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.

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.

High yield synthesis of the bovine leukemia virus (BLV) p24 major internal protein in Saccharomyces cerevisiae.

Bovine leukemia virus (BLV) p24 gene was expressed in Saccharomyces cerevisiae under the control of the PHO5 (encoding repressible acid phosphatase, rAPase) promoter. Yeast cells were transformed by a yeast-E. coli shuttle vector carrying the PHO5 promoter, the p24 gene and the CYC1 transcription terminator. After low inorganic phosphate (Pi) induction of the PHO5 promoter, p24 accumulated in the producing cells up to a concentration representing 10% of total soluble proteins. The expression level of p24 gene was not increased by insertion of the positive regulatory gene PHO4 on the p24 expression vector. The p24 produced in this system and incubated in crude yeast extract showed a remarkably high resistance to proteolytic degradation, a feature that presumably correlates with the compact globular conformation of the protein combined to the stabilizing effect of the N-terminal residue.

Biochemical and immunological characterization of the bovine leukemia virus (BLV) envelope glycoprotein (gp51) produced in Saccharomyces cerevisiae.

The nucleotide sequence coding for bovine leukemia virus (BLV) envelope glycoprotein gp51 was inserted into a yeast-Escherichia coli shuttle vector carrying the promoter and secretion signal sequence of PHO5 (the yeast gene coding for repressible acid phosphatase) and the CYC1 transcriptional terminator. Yeast cells transformed by this construction synthesized gp51 after PHO5 induction by inorganic phosphate deprivation. The yeast-expressed gp51 was partially glycosylated into heterodisperse protein molecules ranging from 40 to 48 kDa. No gp51 was excreted in the culture medium. The amount of protein accumulated in yeast cells was estimated to reach 0.06% of soluble proteins. This modest level of expression seemed to be due to the toxicity of gp51 to the yeast cell. The yeast-expressed gp51 products were used in enzyme-linked immunosorbent assays for the detection of antibodies in sera from BLV-infected animals; they were also screened for the presence of well-defined biological epitopes. In both studies poor reactivity was observed. Rabbits immunized with the recombinant gp51 showed high antibody titers to native BLV gp51. However, these antibodies did not neutralize BLV in vitro.

Cystine fluxes across the isolated jejunal epithelium in cystinuria: increased efflux permeability at the luminal membrane.

In cystinuria, renal clearance of cystine frequently exceeds creatinine clearance, suggesting net cystine secretion; and absorption of the (di)basic amino acid is impaired at the luminal membrane of the jejunal and probably also renal tubular epithelium. We studied cystine transport in vitro in jejunal biopsy specimens of eight subjects with homozygous cystinuria and in 12 controls. Cellular/medium cystine distribution ratio was reduced in cystinuria (1.36 +/- 0.36 versus 5.36 +/- 0.61, p less than 0.001). Cystine influx across the luminal membrane was normal (221 +/- 48 versus 261 +/- 79 pmol X h-1 cm-2). Measurement of transepithelial cystine fluxes showed net absorption in controls but secretion in cystinuria. Apparent permeability coefficients were close to normal in cystinuria except that the efflux permeability at the luminal membrane was significantly increased (0.839 +/- 0.22 versus 0.186 +/- 0.12 X h-1 cm-2), and, accordingly, at the luminal membrane, the influx over efflux permeability ratio was small (1.01 +/- 0.50 versus 4.95 +/- 0.80, p less than 0.001). The defect in cystine transport in cystinuria is apparently not caused by decreased influx but increased efflux of cystine (or cystine) from the cell to the lumen across the luminal membrane.

[Complications and sequelae in the endoscopic follow-up of uretero-sigmoidostomies]. / Incidents et accidents de la surveillance endoscopique des urétérosigmoïdostomies.

Progress in gastrointestinal endoscopy enables it to be used routinely in the diagnosis, treatment and follow-up of colonic tumours. The development of a tumour, especially adenocarcinoma, at the site of anastomosis of uretero-sigmoidostomy is a particular case. The endoscopic examination is delicate and confusion between the ureterocolonic junction and a polyp may have dramatic consequences when biopsy is performed. Two cases are reported: in one case, the error required nephro-ureterectomy and in the other case percutaneous nephrostomy, now a routine procedure, allowed salvage of the single kidney and preservation of the diversion with satisfactory long term function.

Membranoproliferative glomerulonephritis associated with pulmonary sarcoidosis.

A 34-year-old woman with pulmonary and ganglionary sarcoidosis developed a nephritic syndrome. The renal biopsy demonstrated a type-1 membranoproliferative glomerulonephritis. Clinical and histological remission of the renal disease following indomethacin treatment was associated with the remission of sarcoidosis. Prior reports have emphasized the association of a membranous glomerulonephritis with sarcoidosis. Temporal sequence of events and biological data suggest that hypocomplementemic membranoproliferative glomerulonephritis may be linked to sarcoidosis. The role of immune complexes in the pathogenesis of the 2 diseases is also discussed.

[Open heart cardiac surgery in severe renal insufficiency and dialysis patients]. / Chirurgie cardiaque à coeur ouvert chez l'insuffisant rénal grave et le dialysé.

Surgically remediable cardiovascular complications are common in patients with renal failure treated by dialysis. 20 such patients were operated in our department (16 men and 4 women), aged 27 to 61 years (mean 44.5 years). 12 patients had undergone haemodialysis for 1 to 84 months; 4 patients were treated by peritoneal dialysis; the remaining four patients all had severe renal failure with creatinine clearances of less than 10 ml per minute. All patients were operated immediately after a session of dialysis. Particular attention was paid to preserving the peripheral arterial and venous vessels during anaesthesia and cardiopulmonary bypass. The jugular veins were used whenever possible to spare the upper limb veins and the dorsalis pedis arteries were used for the monitoring of systemic blood pressure to spare the radial arteries for eventual arteriovenous fistulae. Weight gain during the operation was limited by cardiopulmonary bypass techniques. The circuit was filled with 200 cc of B 21, 500 cc of isotonic bicarbonate solution and 800 cc of frozen plasma with potassium supplements. Mean weight gain was moderate (1.1 +/- 0.4 kg). 12 patients underwent valve replacement. The surgical indication was acute endocarditis in 6 cases. The aortic valve was replaced in 10 cases and the mitral valve in 2 cases by mechanical valve prostheses because of the high risk of calcification of bioprostheses in severe renal failure. 8 patients underwent coronary bypass graft surgery. Arterial blood pressure was maintained at over 60 mmHg and large doses of heparin were used to protect the arteriovenous shunts.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum concentration and peritoneal transfer of aluminum during treatment by continuous ambulatory peritoneal dialysis.

The evolution of the aluminum (A1) serum levels during a 2-year follow-up and the peritoneal transfer of A1 were studied in 22 patients treated by continuous ambulatory peritoneal dialysis (CAPD), using a dialysate with a very low A1 concentration (r = 0.25 - 0.30 mumoles/liter). Patients were divided in three groups. A transfer of A1 from the patient to the dialysate was observed in all patients. In group 1, patients exclusively treated by CAPD and who have never received aluminum-containing phosphate binders (ACPB), mean level (+/- SD) of serum A1 stabilized within a safe range (0.60 +/- 0.28 mumoles/liter). In group 2 the oral administration of ACPB in patients exclusively treated by CAPD induced a slow and progressive increase of A1 serum concentration despite the increase of the A1 excretion through the peritoneal route. In group 3, patients previously treated by hemodialysis and receiving ACPB, the high serum A1 levels observed before treatment by CAPD decreased rapidly on CAPD. A1 removal through the peritoneum was higher in group 3 than in group 2 despite serum A1 levels not statistically different in both groups. A1 removal through the peritoneum is mainly influenced by serum and dialysate A1 concentration. A1 body stores could play a role in the transfer of A1 through the peritoneum. Three cases of A1 poisoning due to the accidental use of a dialysate with a high A1 content are reported.

The glutathione-dependent glyoxalase pathway in the yeast Saccharomyces cerevisiae.

Glyoxalase I (EC 4.4.1.5), which catalyzes the reaction methylglyoxal + GSH leads to S-lactoylglutathione, is a ubiquitous enzyme for which no clear physiological function has been shown. In the yeast Saccharomyces cerevisiae, methylglyoxal may derive from the spontaneous decay of intracellular glyceraldehyde-3-P, which may accumulate during growth on glycerol as the carbon source. The half-life time for the triose phosphate was found to be 4.6 h under physiological conditions (pH 6.2, 0.05 M phosphate at 30 degrees C). Glyoxalase I is induced by growth on glycerol or by the addition of methylglyoxal to the growth medium. The enzyme is also subject to carbon catabolite repression. A mutant strain, fully defective in glyoxalase I and bearing only one nuclear mutation, was obtained. The strain, which is killed by exposure to glycerol, excretes methylglyoxal into the medium. Growth of the mutant on glucose as carbon source appears to be similar to that of the wild type strain. This investigation has clearly demonstrated a physiological role of glyoxalase I in a eucaryotic cell.

Erythrocyte organic phosphates and whole blood oxygen affinity in patients on continuous ambulatory peritoneal dialysis.

Whole blood oxygen affinity, erythrocyte pH and organic phosphates were studied in five anaemic untransfused patients with end stage renal disease undergoing continuous ambulatory peritoneal dialysis. Decreased whole blood oxygen affinity with increased adenosine triphosphate and normal 2.3 diphosphoglycerate (DPG) values were observed. Normal and stable serum phosphate and permanent mild metabolic acidosis may be important factors contributing to maintain DPG levels within the normal range despite anaemia. Continuous dialysis avoids cyclic fluctuation of blood oxygen affinity as described during and after dialysis sessions in patients on maintenance haemodialysis.

Early anuria prevention in human kidney transplantation. Advantage of fluid load under pulmonary arterial pressure monitoring during surgical period.

In human kidney transplantation, a high blood flow established through the graft immediately upon clamp release is usually associated with immediate satisfactory renal function. One hundred consecutive kidney transplant patients were thus provided with a large volume of fluid during surgery. To avoid pulmonary edema, fluid load was given under mean pulmonary arterial pressure (PAP) monitoring, and controlled ventilation was maintained during the early postoperative period. Whether initial PAP value was within normal range or elevated, all patients required an equivalent fluid load to reach the best hemodynamic condition upon clamp removal. The mean intraoperative fluid load consisted of 2406 +/- 968 ml of water with 22.8 +/- 9.4 g of sodium chloride, 5.9 +/- 1.8 units of albumin, and 2.6 +/- 1.8 units of packed red blood cells. Immediately before clamp release patients were given furosemide and mannitol. During the postoperative period, i.v. infusions consisted of water and sodium chloride (6 g/liter) to match urine output, provided that diuresis was equal to or above 400 ml/hr. If diuresis remained or decreased below this level, diuresis replacement was associated with PAP-controlled infusion of saline, albumin, and red blood cells if needed. Furosemide was eventually given if diuresis did not increase above 400 ml/hr with fluid loading. With this protocol a good early diuresis was established in 95% of the cases. Ten patients required dialysis before the 5th postoperative day, one of them because of fluid overload and anuria. Concurrently, a decreased mortality rate and an increased graft survival rate were observed.

[The prognosis in primary chronic glomerulonephritis in the adult. 298 clinicopathological cases (author's transl)]. / Le pronostic des glomérulonéphrites chroniques primitives de l'adulte. 298 observations anatomo-cliniques.

The study involved 298 cases of chronic glomerulonephritis (GN) in adults. The results of renal biopsy were used to classify the patients into four groups: Membranous GN, 81 cases; focal glomerulo sclerosis, 80 cases; Membrano-proliferative GN, 62 cases; GN with mesangial deposits of IgA, 75 cases. The patients were observed over a period ranging from 1 month to 36 years. The average period of surveillance for each category was between 4 and 6 years. The course in each histological type was assessed on the basis of actuarial tates of renal death, of moderate renal insufficiency (plasma creatinine greater than 1.5 mg%( and of hypertension. Renal survival at 10 years was was arounds 90% for membranous GN. 85% for GN with mesangial deposits of IgA, 70% for focal glomerulo sclerosis and 50% for membrano-proliferative GN. The prognosis should be based upon a combination of histological and clinical findings. Severity of prolonged nephrotic syndrome, regardless of the histological type of the nephropathy, is worthy of emphasis. In the group fo focal glomerulo sclerosis, prognosis differs greatly in relation to the presence or absence of a nephrotic syndrome. Complete remission may be seen in the group of focal glomerulo sclerosis, and in membrano-proliferative GN despite the persistence or worsening of histological lesions seen on repeated biopsies.