Rare EGFR exon 18 and exon 20 mutations in non-small-cell lung cancer on 10 117 patients: a multicentre observational study by the French ERMETIC-IFCT network.

BACKGROUND: There is scarce data available about epidermal growth factor receptor (EGFR) mutations other than common exon 19 deletions and exon 21 (L858R) mutations. PATIENTS AND METHODS: EGFR exon 18 and/or exon 20 mutations were collected from 10 117 non-small-cell lung cancer (NSCLC) samples analysed at 15 French National Cancer Institute (INCa)-platforms of the ERMETIC-IFCT network. RESULTS: Between 2008 and 2011, 1047 (10%) samples were EGFR-mutated, 102 (10%) with rare mutations: 41 (4%) in exon 18, 49 (5%) in exon 20, and 12 (1%) with other EGFR mutations. Exon 20 mutations were related to never-smoker status, when compared with exon 18 mutations (P < 0.001). Median overall survival (OS) of metastatic disease was 21 months [95% confidence interval (CI) 12-24], worse in smokers than in non-smoker patients with exon 20 mutations (12 versus 21 months; hazard ratio [HR] for death 0.27, 95% CI 0.08-0.87, P = 0.03). Under EGFR-tyrosine kinase inhibitors (TKIs), median OS was 14 months (95% CI 6-21); disease control rate was better for complex mutations (6 of 7, 86%) than for single mutations (16 of 40, 40%) (P = 0.03). CONCLUSIONS: Rare EGFR-mutated NSCLCs are heterogeneous, with resistance of distal exon 20 insertions and better sensitivity of exon 18 or complex mutations to EGFR-TKIs, probably requiring individual assessment.

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.

EGFR-TKI and lung adenocarcinoma with CNS relapse: interest of molecular follow-up.

The epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) erlotinib improves survival of lung cancer as second- or third-line therapy. However, after an initial response, most patients will recur, particularly within the central nervous system. The present study reports the case of a 27-yr-old nonsmoking male presenting with a metastatic lung adenocarcinoma with EGFR exon 19 deletion, associated with sensitivity to EGFR-TKI. Gefitinib, followed by chemotherapy and finally erlotinib resulted in prolonged disease control, until multiple liver metastases were detected. After stopping EGFR-TKI, brain metastases with carcinomatous meningitis were diagnosed. A secondary T790M mutation, associated with resistance to EGFR-TKI, was found on the liver biopsy but not in the cerebrospinal fluid. Erlotinib was reintroduced and allowed a quick neurological improvement, even though the extra-cranial disease remained resistant to erlotinib. The present report underscores the interest of molecular monitoring in lung cancer. Persistent cerebral tyrosine kinase inhibitor sensitivity should be considered in patients presenting with an early central nervous system relapse after stopping epidermal growth factor receptor tyrosine kinase inhibitor, even with a T790M-resistant mutation in noncerebral metastases. Questions remain concerning the selection of sub-clones during epidermal growth factor receptor tyrosine kinase inhibitor therapy, which could differ according to metastatic sites, especially in the central nervous system.

[Chronic renal insufficiency. A permanent public health problem]. / L'insuffisance rénale chronique. Un problème permanent de santé publique.

Chronic renal insufficiency raises an ever-increasing public-health problem due to its permanent growth among the general population and the escalating cost of renal replacement therapies. By the end of 1995 there were close to 33,700 patients with end-stage renal failure maintained alive with renal replacement methods in France. About 11,200 had a functioning kidney graft, whereas 22,500 were treated with various dialysis techniques, in and out-of-center hemodialysis and peritoneal dialysis. An optimal health policy should contribute both to prevent renal insufficiency and offer each patient his/her best specific mode of treatment at the lowest cost for the community. Renal transplantation should be much more widely promoted and utilized through measures aiming at reducing the too high refusal rates of organ donation in subjects with brain-death. Promotion and extension of out-of-center dialysis techniques are also necessary. Design of reliable epidemiological studies dealing not only with end-stage renal failure patients but with the early stage and time-course of renal insufficiency is also mandatory. A deeper investigation in the area of renal-risk factors and a qualified follow-up of patients with mild/moderate renal insufficiency are essential to avoid or delay an evolution towards end-stage renal failure. Prevention of renal fibrosis has a central role in such a long-term public health-policy.

[The consumption of psychotropics in France and some european countries]. / La consommation des psychotropes en France et dans quelques pays européens.

The consumption in France of psychotropic drugs, of all classes, except for neuroleptics, is great, even very great, and largely superior to that observed in neighbouring European countries such as Germany, Spain, Italy and the United Kingdom. With regard to hypnotics and anxiolytics, 5 to 7 per cent of the French adult population consumes them regularly. The percentages rise with age, among females, and with the presence of unfavourable medical-social conditions. The consumption of this class of therapeutic drugs has remained stable for about ten years. The consumption of antidepressants regularly grows at a rate of about 5 per cent per year, essentially linked to the consumption of serotonin-recapture inhibitors and especially fluoxetine, currently prescribed by general practitioners in about 60 per cent of cases. The factors responsible for the strong French consumption of psychotropic drugs and the divergences with our European neighbours are complex, numerous, and poorly analysed for lack of valid comparative studies. Better prescribing practices require specific initial and continuous training to take into account the realities of everyday medical practice.

Characterization of genetic exchanges between various highly divergent tbpBs, having occurred in Neisseria meningitidis.

Transferrin-binding protein B (TbpB) from Neisseria is an outer membrane-associated extracellular protein involved in iron capture during bacterial infection. The tbpB genes display extensive divergences throughout the open reading frame (ORF) that have presumably been selected under the pressure of the immune system. Early studies suggested that they could possibly constitute two distantly related groups of genes (sharing less than 57% identical nt). However, the analysis of one tbpB suggested the existence of a greater genetic diversity, and the occurrence of horizontal genetic exchanges leading to rearrangements of highly divergent ORFs. This study has confirmed this and revealed the occurrence of genetic exchanges having involved at least three types of very distantly related tbpBs. These rearrangements resulted from recombination events having occurred at very similar positions within an ORF region encoding a highly structured protein domain, probably due to constraints imposed by protein function and mode(s) of folding. These new data also provide valuable tools for epidemiological studies and evaluation of TbpBs as candidate vaccines.

[Psychotropic drug consumption in France and several European countries]. / La consommation des psychotropes en France et dans quelques pays européens.

The consumption in France of psychotropic drugs, of all classes, except for neuroleptics, is great, even very great, and largely superior to that observed in neighbouring European countries such as Germany, Spain, Italy and the United Kingdom. With regard to hypnotics and anxiolytics, 5 to 7 per cent of the French adult population consumes them regularly. The percentages rise with age, among females, and with the presence of unfavourable medical-social conditions. The consumption of this class of therapeutic drugs has remained stable for about ten years. The consumption of antidepressants regularly grows at a rate of about 5 per cent per year, essentially linked to the consumption of serotonin-recapture inhibitors and especially fluoxetine, currently prescribed by general practitioners in about 60 per cent of cases. The factors responsible for the strong French consumption of psychotropic drugs and the divergences with our European neighbours are complex, numerous, and poorly analysed for lack of valid comparative studies. Better prescribing practices require specific initial and continuous training to take into account the realities of everyday medical practice.

Molecular characterization of hybrid Tbp2 proteins from Neisseria meningitidis.

Transferrin-binding protein 2 (Tbp2) from Neisseria is an outer membrane-associated extracellular lipoprotein that is involved in iron capture within the infected host. The analysis of tbp2 clones isolated from various bacterial strains revealed extensive divergences throughout the open reading frame (ORF), with predicted amino acid (aa) sequences displaying 47% to 83% identity. Such a variability is likely to have resulted from the selective pressure exerted by the host immune system, but raises questions regarding the existing constraints for conservation of protein function. Indeed, the neisserial Tbp2s include a large structured domain, extending throughout the N-terminal half of the protein (approximately 270-290 aa), which is extremely stable and whose conformational integrity is required for efficient binding to human transferrin (hTf). In this work, a functional study of Tbp2s encoded by hybrid genes constructed by reassorting highly divergent tbp2 sequences in the region of the ORF encoding this structured domain was performed. The data demonstrate that the determinant intramolecular interactions allowing formation of a stable Tbp2 structure able to interact efficiently with hTf or/and that the Tbp2 residues involved in the interaction with hTf are not well conserved. However, a number of rearrangements appeared to generate genes encoding proteins which have retained structural stability and hTf-binding capacity. This suggested that despite the extreme aa sequence divergence and the conformational constraints, horizontal genetic exchanges, which are known to occur in neisserial populations, may have contributed significantly to the generation of sequence variation within tbp2 ORFs. The analysis of two tbp2 clones characterized in this work supports this hypothesis.

Production of lipidated meningococcal transferrin binding protein 2 in Escherichia coli.

Neisseria meningitidis strains grown under iron starvation conditions produce transferrin binding proteins (Tbp1 and Tbp2) which have been shown to play a major role in iron acquisition. Recent studies performed with Tbp2 purified from N. meningitidis suggest that this surface protein is a potential vaccine component. In order to further evaluate the immunogenicity of Tbp2, it was essential to develop a heterologous expression system to generate high amounts of purified protein. Tbp2 is produced in Neisseria as a precursor with a signal peptide whose cleavage follows a lipidation step on a cysteine residue which is the first amino acid in the mature protein. When produced in Escherichia coli with its natural signal peptide, a high amount of Tbp2 (about 10% of total cell proteins) was detected. However, most of the protein was nonlipidated precursor and only a small fraction was mature Tbp2. In order to optimize the maturation of the precursor, the natural signal sequence was replaced by several E. coli lipoprotein signal peptides. Expression levels and maturation of the precursor were highly variable depending on the signal peptide used. With one of these, an efficient maturation and a high amount of mature lipidated Tbp2 were obtained (about 3% of total cell proteins). A large-scale production process was then established for this E. coli-produced Tbp2.

Characterization of a highly structured domain in Tbp2 from Neisseria meningitidis involved in binding to human transferrin.

The binding of iron-loaded human transferrin at the surface of Neisseria meningitidis is mediated by two polypeptides, Tbp1 and Tbp2. Predicted Tbp amino acid sequences from N. meningitidis strains are highly divergent. This variability is particularly pronounced throughout the Tbp2 polypeptide. In this study, a highly structured and extremely stable Tbp2 domain of about 270 to 290 amino acids which is involved in the binding to transferrin and whose position is well conserved has been characterized. The conservation of such a remarkable structure in a very divergent protein domain (there is only 43% amino acid identity within this region) suggests that is plays an essential biological role and raises a number of questions regarding tbp2 evolution.

[Evaluation of hospital care. An experiment at the Public Assistance Hospitals in Paris]. / L'évaluation des soins à l'hôpital. Une expérience à l'Assistance Publique-Hôpitaux de Paris.

Since 1990 the Assistance Publique--Hôpitaux de Paris (AP-HP) developed a policy of health care assessment in agreement with the implications of the law of July 31, 1991. The department of Evaluation of health care, in close collaboration with the local Committees which operate in most of the hospitals of AP-HP, performed during the last three years about fifty studies dealing with two main topics: professional procedures and quality of care. Through three specific selected studies, the authors emphasize the requirements of an assessment policy to efficiently contribute to the improvement of quality of care.

Cloning and characterization of Neisseria meningitidis genes encoding the transferrin-binding proteins Tbp1 and Tbp2.

Genes tbp1 and tbp2, encoding the transferrin-binding proteins Tbp1 and Tbp2, have been isolated from two strains of Neisseria meningitidis. The tbp2 and tbp1 open reading frames are tandemly arranged in the genome with an 87-bp intergenic region, and the DNA region upstream from the tbp2-coding sequence contains domains homologous to Escherichia coli promoter consensus motives. Nucleotide sequence analysis suggests the existence of a Tbp1 precursor carrying an N-terminal signal peptide with a peptidase I cleavage site and of a Tbp2 precursor with N-terminal homology to lipoproteins, including a peptidase II cleavage site. Comparison of the Tbp1 deduced amino acid (aa) sequences from both strains showed about 76% aa homology, while those of Tbp2 revealed only about 47% aa homology. These comparisons should be extended to other Neisseria strains in order to evaluate further this genetic divergence further.

Two-dimensional 1H NMR study of recombinant insect defensin A in water: resonance assignments, secondary structure and global folding.

A 500 MHz 2D 1H NMR study of recombinant insect defensin A is reported. This defense protein of 40 residues contains 3 disulfide bridges, is positively charged and exhibits antibacterial properties. 2D NMR maps of recombinant defensin A were fully assigned and secondary structure elements were localized. The set of NOE connectivities, 3JNH-alpha H coupling constants as well as 1H/2H exchange rates and delta delta/delta T temperature coefficients of NH protons strongly support the existence of an alpha-helix (residues 14-24) and of an antiparallel beta-sheet (residues 27-40). Models of the backbone folding were generated by using the DISMAN program and energy refined by using the AMBER program. This was done on the basis of: (i) 133 selected NOEs, (ii) 21 dihedral restraints from 3JNH-alpha H coupling constants, (iii) 12 hydrogen bonds mostly deduced from 1H/2H exchange rates or temperature coefficients, in addition to 9 initial disulfide bridge covalent constraints. The two secondary structure elements and the two bends connecting them involve approximately 70% of the total number of residues, which impose some stability in the C-terminal part of the molecule. The remaining N-terminal fragment forms a less well defined loop. This spatial organization, in which a beta-sheet is linked to an alpha-helix by two disulfide bridges and to a large loop by a third disulfide bridge, is rather similar to that found in scorpion charybdotoxin and seems to be partly present in several invertebrate toxins.