The study protocol was in accordance with the ethical guidelines of the 1995 Declaration of Helsinki and was approved by independent ethics committees at Daping Hospital, Third Military Medical University. Tubacin clinical Writ ten informed consent was obtained from the patient for the publication of this report and any accompanying images. DNA isolation Mutation analysis of these genes was performed by ex traction of genomic DNA from formalin fixed, paraffin embedded tissue slides or sections with the use of the QIAamp DNA FFPE Tissue Kit, according to the manufacturers instruction. Tumor DNA was isolated from areas which were selected under light microscopic control by three senior pathologists and which containing at least 70% tumor cells in paraffin embedded tumor samples.

Mutation detection EGFR, KRAS and PIK3CA mutation analysis Mutational analysis was performed as described previously. For EGFR mutations analysis, we used the EGFR Scor pions kit, which combines Scor pions amplification refractory mutation system and Scorpions technology, to detect Inhibitors,Modulators,Libraries mutations in Real time Polymerase Chain Reaction reactions. Mutant KRAS in exon 2 was detected using a validated KRAS mutation kit Inhibitors,Modulators,Libraries that identifies seven somatic mutations located in codons 12 and 13 using allele specific Real Time PCR. PIK3CA mutations in exons 9 and 20 were Inhibitors,Modulators,Libraries detected using a validated PIK3CA mutation kit that identifies three somatic mutations by Real Time PCR based on ARMS and Scorpion technology. All the analysis of these genes mutations were performed in an ABI Prism 7700 se quence detector. SDS2.

0 software was performed for data analysis ac cording to the manufacturers instructions. Each sample was analyzed Inhibitors,Modulators,Libraries in triplicate or duplicate. PTEN mutation analysis PTEN mutations in exons 5, 6 and 8 were evaluated using a method previously published. High resolution melt ing analysis was performed on genomic DNA prepared from scraped paraffin slides. Two round PCR was done using 6 primer sets covering three exons of the PTEN gene. The following primer sets for Exon 5 were used, PTEN F. All exons were ampli fied with the following PCR conditions, pretreatment at 94 C for 4 minutes, 35 cycles of amplification, and a single 10 minute final extension procedure. Each of these 35 cy cles consisted of a denaturing step at 94 C for 1 minute, an Inhibitors,Modulators,Libraries annealing step of one minute, and an extension step at 72 C for 1 minute.

After the final extension, an additional denatur ation step at 95 C for 30 s was carried out. Subsequently, the PCR products were briefly centrifuged and were used directly for high resolution melting using the LightScanner instrument. LightScanner analyt ical software with Call IT 2. 0 was performed for data analysis according to the tech support manufac turers instructions. All HRM assays were replicated two to three times for each sample. Statistical analysis All the data were processed using SPSS13. 0 software.