No abstract

We present a wrapper-based approach to estimate and control the false discovery rate for peptide identifications using the outputs from multiple commercially available MS/MS search engines. Features of the approach include the flexibility to combine output from multiple search engines with sequence and spectral derived features in a flexible classification model to produce a score associated with correct peptide identifications. This classification model score from a reversed database search is taken as the null distribution for estimating p-values and false discovery rates using a simple and established statistical procedure. Results from 10 analyses of rat sera on an LTQ-FT mass spectrometer indicate that the method is well calibrated for controlling the proportion of false positives in a set of reported peptide identifications while correctly identifying more peptides than rule-based methods using one search engine alone.

In this study, we report a method for direct determination of gemcitabine incorporation into human DNA. Gemcitabine (dFdC), a structural analog of the nucleoside deoxycytidine (dC), derives its primary antitumor activity through interruption of DNA synthesis. Unlike other surrogate measures, DNA incorporation provides a mechanistic end point useful for dose optimization. DNA samples (ca. 25 microg) were hydrolyzed using a two-step enzymatic procedure to release dFdC which was subsequently quantified by LC-ESI-MS/MS using stable isotope labeled internal standards and selected reaction monitoring (SRM). dFdC was quantitated and reported relative to deoxyguanosine (dG) since dG is the complementary base for both dFdC and dC. The SRM channel for dG was detuned using collision energy as the attenuating parameter in order to accommodate the difference in relative abundance for these two analytes (>104) and enable simultaneous quantification from the same injection. The assay was shown to be independent of the amount of DNA analyzed. The method was validated for clinical use using a 3 day procedure assessing precision, accuracy, stability, selectivity, and robustness. The validated ranges for dFdC and dG were 5-7500 pg/mL and 0.1-150 microg/mL, respectively. Results are presented which confirm that the ratio of dFdC to dG in DNA isolated from tumor cells incubated with dFdC increases with increased exposure to the drug and that dFdC can also be quantified from DNA extracted from blood.

The aim of the present study was to investigate the relationship between circulating PCSK9 (proprotein convertase subtilisin kexin type 9) and FCHL (familial combined hyperlipidaemia) and, when positive, to determine the strength of its heritability. Plasma PCSK9 levels were measured in FCHL patients (n=45), NL (normolipidaemic) relatives (n=139) and their spouses (n=72). In addition, 11 FCHL patients were treated with atorvastatin to study the response in PCSK9 levels. PCSK9 levels were higher in FCHL patients compared with NL relatives and spouses: 96.1 compared with 78.7 and 82.0 ng/ml (P=0.004 and P=0.002 respectively). PCSK9 was significantly associated with both TAG (triacylglycerol) and apolipoprotein B levels (P<0.001). The latter relationship was accounted for by LDL (low-density lipoprotein)-apolipoprotein B (r=0.31, P=0.02), not by VLDL (very-low-density lipoprotein)-apolipoprotein B (r=0.09, P=0.49) in a subgroup of subjects (n=59). Heritability calculations for PCSK9 using SOLAR and FCOR software yielded estimates of 67-84% respectively (P<0.0001). PCSK9 increased from 122 to 150 ng/ml in 11 FCHL patients treated with atorvastatin (40 mg) once daily for 8 weeks (P=0.018). In conclusion, plasma PCSK9 is a heritable trait associated with both FCHL diagnostic hallmarks. These results, combined with the significant rise in PCSK9 levels after statin therapy, warrant further studies in order to unravel the exact role of PCSK9 in the pathogenesis and treatment of this highly prevalent genetic dyslipidaemia.

Anaplastic lymphoma kinase (ALK, Chr 2) is an orphan transmembrane receptor tyrosine kinase that is activated in non-small cell lung cancer (NSCLC), neuroblastoma, and anaplastic large cell lymphomas (ALCL). Genetic activation of ALK can occur as a result of gene amplification, kinase-domain point mutations, or by fusion of the kinase domain to structural genes such as echinoderm microtubule associated protein-like 4 (EML4). Ultimately this leads to further activation of downstream effector proteins-including STAT3, AKT or ERK-enhanced cell proliferation, and disease. A study of 602 cancer cell lines found that neuroblastoma, NSCLC and ALCL cells harboring ALK translocations and amplifications were particularly responsive to treatment with ALK inhibitor therapy (McDermott et al, 2008). Recently, somatic and germline missense mutations were also identified in the ALK kinase domain and linked to 8.4% of hereditary and spontaneous pediatric neuroblastomas (Mosse et al., 2008). The ALK mutations were found to cause an increase in ALK expression that when knocked down resulted in an inhibition of cancer cell growth (Mosse et al., 2008). These results suggest that ALK point mutations have similar functional consequences in neuroblastoma as ALK structural variations and could therefore confer cancer cell sensitivity to ALK inhibitor therapy. Although few studies have investigated this to date, the ALK point mutations may result in similar functional consequences for other cancer cell types. If so, the ALK mutations may be ideal genetic biomarkers to identify patients who would be clinically responsive to ALK inhibitor therapy. Therefore, we sought to better understand a subset of these ALK somatic mutations, their frequency in other cancer types, and potential as clinical biomarkers for cancer drug response. We designed and developed custom allelic discrimination assays to genotype three ALK activating mutations; M1166R, F1174L and R1275Q in DNA extracted from 23 cancer cell lines and 47 tumor samples. Although no neuroblastoma samples were included, these 70 samples represented a dozen different cancer types and tissues, including prostate, breast, ovary, lung, liver and pancreas. We also generated synthetic oligonucleotide allele controls to establish the necessary heterozygous and homozygous genotype clusters for automated genotype calling. Our data showed that the ALK mutations were monomorphic wildtype in all 70 cancer samples tested. These results suggest that the 12.4% frequency of the ALK mutations observed in neuroblastoma may be very specific and play a particular role in that disease. More studies are required to elucidate if these ALK mutations may provide tools for additional cancer patient stratification, including predicted disease progression and response beyond the previously identified ALK translocations and amplifications. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2730.