Detecting the spectrum of multigene mutations in non-small cell lung cancer by Snapshot assay.

As molecular targets continue to be identified and more targeted inhibitors are developed for personalized treatment of non-small cell lung cancer (NSCLC), multigene mutation determination will be needed for routine oncology practice and for clinical trials. In this study, we evaluated the sensitivity and specificity of multigene mutation testing by using the Snapshot assay in NSCLC. We retrospectively reviewed a cohort of 110 consecutive NSCLC specimens for which epidermal growth factor receptor (EGFR) mutation testing was performed between November 2011 and December 2011 using Sanger sequencing. Using the Snapshot assay, mutation statuses were detected for EGFR, Kirsten rate sarcoma viral oncogene homolog (KRAS), phosphoinositide-3-kinase catalytic alpha polypeptide (PIK3CA), v-Raf murine sarcoma viral oncogene homolog B1 (BRAF), v-ras neuroblastoma viral oncogene homolog (NRAS), dual specificity mitogen activated protein kinase kinase 1 (MEK1), phosphatase and tensin homolog (PTEN), and human epidermal growth factor receptor 2 (HER2) in patient specimens and cell line DNA. Snapshot data were compared to Sanger sequencing data. Of the 110 samples, 51 (46.4%) harbored at least one mutation. The mutation frequency in adenocarcinoma specimens was 55.6%, and the frequencies of EGFR, KRAS, PIK3CA, PTEN, and MEK1 mutations were 35.5%, 9.1%, 3.6%, 0.9%, and 0.9%, respectively. No mutation was found in the HER2, NRAS, or BRAF genes. Three of the 51 mutant samples harbored double mutations: two PIK3CA mutations coexisted with KRAS or EGFR mutations, and another KRAS mutation coexisted with a PTEN mutation. Among the 110 samples, 47 were surgical specimens, 60 were biopsy specimens, and 3 were cytological specimens; the corresponding mutation frequencies were 51.1%, 41.7%, and 66.7%, respectively (P = 0.532). Compared to Sanger sequencing, Snapshot specificity was 98.4% and sensitivity was 100% (positive predictive value, 97.9%; negative predictive value, 100%). The Snapshot assay is a sensitive and easily customized assay for multigene mutation testing in clinical practice.

Establishment of patient-derived non-small cell lung cancer xenograft models with genetic aberrations within EGFR, KRAS and FGFR1: useful tools for preclinical studies of targeted therapies.

BACKGROUND: Patient-derived tumor xenograft models have been established and increasingly used for preclinical studies of targeted therapies in recent years. However, patient-derived non-small cell lung cancer (NSCLC) xenograft mouse models are relatively few in number and are limited in their degree of genetic characterization and validation. In this study, we aimed to establish a variety of patient-derived NSCLC models and characterize these for common genetic aberrations to provide more informative models for preclinical drug efficacy testing. METHODS: NSCLC tissues from thirty-one patients were collected and implanted into immunodeficient mice. Established xenograft models were characterized for common genetic aberrations, including detection of gene mutations within EGFR and KRAS, and genetic amplification of FGFR1 and cMET. Finally, gefitinib anti-tumor efficacy was tested in these patient-derived NSCLC xenograft models. RESULTS: Ten passable patient-derived NSCLC xenograft models were established by implantation of NSCLC specimens of thirty-one patients into immunodeficient mice. Genetic aberrations were detected in six of the models, including one model with an EGFR activating mutation (Exon19 Del), one model with KRAS mutation, one model with both KRAS mutation and cMET gene amplification, and three models with FGFR1 amplification. Anti-tumor efficacy studies using gefitinib demonstrated that the EGFR activating mutation model had superior sensitivity and that the KRAS mutation models were resistant to gefitinib. The range of gefitinib responses in the patient-derived NSCLC xenograft models were consistent with the results reported from clinical trials. Furthermore, we observed that patient-derived NSCLC models with FGFR1 gene amplification were insensitive to gefitinib treatment. CONCLUSIONS: Ten patient-derived NSCLC xenograft models were established containing a variety of genetic aberrations including EGFR activating mutation, KRAS mutation, and FGFR1 and cMET amplification. Gefitinib anti-tumor efficacy in these patient-derived NSCLC xenografts containing EGFR and KRAS mutation was consistent with the reported results from previous clinical trials. Thus, data from our panel of patient-derived NSCLC xenograft models confirms the utility of these models in furthering our understanding of this disease and aiding the development of personalized therapies for NSCLC patients.

[Relationship of collagen type I alpha 1 and alpha 2 gene polymorphisms with bone mineral density].

OBJECTIVE: To investigate collagen type I alpha 1 (COL1A1) and alpha 2 (COL1A2) gene polymorphisms in Chinese and their relationship with bone mineral density. METHODS: Totalling 628 residents of Han nationality in Guangzhou aged 53.4-15.9 (range 20-79) years were surveyed for COL1A1 and COL1A2 gene genotypes by polymerase chain reaction-restriction fragment length polymorphism. Bone mineral density of the lumbar vertebrae, greater trochanter, femur neck and Ward's triangle was measured by dual-energy X-ray absorptiometry. RESULTS: COL1A1 Sp1 polymorphism was not found in these subjects, and the genotype of all samples were type SS. COL1A2 genotyping revealed the distribution of EE genotype in 49.7%, Ee in 40.9% and ee in 9.4% of the subjects. The frequency distribution of EcoR1 alleles followed the Hardy-Weinberg equilibrium. The mean bone mineral density did no significantly differ among these genotype groups (P>0.05 by analysis of variance). CONCLUSION: COL1A1 Sp1 binding site polymorphism is absent and COL1A2 EcoR1 site polymorphism is not associated with bone mineral density in Chinese of Han nationality.

Cerebral intracellular calcium concentrations in asphyxiated rat fetuses resuscitated with oxygen.

OBJECTIVE: To investigate the effects of resuscitation with three different oxygen concentrations on cerebral intra- and extra-cellular calcium, sodium and potassium changes in asphyxiated rat fetuses. METHODS: Fifty-six fetal rats of gestational age of 20 days were randomly assigned into five study groups: sham operation group (control, n = 11), room-air resuscitation group (n = 10), and 3 oxygen-resuscitated groups (n = 14, 11, and 10 respectively). Different inhaled oxygen concentrations and different timings of oxygen delivery were assigned. Except for control all fetal rats were rendered ischemic and hypoxic in utero by interrupting the placental circulation. After re-circulation, intra- and extra-cellular concentrations of calcium, sodium, and potassium in the brains were measured for each individual group. RESULTS: The mean intracellular free calcium concentration of fetal rat brains was similar for the room-air resuscitation group (552.1 +/- 93.5 nmol/L) and the group resuscitated with 92.8% oxygen (520.6 +/- 79.1 nmol/L) and both were significantly higher than in the control (315.3 +/- 86.9 nmol/L) (P < 0.001). After resuscitation with 65% oxygen, be it instituted before or immediately after hypoxia, their mean intracellular free calcium concentrations in the brain cells (441.5 +/- 47.9 and 452.9 +/- 36.4 nmol/L respectively) were significantly lower than those in the room-air resuscitation (P < 0.01) and 92.8% oxygen group (P < 0.05), though still higher than in the control (P > 0.05). There was no difference in the total concentrations of calcium, sodium, or potassium among all groups. CONCLUSIONS: Resuscitation with 92.8% oxygen or room air exerted a similar effect on the parameters measured, indicating that resuscitation of asphyxiated neonates using 100% oxygen might not be superior to using room air. Resuscitation with 65% oxygen resulted in lower cerebral intracellular calcium concentrations and might produce a better outcome than using 100% oxygen or room air.