Quantification of [18F]afatinib using PET/CT in NSCLC patients: a feasibility study.

INTRODUCTION: Only a subgroup of non-small cell lung cancer (NSCLC) patients benefit from treatment using epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) such as afatinib. Tumour uptake of [18F]afatinib using positron emission tomography (PET) may identify those patients that respond to afatinib therapy. Therefore, the aim of this study was to find the optimal tracer kinetic model for quantification of [18F]afatinib uptake in NSCLC tumours. METHODS: [18F]Afatinib PET scans were performed in 10 NSCLC patients. The first patient was scanned for the purpose of dosimetry. Subsequent patients underwent a 20-min dynamic [15O]H2O PET scan (370 MBq) followed by a dynamic [18F]afatinib PET scan (342 ± 24 MBq) of 60 or 90 min. Using the Akaike information criterion (AIC), three pharmacokinetic plasma input models were evaluated with both metabolite-corrected sampler-based input and image-derived (IDIF) input functions in combination with discrete blood samples. Correlation analysis of arterial on-line sampling versus IDIF was performed. In addition, perfusion dependency and simplified measures were assessed. RESULTS: Ten patients were included. The injected activity of [18F]afatinib was 341 ± 37 MBq. Fifteen tumours could be identified in the field of view of the scanner. Based on AIC, tumour kinetics were best described using an irreversible two-tissue compartment model and a metabolite-corrected sampler-based input function (Akaike 50%). Correlation of plasma-based input functions with metabolite-corrected IDIF was very strong (r2 = 0.93). The preferred simplified uptake parameter was the tumour-to-blood ratio over the 60- to 90-min time interval (TBR60-90). Tumour uptake of [18F]afatinib was independent of perfusion. CONCLUSION: The preferred pharmacokinetic model for quantifying [18F]afatinib uptake in NSCLC tumours was the 2T3K_vb model. TBR60-90 showed excellent correlation with this model and is the best candidate simplified method. TRIAL REGISTRATION: https://eudract.ema.europa.eu/ nr 2012-002849-38.

An intronic polymorphic deletion in the PTEN gene: implications for molecular diagnostic testing.

BACKGROUND: A cohort of 629 patients with suspected Bannayan-Riley-Ruvalcaba syndrome or Cowden syndrome was tested for mutations in the PTEN gene. METHODS: Dosage analysis of PTEN was carried out using a PTEN-specific multiplex ligation-dependent probe amplification (MLPA) kit, whereas point mutation analysis was performed using direct sequencing. RESULTS: Approximately 4% of the patients from the testing cohort were heterozygously deleted for the two MLPA probe-binding sites situated in intron 1. The same deletion was subsequently seen in ∼3% of 220 normal controls, and in patients from the testing cohort with a causative mutation elsewhere in the PTEN gene. Sequencing of the variant revealed an 899 bp deletion, the 3' breakpoint of which is only 58 bp from the start of exon 2. CONCLUSION: Although all evidence suggests that the 899 bp deletion is a polymorphism with no clinical effect, it removes the binding sites of almost all published PTEN exon 2 forward primers, resulting in allelic loss during PCR.

Myoclonus-dystonia: significance of large SGCE deletions.

Myoclonus-dystonia (M-D) is an autosomal-dominant movement disorder caused by mutations in SGCE. We investigated the frequency and type of SGCE mutations with emphasis on gene dosage alterations and explored the associated phenotypes. We tested 35 M-D index patients by multiplex ligation-dependent probe amplification (MLPA) and genomic sequencing. Mutations were found in 26% (9/35) of the cases, all but three with definite M-D. Two heterozygous deletions of the entire SGCE gene and flanking DNA and a heterozygous deletion of exon 2 only were detected, accounting for 33% (3/9) of the mutations found. Both large deletions contained COL1A2 and were additionally associated with joint problems. Further, we discovered one novel small deletion (c.771_772delAT, p.C258X) and four recurrent point mutations (c.289C>T, p.R97X; c.304C>T, p.R102X; c.709C>T, p.R237X; c.1114C>T, p.R372X). A Medline search identified 22 articles on SGCE mutational screening. Sixty-four unrelated M-D patients were described with 41 different mutations. No genotype-phenotype association was found, except in patients with deletions encompassing additional genes. In conclusion, a rigorous clinical preselection of patients and careful accounting for non-motor signs should precede mutational tests. Gene dosage studies should be included in routine SGCE genetic testing.

Rapid and quantitative analysis of unconjugated C(27) bile acids in plasma and blood samples by tandem mass spectrometry.

A subgroup of peroxisomal disorders, peroxisome biogenesis defects (PBD), can be differentiated by elevated levels of C(27) bile acids in plasma and bile. Patients with peroxisomal disorders, who lack the ability to chain-shorten the C(27) bile acid intermediates into C(24) bile acids, show elevated levels of C(27) bile acids, notably 3 alpha,7 alpha-dihydroxy-5 beta-cholest-26-oic acid and 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan-26-oic acid. C(27) bile acids are normally estimated against other bile acid standards, by time-consuming gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry methods, in plasma (minimum of 50 microl). In this article we describe the quantitation of unconjugated di- and trihydroxy C(27) bile acids in 5-microl plasma samples and 3-mm blood spots, using deuterium-labeled internal standards. The synthesis of (2)H(3)-labeled di- and trihydroxycoprostanic acids is described. The sample preparation and analysis by electrospray tandem mass spectrometry (ES-MS/MS) takes less than 1 h and features dimethylaminoethyl ester derivatives. The levels of the di- and trihydroxy bile acids are significantly higher in PBD patients than in age-matched control subjects for both plasma and blood spots collected at birth (some stored for up to 18 years). Excellent correlation is observed between the C(26:0)/C(22:0) very long chain fatty acid (VLCFA) ratio and the levels of trihydroxy C(27) bile acids in plasma from PBD patients. The ES-MS/MS method can be used to rapidly screen for PBD patients in plasma samples with elevated C(26:0)/C(22:0) VLCFA ratios and in archived collections of neonatal blood spots. - Johnson, D. W., H. J. ten Brink, R. C. Schuit, and C. Jakobs. Rapid and quantitative analysis of unconjugated C(27) bile acids in plasma and blood samples by tandem mass spectrometry. J. Lipid Res. 2001. 42: 9;-16.