurred on Ocotea sp. (Lauraceae). Finally Parastenella callisthenis-fasciculatae was collected on a Vochysiaceae (viz. Callisthene fasciculate) endemic to the cerrado.     

Proteomic analysis of human very low-density lipoprotein by two-dimensional gel electrophoresis and MALDI-TOF/TOF

Biochemical studies of lipoproteins have shed light on their composition, highly contributing to the comprehension of their function. Due to the complexity of their structure, however, an in-depth structural analysis, in terms of components and PTMs, may still unravel important players in physiological and pathological processes of lipid metabolism. In this study, we performed a protein map of very low-density lipoprotein (VLDL) using a 2-DE MALDI-TOF/TOF proteomic approach. Several VLDL-associated apolipoproteins were identified, including five isoforms of apoE, three isoforms of apoC-IV, and one isoform each of apoC-III, apoM, apoA-I, and apoA-IV. Notably, we also identified seven isoforms of apoL-I and two isoforms of prenylcysteine lyase as new VLDL-associated proteins. Furthermore, we were able to identify PTM of apoE, which was found to be differently O-glycosylated at Thr212 residue, and PTM of apoL-I which we described, for the first time, to be phosphorylated at Ser296. While the physiological relevance of our finding remains to be assessed, we believe that our results will be useful as reference for future studies of VLDL structure in specific physiopathological conditions.     

Isolation and Characterization of Circulating Tumor Cells in Squamous Cell Carcinoma of the Lung Using a Non-EpCAM-Based Capture Method

Introduction

The exclusion of circulating tumor cells (CTCs) that have lost epithelial antigens during the epithelial-to-mesenchymal transition (EMT) process by using Epithelial Cell Adhesion Molecule (EpCAM) based capture methods is still a matter of debate. In this study, cells obtained after depletion procedure from blood samples of squamous cell lung cancer (SQCLC) patients were identified based on morphology and characterized with the combination of FISH assessment and immunophenotypic profile.

Materials and Methods

Five mL blood samples, collected from 55 advanced SQCLC patients, were analyzed by a non-EpCAM-based capture method. After depletion of leukocytes and erythroid cells, the negative fraction was characterized by both FISH using a fibroblast growth factor receptor 1 (FGFR1) probe and by immunocytochemistry. Thirty healthy donors were also tested.

Results

Based on morphology (nuclear dimension ≥10 μm, shape and hypercromatic aspect) suspicious circulating cells clearly distinguishable from contaminant leukocytes were observed in 49/55 (89%) SQCLC patients. Thirty-four of the 44 (77%) samples evaluable for FGFR1 FISH showed ≥ 6 FGFR1 gene copy number on average per cell. Vimentin expression involved 43% (18/42) of pooled circulating SQCLC cells, whereas only 29% (14/48) were EpCAM positive. Confocal microscopy confirmed the localization of FGFR1 probe in suspicious circulating cells. Suspicious circulating elements were also observed in healthy donors and did not show any epithelial associated antigens. A significantly lower number of suspicious circulating cells in healthy donors compared to SQCLC patients was found.

Conclusions

Among the heterogeneous cell population isolated by depletion procedure, the coexistence of cells with epithelial and/or mesenchymal phenotype suggests that EMT may participate to transendothelial invasion and migration of tumor cells in advanced SQCLC. The finding of cells with neither EpCAM or EMT phenotype, retrieved after non-EpCAM-based systems, underlines the presence of suspicious elements in the blood of both SQCLC patients and healthy donors. Further phenotyping and molecular analyses are necessary to fully characterize these circulating elements.     

Interaction of the N-(3-Methylpyridin-2-yl)amide Derivatives of Flurbiprofen and Ibuprofen with FAAH: Enantiomeric Selectivity and Binding Mode

Background

Combined fatty acid amide hydrolase (FAAH) and cyclooxygenase (COX) inhibition is a promising approach for pain-relief. The Flu-AM1 and Ibu-AM5 derivatives of flurbiprofen and ibuprofen retain similar COX-inhibitory properties and are more potent inhibitors of FAAH than the parent compounds. However, little is known as to the nature of their interaction with FAAH, or to the importance of their chirality. This has been explored here.

Methodology/Principal Findings

FAAH inhibitory activity was measured in rat brain homogenates and in lysates expressing either wild-type or FAAH^T488A-mutated enzyme. Molecular modelling was undertaken using both docking and molecular dynamics. The (R)- and (S)-enantiomers of Flu-AM1 inhibited rat FAAH with similar potencies (IC₅₀ values of 0.74 and 0.99 μM, respectively), whereas the (S)-enantiomer of Ibu-AM5 (IC₅₀ 0.59 μM) was more potent than the (R)-enantiomer (IC₅₀ 5.7 μM). Multiple inhibition experiments indicated that both (R)-Flu-AM1 and (S)-Ibu-AM5 inhibited FAAH in a manner mutually exclusive to carprofen. Computational studies indicated that the binding site for the Flu-AM1 and Ibu-AM5 enantiomers was located between the acyl chain binding channel and the membrane access channel, in a site overlapping the carprofen binding site, and showed a binding mode in line with that proposed for carprofen and other non-covalent ligands. The potency of (R)-Flu-AM1 was lower towards lysates expressing FAAH mutated at the proposed carprofen binding area than in lysates expressing wild-type FAAH.

Conclusions/Significance

The study provides kinetic and structural evidence that the enantiomers of Flu-AM1 and Ibu-AM5 bind in the substrate channel of FAAH. This information will be useful in aiding the design of novel dual-action FAAH: COX inhibitors.     

Radiation-induced circulating miRNA expression in blood of head and neck cancer patients

In recent years, scientists have found evidence confirming the aberrant expression of miRNAs in cancer patients compared to healthy individuals. The growing interest in the identification of non-invasive and specific diagnostic and prognostic molecular markers has identified microRNAs as potential candidates in cancer diagnosis, prognosis and treatment response. In the present study, we have analyzed the expression profile of circulating miR-21, -191 and -421 in peripheral blood of head and neck cancer patients (HNC) to investigate a possible modulation of mRNA levels by radiation and to identify the role of mRNA as biomarkers of cancer prognosis. Results showed a modulation of the microRNA expression at different time points after radiotherapy, suggesting that treatment may influence the release of circulating miRNAs depending also on the time interval elapsed since radiotherapy. The expression levels of miR-21, -191 and -421 were higher in blood of patients treated with radiotherapy alone after 6 months from the end of therapy and high levels of them seemed to correlate with the remission of the disease. The trends shown in this study confirmed that miRNAs could be useful prognosis markers and could provide preliminary data for further evaluation in predicting patients’ response to radiotherapy by developing miRNA-based treatments to improve the sensitivity of cancer cells to radiotherapy.     

Purification of a GSH-affinity binding protein from Bacteroides fragilis devoid of glutathione transferase activity

For the location of the aminoglycoside-(3)-N-acetyltransferase isoenzyme II (AAC(3)-II) in the bacterial cell, two strains were studied: Escherichia coli HB101(pJV03), producing the 31-kDa AAC (3)-II enzyme, and E. coli HB101, which served as a control. From each strain five protein fractions were prepared: culture supernatant, and proteins occurring in the periplasm, cytoplasm, inner membrane and outer membrane. All fractions were tested for enzymatic activity of AAC(3)-II. Most of the acetylating activity was found in the cytoplasmic fraction. The distribution of marker enzymes showed a good separation between the periplasmic and the cytoplasmic fraction.     

Molecular basis underlying the biological effects elicited by extremely low-frequency magnetic field (ELF-MF) on neuroblastoma cells

Extremely low-frequency magnetic fields (ELF-MFs) may affect human health because of the possible associations with leukemia but also with cancer, cardiovascular, and neurological disorders. In the present work, human SH-SY5Y neuroblastoma cells were exposed to a 50 Hz, 1 mT sinusoidal ELF-MF at three different times, that is, 5 days (T5), 10 days (T10), and 15 days (T15) and then the effects of ELF-MF on proteome expression and biological behavior were investigated. Through comparative analysis between treated and control samples, we analyzed the proteome changes induced by ELF-MF exposure. Nine new proteins resolved in sample after a 15-day treatment were involved in a cellular defense mechanism and/or in cellular organization and proliferation such as peroxiredoxin isoenzymes (2, 3, and 6), 3-mercaptopyruvate sulfurtransferase, actin cytoplasmatic 2, t-complex protein subunit beta, ropporin-1A, and profilin-2 and spindlin-1. Our results indicated that ELF-MFs exposure altered the proliferative status and other important cell biology-related parameters, such as cell growth pattern, and cytoskeletal organization. These findings support our hypothesis that ELF radiation could trigger a shift toward a more invasive phenotype.