Thomas Lavaux

Detection of K-Ras mutations in tumour samples of patients with non-small cell lung cancer using PNA-mediated PCR clamping

Non-small cell lung cancers (NSCLC), in particular adenocarcinoma, are often mixed with normal cells. Therefore, low sensitivity of direct sequencing used for K-Ras mutation analysis could be inadequate in some cases. Our study focused on the possibility to increase the detection of K-Ras mutations in cases of low tumour cellularity. Besides direct sequencing, we used wild-type hybridisation probes and peptide-nucleic-acid (PNA)-mediated PCR clamping to detect mutations at codons 12 and 13, in 114 routine consecutive NSCLC frozen surgical tumours untreated by targeted drugs. The sensitivity of the analysis without or with PNA was 10 and 1% of tumour DNA, respectively. Direct sequencing revealed K-Ras mutations in 11 out of 114 tumours (10%). Using PNA-mediated PCR clamping, 10 additional cases of K-Ras mutations were detected (21 out of 114, 18%, P<0.005), among which five in samples with low tumour cellularity. In adenocarcinoma, K-Ras mutation frequency increased from 7 out of 55 (13%) by direct sequencing to 15 out of 55 (27%) by clamped-PCR (P<0.005). K-Ras mutations detected by these sensitive techniques lost its prognostic value. In conclusion, a rapid and sensitive PCR-clamping test avoiding macro or micro dissection could be proposed in routine analysis especially for NSCLC samples with low percentage of tumour cells such as bronchial biopsies or after neoadjuvant chemotherapy.

Influence of drotrecogin alpha (activated) infusion on the variation of Bax/Bcl-2 and Bax/Bcl-xl ratios in circulating mononuclear cells: a cohort study in septic shock patients.

Objective: Drotrecogin alpha (activated) (DAA), or recombinant human activated protein C, is a new treatment in sepsis‐induced multiple organ failure, leading to significant reduction in the mortality rate, thanks to its anticoagulant properties. It has been suggested that DAA has anti‐inflammatory and antiapoptotic effects in sepsis animal models. This study investigates the potential actions of DAA on circulating mononuclear cells apoptosis in human septic shock. Design: Prospective, cohort study. Setting: Two intensive care wards and two research laboratories in a university hospital. Patients: Twenty‐two septic shock patients with DAA treatment (DAA+), 19 septic shock patients without DAA treatment (DAA−), and 14 healthy controls were successively enrolled, but only 20 DAA+ and 16 DAA− patients fulfilled criteria for statistical analysis. Interventions: Blood samples were collected at inclusion and 24 hrs later. Measurements and Main Results: Circulating mononuclear cell apoptosis levels were assessed by flow cytometry with annexin V, and variations of the apoptotic rheostats (Bax/Bcl‐2 and Bax/Bcl‐xl ratios) were analyzed by real‐time reverse transcription‐polymerase chain reaction. Apoptosis was significantly increased in septic shock patients (DAA+, 12 ± 6.4%; DAA−, 10.4 ± 5%) vs. healthy patients (3.4 ± 2.1%, p < .001). Twenty‐four hours after DAA infusion, apoptosis was significantly lower in the DAA+ group compared with DAA− ones (respectively, 11.7 ± 5.3% and 16.2 ± 7.6%, p < .001). At inclusion, DAA+ and DAA− groups showed comparable Bax/Bcl‐2 ratio (DAA+, 0.92 ± 0.9; DAA−, 1.32 ± 0.87) and Bax/Bcl‐xl ratio (DAA+, 2 ± 1.04; DAA−, 1.31 ± 0.93). In contrast, 24 hrs later we observed a significant decrease in these ratios, indicating an antiapoptotic effect in the DAA+ group (Bax/Bcl‐2, 0.39 ± 0.27; Bax/Bcl‐xl, 0.68 ± 0.35) compared with the DAA− group (Bax/Bcl‐2, 1.81 ± 1.1; Bax/Bcl‐xl, 1.22 ± 0.92, p = .001 and p = .039, respectively). Conclusions: In vivo, in human septic shock, DAA has antiapoptotic effects on circulating mononuclear cells, assessed by a significant decrease of both the Bax/Bcl‐2 and Bax/Bcl‐xl ratios.

Endogenous Morphine Levels Are Increased in Sepsis: A Partial Implication of Neutrophils

Background Mammalian cells synthesize morphine and the respective biosynthetic pathway has been elucidated. Human neutrophils release this alkaloid into the media after exposure to morphine precursors. However, the exact role of endogenous morphine in inflammatory processes remains unclear. We postulate that morphine is released during infection and can be determined in the serum of patients with severe infection such as sepsis. Methodology The presence and subcellular immunolocalization of endogenous morphine was investigated by ELISA, mass spectrometry analysis and laser confocal microscopy. Neutrophils were activated with Interleukin-8 (IL-8) or lipopolysaccharide (LPS). Morphine secretion was determined by a morphine-specific ELISA. μ opioid receptor expression was assessed with flow cytometry. Serum morphine concentrations of septic patients were determined with a morphine-specific ELISA and morphine identity was confirmed in human neutrophils and serum of septic patients by mass spectrometry analysis. The effects of the concentration of morphine found in serum of septic patients on LPS-induced release of IL-8 by human neutrophils were tested. Principal Findings We confirmed the presence of morphine in human neutrophil extracts and showed its colocalisation with lactoferrin within the secondary granules of neutrophils. Morphine secretion was quantified in the supernatant of activated human polymorphonuclear neutrophils in the presence and absence of Ca2+. LPS and IL-8 were able to induce a significant release of morphine only in presence of Ca2+. LPS treatment increased μ opioid receptor expression on neutrophils. Low concentration of morphine (8 nM) significantly inhibited the release of IL-8 from neutrophils when coincubated with LPS. This effect was reversed by naloxone. Patients with sepsis, severe sepsis and septic shock had significant higher circulating morphine levels compared to patients with systemic inflammatory response syndrome and healthy controls. Mass spectrometry analysis showed that endogenous morphine from serum of patient with sepsis was identical to poppy-derived morphine. Conclusions Our results indicate that morphine concentrations are increased significantly in the serum of patients with systemic infection and that morphine is, at least in part, secreted from neutrophils during sepsis. Morphine concentrations equivalent to those found in the serum of septic patients significantly inhibited LPS-induced IL-8 secretion in neutrophils.

Prognostic Value of Chromogranin A at Admission in Critically Ill Patients: A Cohort Study in a Medical Intensive Care Unit

BACKGROUND Risk assessments of patients should be based on objective variables, such as biological markers that can be measured routinely. The acute response to stress causes the release of catecholamines from the adrenal medulla accompanied by chromogranin A (CGA). To date, no study has evaluated the prognostic value of CGA in critically ill intensive care unit patients. METHODS We conducted a prospective study of intensive care unit patients by measuring serum procalcitonin (PCT), C-reactive protein (CRP), and CGA at the time of admission. Univariate and multivariate analyses were performed to evaluate the ability of these biomarkers to predict mortality. RESULTS In 120 consecutive patients, we found positive correlations between CGA and the following: CRP (r(2) = 0.216; P = 0.02), PCT (r(2) = 0.396; P < 0.001), Simplified Acute Physiologic Score II (SAPS II) (r(2) = 0.438; P < 0.001), and the Logistic Organ Dysfunction System (LODS) score (r(2) = 0.374; P < 0.001). Nonsurvivors had significantly higher CGA and PCT concentrations than survivors [median (interquartile range): 293.0 microg/L (163.5-699.5 microg/L) vs 86.0 microg/L (53.8-175.3 microg/L) for CGA, and 6.78 microg/L (2.39-22.92 microg/L) vs 0.54 microg/L (0.16-6.28 microg/L) for PCT; P < 0.001 for both comparisons]. In a multivariable linear regression analysis, creatinine (P < 0.001), age (P < 0.001), and SAPS II (P = 0.002) were the only significant independent variables predicting CGA concentration (r(2) = 0.352). A multivariate Cox regression analysis identified 3 independent factors predicting death: log-normalized CGA concentration [hazard ratio (HR), 7.248; 95% confidence interval (CI), 3.004-17.487], SAPS II (HR, 1.046; 95% CI, 1.026-1.067), and cardiogenic shock (HR, 3.920; 95% CI, 1.731-8.880). CONCLUSIONS CGA is a strong and independent indicator of prognosis in critically ill nonsurgical patients.

KIT gene in pediatric osteosarcomas: could it be a new therapeutic target?

In our previous study, a frequent rearrangement at 4q12 has been identified by allelotyping in our large and homogeneous population of pediatric osteosarcomas and it was significantly linked to c‐kit protein overexpression. To confirm and understand the involvement of KIT in this tumor, the next step of the study was designed to detect the potential mutations of KIT gene by sequencing the frequently mutated exons 6, 8, 10, 11, 13, 17 and 21 and, in case of unmutated samples, to confirm the genomic amplifications of the wild‐type receptor by real‐time quantitative PCR (QPCR). A new microsatellite and QPCR targeting PDGFRA was also added to check the accuracy of the 4q11‐12 locus. These techniques were performed in 74 pediatric high‐grade osteosarcomas treated with the OS94 protocol. Surprisingly, no mutations were found, but, only DNA amplification of KIT gene in the entire population. PDGFRA gene QPCR revealed an unexpected result of predominant deletions in the rearranged tumors. All these results confirm the major role of the 4q11‐12 locus and specifically the involvement of c‐kit wild‐type receptor overexpression in pediatric osteosarcomas and leads us to believe that inhibitors targeting this receptor could have a therapeutic effect in a selected group of patients.

Emergency step-by-step specific immunotherapy in severe digoxin poisoning: an observational cohort study.

Objective To evaluate the efficacy and safety of a step-by-step fixed dose of specific immunotherapy protocol in case of severe digoxin poisoning in an open uncontrolled prospective study. Methods Twenty consecutive patients were admitted because of severe digoxin poisoning. The inclusion criteria were: digoxin overdose and either life-threatening arrhythmia; high-degree atrioventricular block, ventricular arrhythmia, or bradycardia less than 50 bpm and hyperkalaemia (>5.5 mmol/l). A two-step protocol of antidigoxin antibodies treatment was carried out. At admission, every patient received two vials of specific Fab-fragments. If after 1 h following infusion ECG signs regressed, no more treatment was given. If ECG signs did not regress, patients were given two more vials. At inclusion and 6 h after immunotherapy, clinical (cardiac rhythm, ECG records) and biological (serum digoxin concentration, potassium) findings were recorded. Results Patients had a median (interquartile range) age of 83 (75–90) years. Four patients had acute poisoning and 16 chronic overdoses. Eleven patients showed ventricular arrhythmia, and five had high-degree atrioventricular block. Seventy percent of the patients needed only the first step. Significant decreases were observed in the number of cardiac dysrhythmia (16 vs. three patients), in the median (interquartile range) of serum digoxin concentration [5 μg/l (3.8–6.2) vs. 0.4 μg/l (0.3–2.2)] and in serum potassium [4.6 mmol/l (4.1–5.5) vs. 3.85 mmol/l (3.7–4.55)] before and after immunotherapy. The digoxin-related mortality was 5%. Conclusion This protocol of step-by-step digoxin-specific immunotherapy seems to be as effective as the equimolar treatment, and there was significant cost reduction in case of acute poisoning.

Carbidopa Effect on 18F-FDOPA Uptake in Insulinoma: from Cell Culture to microPET Imaging

Patient premedication with carbidopa seems to improve the accuracy of 6-18F-fluoro-3,4-dihydroxy-l-phenylalanine (18F-FDOPA) PET for insulinoma diagnosis. However, the risk of PET false-negative results in the presence of carbidopa is a concern. Consequently, we aimed to evaluate the effect of carbidopa on 18F-FDOPA uptake in insulinoma β-cells and an insulinoma xenograft model in mice. Methods: 18F-FDOPA in vitro accumulation was assessed in the murine β-cell line RIN-m5F. In vivo small-animal PET experiments were performed on tumor-bearing nude mice after subcutaneous injection of RIN-m5F cells. Experiments were conducted with and without carbidopa pretreatment. Results: Incubation of RIN-m5F cells with 80 μM carbidopa did not significantly affect the cellular accumulation of 18F-FDOPA. Tumor xenografts were clearly detectable by small-animal PET in all cases. Insulinoma xenografts in carbidopa-treated mice showed significantly higher 18F-FDOPA uptake than those in nontreated mice. Regardless of carbidopa premedication, the xenografts were characterized by an early increase in 18F-FDOPA uptake and then a progressive reduction over time. Conclusion: Carbidopa did not influence in vitro 18F-FDOPA accumulation in RIN-m5F cells but improved insulinoma imaging in vivo. Our findings increase current knowledge about the 18F-FDOPA uptake profile of RIN-m5F cells and a related xenograft model. To our knowledge, the present work represents the first preclinical research specifically focused on insulinomas, with potential translational implications.

Two chromogranin A-derived peptides, chromofungin and catestatin, induce neutrophil activation via a store-operated channel-dependent mechanism

New endogenous antimicrobial peptides derived from the natural processing of chromogranin A (CgA) are co-secreted with catecholamines upon stimulation of chromaffin cells. Since PMNs play a central role in innate immunity, we examine responses by PMNs following stimulation by two antimicrobial CgA-derived peptides.

Interleukin-10 gene down-expression in circulating mononuclear cells during infusion of drotrecogin-α activated: a pilot study.

IntroductionThe purpose of this study was to investigate the gene expression of interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α) and interleukin-10 (IL-10) in circulating mononuclear cells harvested from septic shock patients on drotrecogin-α activated (DAA) in order to determine whether this treatment has any effect on the inflammation phase.MethodsWe conducted a prospective cohort study in two intensive care departments. Blood samples were collected at inclusion (T1) and 36 hours later (T2) to measure plasma cytokines and the changes in intracellular TNF-α, IL-10 and IFN-γ mRNA expressions using the real-time quantitative polymerase chain reaction (RT-qPCR). Thirty-two septic shock patients were included: 16 with DAA at 24 μg/kg/h for 96 hours (DAA+) and 16 control (DAA-) eligible but contraindicated for DAA because of low platelet count.ResultsThe basal characteristics were similar in both groups: mortality (50%), plasma cytokine concentrations, and baseline IFN-γ, TNF-α and IL-10 mRNA expressions (DAA+ vs. DAA-). At T2, there was a significant IFN-γ gene down-regulation in DAA+ but not in DAA- patients (-0.34 (-0.62; +1.54) vs. +1.41 (+0.35; +5.87), P = 0.008). In survivors, DAA administration was associated with a down-expression of both IFN-γ (-0.65 (-0.93; 0.48) vs. +0.7 (-0.04; +1.26), P = 0.01) and IL-10 (-0.78 (-0.92; -0.6) vs. -0.18 (-0.68; +0.46), P = 0.038). In the non-survivors, DAA infusion was associated with IL-10 over-expression when compared with survivors (+0.54 (-0.35; +11.52) vs. -0.78 (-0.92; -0.6), P < 0.001).ConclusionsIn this study, lack of IL-10 gene down-expression despite a 36-hour infusion of DAA is an ominous sign in septic shock patients suggesting that DAA is not able to reverse the outcome. Our results suggest that DAA can decrease the expression of anti-inflammatory cytokines in septic shock patients. IL-10 or IFN-γ gene down-expression could represent markers of DAA response.