Preselection of Lung Cancer Cases Using FGFR1 mRNA and Gene Copy Number for Treatment With Ponatinib.

INTRODUCTION: Preclinically, high epidermal growth factor receptor 1 (FGFR1) messenger RNA (FGFR1-MRNA) and FGFR1 amplification (FGFR1-AMP) predicted sensitivity to fibroblast growth factor receptor inhibitors in non-small-cell lung cancer and small-cell lung cancer cell lines. KRAS mutations did not preclude sensitivity. PATIENTS AND METHODS: Metastatic EGFR- and ALK-negative lung cancers were screened for FGFR1-MRNA by in-situ hybridization (ISH) and FGFR1-AMP by silver in-situ hybridization (SISH). Patients with positive findings were offered ponatinib, a multi-kinase inhibitor of FGFR1-4. Differences in overall survival (OS) between cohorts were assessed by the log-rank test. Association of FGFR1 positivity with clinicopathologic features were assessed by Fisher exact test and Kruskal-Wallis rank sum test. RESULTS: A total of 171 cases were prescreened: 9 (7.3%) of 123 SISH+; 53 (42.1%) of 126 ISH+; and 6 cases concordantly positive for SISH and ISH. SISH+ cases had fewer coincident KRAS mutations (P = .03) than SISH- cases, and ISH+ cases had worse OS (P = .020) than ISH- cases. Data distributions suggested a distinct higher positivity cut point for FGFR1 ISH (≥ 20%), occurring in 29 (23%) of 126 cases, was associated with small-cell lung cancer histology (P = .022), soft tissue metastases (P = .050) and shorter OS (P = .031). Four patients received ponatinib on study: All ISH+ by the initial cut point, 2 of 4 by higher cut point, 1 of 4 SISH+. Tolerability was poor. The best response for the 2 higher ISH cases was stable disease and progressive disease for the 2 lower ISH cases. CONCLUSION: Elevated FGFR1-MRNA is more common than FGFR1-AMP and associated with worse OS. Higher FGFR1 mRNA expression may be associated with a specific phenotype and is worthy of further exploration. Ponatinib's poor tolerance suggests further fibroblast growth factor receptor exploration in ISH+ cases should utilize more selective FGFR1 inhibitors.

Antiretroviral therapy provided to HIV-infected Malawian women in a randomized trial diminishes the positive effects of lipid-based nutrient supplements on breast-milk B vitamins.

BACKGROUND: Little information is available on B vitamin concentrations in human milk or on how they are affected by maternal B vitamin deficiencies, antiretroviral therapy, or maternal supplementation. OBJECTIVE: The objective was to evaluate the effects of antiretroviral therapy and/or lipid-based nutrient supplements (LNSs) on B vitamin concentrations in breast milk from HIV-infected women in Malawi. DESIGN: Breast milk was collected from 537 women recruited within the Breastfeeding, Antiretrovirals, and Nutrition study at 2 or 6 wk and 24 wk postpartum. Women were assigned to receive antiretrovirals and LNSs, antiretrovirals only, LNSs only, or a control. Antiretrovirals and LNSs were given to the mothers from weeks 0 to 28. The antiretrovirals were zidovudine/lamivudine and nelfinavir or lopinavir/ritonavir. LNSs provided 93-118% of the Recommended Dietary Allowances of thiamin, riboflavin, niacin, pyridoxine, and vitamin B-12. Infants were exclusively breastfed. RESULTS: LNSs increased milk concentrations of all vitamins except thiamin, whereas antiretrovirals lowered concentrations of nicotinamide, pyridoxal, and vitamin B-12. Although antiretrovirals alone had no significant effect on riboflavin concentrations, they negatively affected the LNS-induced increase in this vitamin. Thiamin was not influenced by the study interventions. Concentrations of all B vitamins were much lower than usually accepted values. CONCLUSIONS: All B vitamins were low in milk, and all but thiamin were increased by maternal supplementation with LNSs. Antiretrovirals alone decreased concentrations of some B vitamins in milk. When LNS was given in addition to antiretrovirals, the negative effect of antiretrovirals offset the positive effect of LNSs for all vitamins except thiamin. This trial was registered at clinicaltrials.gov as NCT00164762.

Ultra-performance liquid chromatography tandem mass-spectrometry (UPLC-MS/MS) for the rapid, simultaneous analysis of thiamin, riboflavin, flavin adenine dinucleotide, nicotinamide and pyridoxal in human milk.

A novel, rapid and sensitive ultra-performance liquid-chromatography tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous determination of several B-vitamins in human milk was developed. Resolution by retention time or multiple reaction monitoring (MRM) for thiamin, riboflavin, flavin adenine dinucleotide (FAD), nicotinamide and pyridoxal (PL) has been optimized within 2 min using a gradient of 10 mM ammonium formate (aq) and acetonitrile. Thiamin-(4-methyl-¹³C-thiazol-5-yl-¹³C3) hydrochloride, riboflavin-dioxo-pyrimidine-¹³C4,¹5N2, and pyridoxal-methyl-d3 hydrochloride were used as internal standards. A sample-like matrix was found to be mandatory for the external standard curve preparation. ¹³C3-caffeine was added for direct assessment of analyte recovery. Intra- and inter-assay variability for all analytes ranged from 0.4 to 7.9% and from 2.2 to 5.2%, respectively. Samples were subjected to protein precipitation and removal of non-polar constituents by diethyl ether prior to analysis. Quantification was done by ratio response to the stable isotope labeled internal standards. The standard addition method determined recovery rates for each vitamin (73.0-100.2%). The limit of quantitation for all vitamins was between 0.05 and 5 ppb depending on the vitamin. Alternative approaches for sample preparation such as protein removal by centrifugal filter units, acetonitrile or trichloroacetic acid revealed low recovery and a greater coefficient of variation. Matrix effect studies indicated a significant influence by matrix constituents, showing the importance of stable isotope labeled internal standards for analyte quantitation in complex matrices.

Development of a rapid, cost-effective TaqMan Real-Time PCR Assay for identification and differentiation of Coccidioides immitis and Coccidioides posadasii.

Coccidioidomycosis is an infection caused by Coccidioides immitis or C. posadasii. We developed a TaqMan real-time PCR assay that rapidly and accurately differentiates the species. This assay can be used as a tool to improve disease surveillance, increase understanding of the natural history of the infection, and assist in clinical differentiation studies.