Screening of the copy number increase of AKT in lung carcinoma by custom-designed MLPA.

Treatments for lung cancer include therapies targeting aberrant oncoproteins, but there remains a high medical need for novel therapies. Our previous studies showed that gene amplification/high-level polysomy of AKT1/2 occurs in more than 10% of lung carcinomas. Here, we describe multiplex ligation-dependent probe amplification analysis (MLPA) as a high-throughput method to evaluate copy number increases (CNIs) of AKT1/2 in lung carcinomas. The performance of MLPA using custom-made probes in formalin-fixed paraffin-embedded tissue was evaluated by comparing it to immunohistochemistry and fluorescence in situ hybridization analysis (FISH). By MLPA, we found 4 out of 30 samples harboring gene "gain" when the conventional cutoff value (> 1.3) was used. Two samples with gene amplification by FISH had MLPA values of 1.85 and 1.75, which were lower than the conventional cutoff for "amplification" (> 2.0). Moreover, samples with CNIs due to polysomy by FISH gave MLPA values between 1.13 and 1.47, so some samples had lower values than 1.3. The reasons appeared to be stromal contamination and the presence of carcinoma cells without CNIs. However, when we changed the cutoff for "gain" to the "average+2xstandard error", we detected CNIs in 10 samples, with only one each of false-positive and false-negative results. The sensitivity was 90% and the specificity was 98%. Consistently, all cases exhibiting CNI by this criteria revealed Akt activation. In conclusion, MLPA implemented with custom-made probes and an optimized cutoff value is a feasible screening method to semi-quantitatively detect oncogene aberrations, and may contribute to the design of individualized, molecularly targeted therapies against lung carcinoma.

Asymmetric Total Synthesis of Kopsiyunnanine K, a Monoterpenoid Indole Alkaloid with a Rearranged Skeleton.

A new monoterpenoid indole alkaloid, kopsiyunnanine K, was isolated from Kopsia arborea. Its intriguing rearranged structure and absolute configuration, which were inferred from spectral data and a possible biosynthetic pathway, were determined on the basis of a 13-step asymmetric total synthesis.