Optimizing Mutation and Fusion Detection in NSCLC by Sequential DNA and RNA Sequencing.

INTRODUCTION: Frequently, patients with locally advanced or metastatic NSCLC are screened for mutations and fusions. In most laboratories, molecular workup includes a multitude of tests: immunohistochemistry (ALK, ROS1, and programmed death-ligand 1 testing), DNA sequencing, in situ hybridization for fusion, and amplification detection. With the fast-emerging new drugs targeting specific fusions and exon-skipping events, this procedure harbors a growing risk of tissue exhaustion. METHODS: In this study, we evaluated the benefit of anchored, multiplexed, polymerase chain reaction-based targeted RNA sequencing (RNA next-generation sequencing [NGS]) in the identification of gene fusions and exon-skipping events in patients, in which no pathogenic driver mutation was found by DNA-based targeted cancer hotspot NGS (DNA NGS). We analyzed a cohort of stage IV NSCLC cases from both in-house and referral hospitals, consisting 38.5% cytology samples and 61.5% microdissected histology samples, mostly core needle biopsies. We compared molecular findings in a parallel workup (DNA NGS and RNA NGS, cohort 1, n = 198) with a sequential workup (DNA NGS followed by RNA NGS in selected cases, cohort 2, n = 192). We hypothesized the sequential workup to be the more efficient procedure. RESULTS: In both cohorts, a maximum of one oncogenic driver mutation was found per case. This is in concordance with large, whole-genome databases and suggests that it is safe to omit RNA NGS when a clear oncogenic driver is identified in DNA NGS. In addition, this reduced the number of necessary RNA NGS to only 53% of all cases. The tumors of never smokers, however, were enriched for fusions and exon-skipping events (32% versus 4% in former and current smokers, p = 0.00), and therefore benefited more often from the shorter median turnaround time of the parallel approach (15 d versus only 9 d in the parallel workup). CONCLUSIONS: We conclude that sequentially combining DNA NGS and RNA NGS is the most efficient strategy for mutation and fusion detection in smoking-associated NSCLC, whereas for never smokers we recommend a parallel approach. This approach was shown to be feasible on small tissue samples including for cytology tests, can drastically reduce the complexity and cost of molecular workup, and also provides flexibility in the constantly evolving landscape of actionable targets in NSCLC.

Pharmacokinetics of aztreonam in healthy subjects and patients with cystic fibrosis and evaluation of dose-exposure relationships using monte carlo simulation.

Aztreonam (AZM) is a monobactam antibiotic with a high level of activity against gram-negative micro-organisms, including Pseudomonas aeruginosa. We evaluated AZM pharmacokinetics and pharmacokinetic-pharmacodynamic relationships in patients with cystic fibrosis (CF) and healthy subjects. Pharmacokinetic data in eight CF patients and healthy subjects that were matched for age, gender, weight, and height were obtained and analyzed by using the nonparametric adaptive grid algorithm. Probabilities of target attainment using percentages of time of unbound concentration above the MIC (fT>MIC) were obtained by using a Monte Carlo simulation. AZM total body clearance was significantly higher in CF patients (100.1 +/- 17.1 versus 76.2 +/- 7.4 ml/min in healthy subjects; P < 0.01). The pharmacokinetic parameter estimates for terminal half-life (1.54 +/- 0.17 h [mean +/- the standard deviation]) and volume of distribution (0.20 +/- 0.02 liters/kg in patients with CF patients were not different from those in healthy subjects. Monte Carlo simulations with a target of a fT>MIC of 50 to 60% at a dose of 1,000 mg every 8 h indicated a clinical breakpoint of 4 mg/liter and 1 to 2 mg/liter for healthy subjects and CF patients, respectively. This study using matched controls showed that AZM total body clearance and not the volume of distribution is higher in CF patients as a result of increased renal clearance. Pharmacokinetic parameter estimates in healthy subjects resulted in a clinical susceptibility breakpoint of < or =4 mg/liter for a dose of 1,000 mg every 8 h. Patients suspected of having high clearance rates, such as CF patients, should be monitored closely, with dosing regimens adjusted accordingly.