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.

Studies on the structure, expression and function of the yeast regulatory gene PHO2.

The yeast regulatory gene PHO2 encodes a protein assumed to activate, in concert with the PHO4 protein, the transcription of the repressible acid phosphatase-coding gene PHO5. The PHO2 gene was cloned and sequenced. Northern-blot analysis revealed a low and Pi-independent transcription level. We stress the presence of two potential DNA-binding structures, one of them as part of a homeodomain-similar sequence, in the deduced PHO2 protein. Our data indicate that quite a large portion of the C-terminal end of the PHO2 activator is dispensable for derepression of PHO5. Disruption analysis suggests the presence of a nuclear address signal in the N-terminal region. We show that the PHO2 function in the regulation of phosphate metabolism can be partially fulfilled by overproduced PHO4. Inability of pho2 mutants to sporulate indicates a possible involvement of PHO2 products in the regulation of genes related to the life cycle.

Negative regulatory elements of the Saccharomyces cerevisiae PHO system: interaction between PHO80 and PHO85 proteins.

The negative regulatory genes, PHO80 and PHO85, involved in transcriptional regulation of the yeast repressible acid-phosphatase-encoding gene, PHO5, have been cloned. Expression of PHO80 and PHO85 has been studied by means of lacZ fusions. We show here that these expressions are inorganic phosphate (Pi) independent and that they are controlled by the PHO80 gene product; moreover, PHO80 expression is controlled by PHO85. We also present genetic evidence for an interaction between the PHO80 and PHO85 proteins: increased PHO85 gene dosage partially compensates for the pho80-1 mutation and this effect is allele-specific. The pho80-1 allele has been cloned and sequenced. The mutation changes Gly229 to Asp. This region was shown to be essential for PHO80 function by C-terminal deletion analysis.

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.

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.

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.

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.

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.

Early monitoring of human renal transplantations by N-acetyl-beta-D-glucosaminidase isoenzyme activities in urines.

Monitoring of variations in N-acetyl-beta-D-glucosaminidase (NAG) urinary activity, following renal transplantation, has been proposed for the early diagnosis of rejection episodes. In this study, the measurement of urinary NAG-B activity was conducted as a complement to total NAG (A + B) measurement, which is normally used alone. Selective measurement of NAG-B activity is carried out after fixation of NAG-A on ion exchanger in test tubes. Results of NAG (A + B) activity confirm that the assay of urinary NAG is a useful indicator of rejection, but a positive correlation between NAG-B and NAG (A + B) activities was observed during the various complications which can occur after transplantation. The specific measurement of this isoenzyme does not, therefore, seem to provide additional information in the early monitoring of human renal transplantations. Apart from rejection episodes, other factors are likely to produce marked NAG-B excretion, e.g. gentamicin therapy.

Hemodynamic evaluation of hypotension during chronic hemodialysis.

Hypotensive episodes occur frequently during hemodialysis; they are often sudden and difficult to prevent despite careful clinical control. Their etiology was studied by investigating the hemodynamic response of five patients submitted to ultrafiltration during their three first dialyses. A Swan Ganz catheter was inserted and left in position for 5 days. Simultaneous determination of cardiac output, mean pulmonary artery (PAP) and capillary and systemic arterial pressures were recorded. 10 hypotensive episodes were observed. In 3 patients in whom the first hypotensive episode occurred 10 minutes after the start of dialysis, there was a significant drop in PAP, cardiac index and stroke index while heart rate and peripheral resistance remained unchanged. Paradoxical bradycardia was observed. In 4 patients hypotension was observed more than one hour after initiation of dialysis. Before the hypotensive episode there was moderate elevation of heart rate and peripheral resistance and an insignificant reduction in PAP. Cardiac index and stroke index were diminished. The decrease in MAP was only 2 mm Hg. Hypovolemia is the most important factor in hemodialysis-induced hypotension but other factors such as vagal stimulation, autonomic neuropathy and osmotic disequilibrium can interfere with blood pressure control and trigger hypotension. Methods of preventing hypotension during dialysis, including the infusion of low molecular weight dextran, are discussed.

Dense deposit disease: long term follow-up of three cases of recurrence after transplantation.

The incidence and early recurrence after transplantation prove the specificity of the appearance of an electron dense alteration of kidney basement membrane often called dense intra-membranous deposit disease. Three new cases with dense deposit disease affecting the original kidneys have been followed-up after transplantation for periods ranging from 4 to 8 years and illustrate the natural history of the recurrence. Serial kidney biopsies showed the predominance of dense deposits near the mesangial area and the vascular pole. These deposits were also seen in some tubular basement membranes. Absence of cell proliferation was noted in all biopsies performed. Immunofluorescence studies revealed fixation of C3 alone. Histological signs of recurrence are compatible with the absence of clinical and biological signs. Transient or permanent proteinuria and microhematuria were common findings. Serum complement levels, measured after transplantation, were low in all three cases. Despite recurrence of the original glomerulonephritis, long-term survival of the graft was commonly observed, two cases being followed-up for 7 and 8 years. Patients with dense intra-membranous deposits glomerulonephritis should not be excluded from a transplantation program. One of the three cases reported here illustrates the exceptional association of recurrence of dense intramembranous deposits, de novo membranous glomerulonephritis and chronic rejection.

Is cryoglobulin detection of clinical significance in chronic glomerulonephritis not related to systemic disease?

Sera from 105 adult patients with idiopathic chronic glomerulonephritis (GN) were investigated to assess the diagnostic and prognostic value of cryoglobulin (CG) detection. CG+ sera were found in 49% of cases but also in 34% of normal donors. CG composition in these two groups was different. Concentrations of CG greater than micron g/ml were considered as abnormal, since concentrations did not exceed this value in the controls. Using this criterion 30% of patients with GN had CG; most had membrano-proliferative GN, GN with mesangial IgA deposits, and membranous GN. CG occurred with the same frequency but at higher concentrations in the group of patients with diffuse proliferative GN than in other groups; however, there was no difference in the frequency and concentration of CG when GN was thought to be mediated by immune complexes (IC) than when it was not. No relationship was observed with disease activity. In conclusion low CG might represent an in vitro artifact of a common in vivo immunoregulatory mechanism in normal donors. Although they might be a marker for the presence of IC in adult patients with GN not related to systemic disease, CG detection appears to be of little help in diagnosis, assessment of disease activity, or therapeutic monitoring.

Dialysis treatment of insulin dependent diabetic patients: ten years experience.

From January 1973 to March 1983, 108 IDD patients with a mean age of 46 years were accepted to the dialysis program of the Hôpital de la Pitié. Since January 1973, 67 patients have been treated by hemodialysis. Since August 1978, 38 patients have been treated by CAPD. Three patients have been treated by intermittent peritoneal dialysis. Although diabetic patients remain at a higher risk compared to patients of the same age group, very encouraging results are observed including a 75% survival rate at three years among hemodialyzed patients less than 50 years old. Since 1978, CAPD, when home dialysis was possible, was selected as a first choice treatment. Some severe peritoneal complications still jeopardize the advantages of this method. Diabetics with ESRD, even in the older age group, should not be excluded from treatment. They should be offered within an integrated program all dialysis methods and transplantation.

[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)

[Criteria of detection and treatment of rejection crises following human cardiac transplantation in a series of 10 cases]. / Critères de détection et traitement des crises de rejet après transplantation cardiaque humaine sur une série de 10 cas

Analysis of a series of 10 cardiac transplantations confirmed that the reject phenomena have a paroxysmal course, the diagnosis of which is different according to the time occurence of the reject crises, whether early (acute early rejection) or late (late acute rejection or chronic rejection). Because of the early diagnosis based on the follow-up of the electrocardiogram, of coagulation, of the graft flow, overcome eight rejection crises on the 3 patients with middle-term survival.

[Quantification of proteinuria by measurement of the protein/creatinine ratio]. / Quantification de la protéinurie par la mesure du rapport protéine sur créatinine.

Accurate quantification of urinary protein excretion is difficult due to problems in 24-hour urine collection. We have evaluated the value of the protein/creatinine ratio in one single urine sample. There was an excellent correlation between this ratio and the protein content of a 24-hour urine collection [Pu (g/24 h) = 11.7 Pu (g/l) Cr (mmol/l); r = 0.96; P less than 0.001]. The best correlation was found with the sample collected after the first voided morning specimen. In our experience the ratio is more accurate for the quantification of urinary protein excretion than measurement of protein excretion in the same urine sample and also determination of protein excretion in 24-hour urine collected under the usual ambulatory conditions. We suggest that the protein/creatinine ratio should be used in ambulatory and hospitalized patients, especially for multicentric clinical trials in nephrology.

[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.

[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.