Clinical and molecular characteristics of non-small-cell lung cancer (NSCLC) harboring EGFR mutation: results of the nationwide French Cooperative Thoracic Intergroup (IFCT) program.

BACKGROUND: EGFR mutations cause inconsistent response to EGFR tyrosine-kinase inhibitors (TKI). To better understand these features, we reviewed all cases of EGFR-mutated non-small-cell lung cancer collected in the Biomarkers France database. PATIENTS AND METHODS: Of 17 664 patients, 1837 (11%) with EGFR-mutated non-small-cell lung cancer were retrospectively analyzed for clinical and molecular characteristics. Results were correlated with survival and treatment response for the 848 stage IV patients. RESULTS: EGFR exon 18, 19, 20 and 21 mutations were found in 102 (5.5%), 931 (51%), 102 (5.5%) and 702 (38%) patients, respectively. Over 50% of exon 18 and 20 mutated patients were smokers. The median follow-up was 51.7 months. EGFR mutation type was prognostic of overall survival (OS) versus wild-type {exon 19: hazard ratio (HR)=0.51 [95% confidence interval (CI): 0.41-0.64], P < 0.0001; exon 21: HR = 0.76 (95% CI: 0.61-0.95), P = 0.002; exon 20: HR = 1.56 (95% CI: 1.02-2.38), P = 0.004}. EGFR mutation type was prognostic of progression-free survival versus wild-type [exon 19: HR = 0.62 (95% CI: 0.49-0.78), P < 0.0001; exon 20: HR = 1.46 (95% CI: 0.96-2.21), P = 0.07]. First-line treatment choice did not influence OS in multivariate analysis. First-line TKI predicted improved progression-free survival versus chemotherapy [HR = 0.67 (95% CI: 0.53-0.85), P = 0.001]. OS was longer for del19 versus L858R, which was associated with better OS compared with other exon 21 mutations, including L861Q. TKI improved survival in patients with exon 18 mutations, while chemotherapy was more beneficial for exon 20-mutated patients. CONCLUSION: EGFR mutation type can inform the most appropriate treatment. Therapeutic schedule had no impact on OS in our study, although TKI should be prescribed in first-line considering the risk of missing the opportunity to use this treatment.

Prognostic and predictive values of oncogenic BRAF, NRAS, c-KIT and MITF in cutaneous and mucous melanoma.

BACKGROUND: Mutations of BRAF, NRAS and c-KIT oncogenes are preferentially described in certain histological subtypes of melanoma and linked to specific histopathological features. BRAF-, MEK- and KIT-inhibitors led to improvement in overall survival of patients harbouring mutated metastatic melanoma. OBJECTIVES: To assess the prevalence and types of BRAF, NRAS, c-KIT and MITF mutations in cutaneous and mucous melanoma and to correlate mutation status with clinicopathological features and outcome. METHODS: Clinicopathological features and mutation status of 108 samples and of 98 consecutive patients were, respectively, assessed in one retrospective and one prospective study. Clinicopathological features were correlated with mutation status and the predictive value of these mutations was studied. RESULTS: This work identified significant correlations between BRAF mutations and melanoma occurring on non-chronic sun-damaged skin and superficial spreading melanoma (P < 0.05) on one hand, and between NRAS mutations and nodular melanoma (P < 0.05) on the other hand. Younger age (P < 0.05), microscopic (P < 0.05) and macroscopic (P < 0.05) lymphatic involvement at diagnosis of primary melanoma were significantly linked to BRAF mutations. A mutated status was a positive predictive factor of a response to BRAF inhibitors (OR = 3.44). Mutated melanoma showed a significantly (P = 0.038) higher objective response rate to cytotoxic chemotherapy (26.3%) than wild-type tumours (6.7%). CONCLUSION: Clinical and pathological characteristics of the primary melanoma differed between wild-type and BRAF- or NRAS-mutated tumours. Patients with BRAF-mutated tumours were younger at diagnosis of primary melanoma. Patients carrying mutations showed better responses better to specific kinase inhibitors and interestingly also to systemic cytotoxic chemotherapy.

Iron excess limits HHIPL-2 gene expression and decreases osteoblastic activity in human MG-63 cells.

UNLABELLED: In order to understand mechanisms involved in osteoporosis observed during iron overload diseases, we analyzed the impact of iron on a human osteoblast-like cell line. Iron exposure decreases osteoblast phenotype. HHIPL-2 is an iron-modulated gene which could contribute to these alterations. Our results suggest osteoblast impairment in iron-related osteoporosis. INTRODUCTION: Iron overload may cause osteoporosis. An iron-related decrease in osteoblast activity has been suggested. METHODS: We investigated the effect of iron exposure on human osteoblast cells (MG-63) by analyzing the impact of ferric ammonium citrate (FAC) and iron citrate (FeCi) on the expression of genes involved in iron metabolism or associated with osteoblast phenotype. A transcriptomic analysis was performed to identify iron-modulated genes. RESULTS: FAC and FeCi exposure modulated cellular iron status with a decrease in TFRC mRNA level and an increase in intracellular ferritin level. FAC increased ROS level and caspase 3 activity. Ferroportin, HFE and TFR2 mRNAs were expressed in MG-63 cells under basal conditions. The level of ferroportin mRNA was increased by iron, whereas HFE mRNA level was decreased. The level of mRNA alpha 1 collagen type I chain, osteocalcin and the transcriptional factor RUNX2 were decreased by iron. Transcriptomic analysis revealed that the mRNA level of HedgeHog Interacting Protein Like-2 (HHIPL-2) gene, encoding an inhibitor of the hedgehog signaling pathway, was decreased in the presence of FAC. Specific inhibition of HHIPL-2 expression decreased osteoblast marker mRNA levels. Purmorphamine, hedgehog pathway activator, increased the mRNA level of GLI1, a target gene for the hedgehog pathway, and decreased osteoblast marker levels. GLI1 mRNA level was increased under iron exposure. CONCLUSION: We showed that in human MG-63 cells, iron exposure impacts iron metabolism and osteoblast gene expression. HHIPL-2 gene expression modulation may contribute to these alterations. Our results support a role of osteoblast impairment in iron-related osteoporosis.

Identification of 96 single nucleotide polymorphisms in eight genes involved in iron metabolism: efficiency of bioinformatic extraction compared with a systematic sequencing approach.

Single nucleotide polymorphisms (SNPs) can significantly contribute to the characterization of the genes predisposing to iron overloads or deficiencies. We report an SNP survey of coding and non-coding regions of eight genes involved in iron metabolism, by two successive methods. First, we made use of the public domain sequence data, by using assembled expressed sequence tags, non-redundant sequences, and SNP database screening. We extracted 77 potential SNPs of which only 31 could be further validated by sequencing DNA from 44 unrelated multi-ethnic individuals. Our results indicate that a bioinformatic approach may be effective only in those cases where candidate SNPs are extracted from two different data sources or in cases of experimentally confirmed SNPs. Second, additional systematic sequencing of DNA from 24 unrelated Breton subjects increased the number of SNPs over a total length of 86 kb to 96. The average distance between the SNPs and minor allele frequencies were higher than reported by others authors; this discrepancy may reflect the nature of the genes studied and the ethnic homogeneity of our test population.

Identification of an endogenous RNA transcribed from the antisense strand of the HFE gene.

Hereditary haemochromatosis is an autosomal recessive disease which results in iron overload, and it is the most frequently inherited disorder in Caucasian populations. The gene involved (HFE) has recently been identified, and it encodes an MHC class I-like molecule. A 2.7 kb cDNA has been isolated, whereas the HFE gene expression is characterized by an almost ubiquitous mRNA of 4.1 kb in size. The difference between this transcript and the isolated cDNA has not yet been explained. Thus, the 5' end of the HFE gene is still undefined and very little is known about the regulation of its expression. By searching this end, we isolated an antisense transcript originating from the same gene locus. Further investigations (rapid amplification of cDNA ends, RT-PCR experiments and dbEST screening) indicated that this RNA spans exon 1, exon 2, part of intron 1 of the HFE gene and approximately 1 kb upstream of it. This HFE antisense transcript is polyadenylated but displays no open reading frame. A ribonuclease A protection assay definitively demonstrated the biological existence of the HFE antisense RNA, which appears to be expressed in all of the tissues and cell lines tested. Furthermore, in vitro coupled transcription-translation experiments revealed that the HFE expression is decreased by this antisense RNA, indicating that it may play a critical role in the regulation of the HFE gene expression.

The HFE gene undergoes alternate splicing processes.

The MHC class I-related HFE gene appears to be involved in iron metabolism, but its pathogenic mechanism in hemochromatosis remains unknown. Furthermore, very little is known about the regulation of its expression. Hybridization of human tissue Northern blots revealed five different HFE mRNAs, indicating that HFE gene transcription is subject to alternative processes. cDNA selection and RT-PCR performed on HeLa cells clearly demonstrated the occurrence of either differential termination or splicing in HFE transcription. Among the numerous molecules identified, two may have a genuine biological significance.

Systematic sequencing of the human HLA-A/HLA-F region: establishment of a cosmid contig and identification of a new gene cluster within 37 kb of sequence.

The class I region of the human histocompatibility complex is characterized by a high density of genes and pseudogenes and a complex structural organization. To elucidate the complete structure of the HLA-A/HLA-F region with a view to defining its contents in genes and pseudogenes, we developed a strategy of systematic sequencing. This report describes the establishment of a cosmid contig spanning most of the region and the analysis of a 37-kb sequence from one of the cosmids. Four new genes, organized with the HCG-V gene in a clustered structure, have been identified. Two of these contain a zinc finger motif characteristic of DNA-binding proteins. The former, a member of the C3HC4 protein family, is highly expressed in prostate and contains a B30-2-like sequence identified in several genes mapped within the class I region. The latter, which is ubiquitously expressed, is the human equivalent of the yeast polymerase IA12.2 subunit and of the murine tctex6 gene. Of the two other genes, one remains an anonymous gene with no particular feature, while the fourth, specifically expressed in testis, is the human equivalent of the murine tctex4 gene. This cluster, located in a region corresponding to a syntenic unit between mouse and human, appears to be highly conserved.

Retroviral-mediated gene transfer corrects very-long-chain fatty acid metabolism in adrenoleukodystrophy fibroblasts.

Adrenoleukodystrophy (ALD), a lethal demyelinating disease of the brain, is caused by mutations of a gene encoding an ATP-binding transporter, called ALDP, localized in the peroxisomal membrane. It is associated with a defective oxidation of very-long-chain fatty acids, leading to their accumulation in many tissues. This study reports that the retroviral-mediated transfer of the ALD cDNA restored very-long-chain fatty acid oxidation in ALD fibroblasts in vitro following abundant expression and appropriate targeting of the vector-encoded ALDP in peroxisomes. The same method may be used in hematopoietic cells as a further step of a gene therapy approach of ALD.

The gene responsible for adrenoleukodystrophy encodes a peroxisomal membrane protein.

Adrenoleukodystrophy is a severe genetic demyelinating disease associated with an impairment of beta-oxidation of very long chain fatty acids (VLCFA) in peroxisomes. Earlier studies had suggested that a deficiency in VLCFA CoA synthetase was the primary defect. A candidate adrenoleukodystrophy gene has recently been cloned and was found unexpectedly to encode a putative ATP-binding cassette transporter. We have raised monoclonal antibodies against this protein, that detect a 75kDa band. This protein was absent in several patients with adrenoleukodystrophy. Immunofluorescence and immunoelectron microscopy showed that the adrenoleukodystrophy protein (ALDP) is associated with the peroxisomal membrane. Distinct immunofluorescence patterns were observed in cell lines from patients with Zellweger syndrome (a peroxisomal biogenesis disorder) belonging to different complementation groups.

Pharmacokinetics and tissue localization of doxycycline polyphosphate and doxycycline hydrochloride in the rat.

Two doxycycline derivatives Doxycycline polyphosphate and Doxycycline hydrochloride were administered to rats at a dose of 20 mg/kg body weight. Doxycycline tissue levels were determined using a microbiological assay. Only an insignificant fraction of the antibiotics was found to cross the blood brain barrier. Doxycycline was highly concentrated in excretory organs: liver, kidneys and caecum. The high intestinal drug level observed is probably related to the entero-hepatic cycle of this antibiotic. There was a good correlation between serum and heart doxycycline concentration; heart level was about twice that of serum. In lung, antibiotic level was always higher than in serum.

Pharmacokinetic study of doxycycline polyphosphate+ after simultaneous ingestion of milk.

Pharmacokinetic parameters of doxycycline polyphosphate were studied in healthy volunteers after oral administration of a single 200 mg dose of this antibiotic with a breakfast containing or not 200 ml of whole milk. Ingestion of milk had only mild effect upon absorption parameters of doxycycline; only a moderate increase of the lag-time was significant. Elimination parameters of doxycycline were impaired by milk; a decrease of the terminal half-life from 28 h to 15 h, and apparent decrease of the enterohepatic circulation and an increase in total body clearance from 40 to 62 ml/min. were observed.

Pharmacokinetics of doxycycline polyphosphate after oral multiple dosing in humans.

Nine healthy volunteers received oral multiple doses of doxycycline polyphosphate for 6 days. Three different dosage schedules were given and the time concentration data obtained was used to determine the best protocol for producing effective serum antibiotic levels during a complete period of treatment with the aid of a mathematical simulation programme. This protocol consisted of the administration of a 200 mg loading dose on the first day, followed by a 100 mg maintenance dose every twelve hours. Using this dosage schedule a steady state was obtained on the first day of treatment, 3 mg/l was the maximum serum level reached, and the lowest serum concentration was more than 1 mg/l which was assumed to be a therapeutically effective serum concentration.

Pharmacokinetic study of doxycycline polyphosphate (PPD), hydrochloride (CHD) and base (DB).

Six healthy volunteers received the same oral dose of doxycycline, base (200 mg). Each received two of the three preparations at two-week intervals. Experimental results were interpreted on the basis of one or two-compartment models. The three preparations gave the elimination constants of the same order of magnitude (0.045 h-1 to 0.051 h-1). The plasma half-life t 1/2 beta was 14.143 h for DP, 15.400 h for DHC and 13.588 h for DB. Vd is higher for DB (91.955 L) than for DPP (73.401 L) and DHC (64.827 L). Total plasma clearance is 52.767 ml/min for DPP, 48.728 ml/min for DHC and 60.174 ml/min for DB. Urinary elimination 72 hours after administration is 29.24% for DPP, 35.60% and 28.15% for DB. Fluorimetric analysis of some of the samples confirmed the values obtained, with the exception of a few parameters such as Vd and clearance, which were lower. This may result from the fact that this method of determination is more broadly responsive, and is not limited to the evaluation of the active fraction. Relative bioavailability of the capsule form of DPP is 111.15% of that of DHC.

Serum kinetics of doxycycline polyphosphate in dogs.

Serum kinetics of Doxycycline polyphosphate (DPP) have been studied in dogs after oral administration of 10 mg.kg-1 by measurement of total serum concentration (Ct) of tetracycline derivatives by a chemical assay and active concentration (Ca) by a microbiological method. Kinetics have been studied using a one compartment open model with absorption by oral route. DPP is rapidly absorbed, the peak serum level is reached three hours after absorption and slowly eliminated (elimination half-life = 12 hours). The main differences observed between Ct and Ca kinetics are in the values of the areas under the curves (AUC) and the peak serum level. The values obtained for these parameters for Ca kinetics were found to be 50% of those obtained for Ct, the volumes of distribution being in inverse proportion. These results are in a good agreement with the correlation and linear regression observed between Ca and Ct showing that 55% of total serum Doxycycline possesses immediate antibacterial activity. It is postulated that this difference between Ct and Ca kinetics is essentially a reflection of the ratios of bound and free drug. Similar results were obtained with the finished pharmaceutical form except for a 15% increase of AUC indicating improvement of the bioavailability of the drug.

[Compression of the posterior branch of the radial nerve by a benign tumour of the elbow region. Report of three cases (author's transl)]. / Compression de la branche postérieure du nerf radial par une tumeur bénigne de la région du coude. Trois observations.

The authors report three cases of a rare disorder : compression of the posterior motor branch of the radial nerve (ramus profundus n. radialis) by a benign tumor of the region of the elbow, in one case by a lipoma, in the other cases by a pseudocyst. Surgical removal was successful in all three cases. The authors review the literature concerning compression of the posterior interosseous nerve (ramus profundus n. radialis) by lipomas and pseudocysts. Apart from two cases treated at a late stage, surgery permitted complete functional recovery in all the other published cases. This emphasises the interest of early radiological diagnosis and of routine operation even the etiology does not appear obvious, especially as surgery is beneficial in the treatment of the tumour itself.