Large scale, prospective screening of EGFR mutations in the blood of advanced NSCLC patients to guide treatment decisions.

BACKGROUND: In a significant percentage of advanced non-small-cell lung cancer (NSCLC) patients, tumor tissue is unavailable or insufficient for genetic analyses. We prospectively analyzed if circulating-free DNA (cfDNA) purified from blood can be used as a surrogate in this setting to select patients for treatment with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs). PATIENTS AND METHODS: Blood samples were collected in 119 hospitals from 1138 advanced NSCLC patients at presentation (n = 1033) or at progression to EGFR-TKIs (n = 105) with no biopsy or insufficient tumor tissue. Serum and plasma were sent to a central laboratory, cfDNA purified and EGFR mutations analyzed and quantified using a real-time PCR assay. Response data from a subset of patients (n = 18) were retrospectively collected. RESULTS: Of 1033 NSCLC patients at presentation, 1026 were assessable; with a prevalence of males and former or current smokers. Sensitizing mutations were found in the cfDNA of 113 patients (11%); with a majority of females, never smokers and exon 19 deletions. Thirty-one patients were positive only in plasma and 11 in serum alone and mutation load was higher in plasma and in cases with exon 19 deletions. More than 50% of samples had <10 pg mutated genomes/µl with allelic fractions below 0.25%. Patients treated first line with TKIs based exclusively on EGFR positivity in blood had an ORR of 72% and a median PFS of 11 months. Of 105 patients screened after progression to EGFR-TKIs, sensitizing mutations were found in 56.2% and the p.T790M resistance mutation in 35.2%. CONCLUSIONS: Large-scale EGFR testing in the blood of unselected advanced NSCLC patients is feasible and can be used to select patients for targeted therapy when testing cannot be done in tissue. The characteristics and clinical outcomes to TKI treatment of the EGFR-mutated patients identified are undistinguishable from those positive in tumor.

Zinc-imidazole positive: a new method for DNA detection after electrophoresis on agarose gels not interfering with DNA biological integrity.

Nucleic acids separated by gel electrophoresis are commonly detected within the gel matrix with ethidium bromide staining, followed by gel irradiation with ultraviolet (UV) light. When the separated nucleic acids are to be recovered for further characterization or use, this methodology is unsuitable (i) because a significant number of chemical lesions to the nucleic acid molecules are caused, heavily compromising their biological activity, and (ii) because of health hazards due to accumulative direct contact with ethidium bromide and exposure to UV-light. As an alternative, for preparative purposes, a new nontoxic detection method employing zinc and imidazole salts is described. After electrophoresis, the gel is first washed in distilled water to substantially remove remaining electrophoresis reagents, then incubated in 40 mM zinc sulfate for 10 min to allow binding of Zn2+ to the DNA, and subsequently washed with distilled water to remove unbound Zn2+ from gel regions devoid of DNA. On soaking in 0.2 M imidazole for a few minutes, zinc-DNA complexes are visualized as deep-white (positive) stained bands against a slightly opaque background. The sensitivity is similar to that of ethidium bromide. Gels can be kept in distilled water for months without loss of staining. After zinc chelation, e.g. with EDTA, it is feasible to quantitatively recover chemically intact and biologically active DNA from the gels, as shown by reelectrophoresis and transformation experiments.

Negative staining with zinc-imidazole of gel electrophoresis-separated nucleic acids.

Zinc and imidazole salts were applied for the detection of nucleic acids on either polyacrylamide of agarose gels. After electrophoresis, polyacrylamide gels are washed in distilled water to remove most of the residual electrophoresis reagents, then incubated in 10 mM zinc sulfate for 10 min, and subsequently immersed in 0.2 M imidazole for 3 min. As a result, zinc salts precipitate on the gel surface, except in the positions occupied by nucleic acids, which appear as transparent, colorless bands. Staining of nucleic acids on agarose gels can be performed by incubation in 40 mM zinc sulfate for 10 min, followed by immersion in 0.2 M imidazole for 5 min to form a deep white-stained background. On soaking in 2 M imidazole for 45 min, the imidazole-induced zinc precipitate is removed from the positions were nucleic acids are located resulting in a negative image of colorless and transparent nucleic acid bands against a white background. The sensitivity of this stain ranges from 5 to 7 ng/band for small (from 1 to 0.2 kbp) DNA, from 7.8 to 13 ng/band for different 22-base oligonucleotides, from 62 to 125 ng/band for large (from 20 to 2 kbp) DNA, and is 1 microgram/band for human peripheral-blood monocyte RNA. After chelation of zinc with EDTA, the nucleic acids can be quantitatively recovered from the gel. The principal advantage of this technique over ethidium bromide staining is evident for preparative purposes. Using zinc-imidazole in the detection of purified pBACIB.1 (2.8 kbp) plasmid DNA and anti-HBsAg single chain Fv antibody fragment (0.7 kbp) DNA, followed by elution from gel slices, ligation and transformation of competent E. coli XL-1 Blue cells, the number of transformants notably increased from 280 (obtained with conventional ethidium bromide staining plus UV-irradiation at 312 nm) to 10,000.