PIK3CA mutations in nasopharyngeal carcinoma

Dear Sir, By molecular cytogenetic studies, we have previously identified high frequency of copy number gain or amplification on chromosomes 3q (70%) in nasopharyngeal carcinoma (NPC), a prevalence cancer in Hong Kong and Southern China. The PIK3CA gene at 3q26.32 was found to be one of the candidate oncogenes in this cancer. Amplification and overexpression of PIK3CA were frequently detected in NPC. PIK3CA encodes for the p110 catalytic subunit of the phosphatidylinositol 3-kinase (PI3K), which is involved in the cell signaling through catalyzing the production of the phosphatidylinositol 3,4,5-triphosphate (PIP3) from phosphatidylinositol 4,5 bisphosphate. The gene is important in regulating the cell growth and cell survival, and its activation was proposed to play a role in oncogenesis. Studies have suggested PIK3CA as an oncogene in several cancers, such as ovarian cancer and cervical cancer, being activated via gene amplification. Strikingly, somatic mutations of PIK3CA were recently discovered in a large-scale mutational analysis in different cancers, including 25–30% of colorectal cancers, gastric cancers and brain tumors. Other studies also showed PIK3CA mutation in about 20–40% of breast and colon cancers, and small portion (6%) of ovarian cancers. These mutations were single nucleotide substitution mutation or deletion mainly found in 2 ‘‘hot spots’’ locating at the helical (exon 9) and kinase (exon 20) domains. Furthermore, the in vitro studies demonstrated that the PIK3CA mutant (H1047R) had increased kinase activity, which is a gain-of-function mutation. This indicated that PIK3CAmutation might be an important mechanism in activating the kinase and drive the signaling pathway to increase cell growth and survival. In the present studies, we have performed mutational analysis on NPC samples to determine whether PIK3CA mutations also involve in NPC tumorigenesis. Mutation analysis of all 20 exons of PIK3CA gene was performed in a normal nasopharyngeal outgrowths (NP4), 4 NPC cell lines (C6661, HK1, CNE2 and HONE1) and 2 xenografts (Xeno-2117 and Xeno-NPC8). The primers and conditions for PCR amplification and DNA sequencing are as previously described. Four pairs of primers for exon 20 were newly designed for DNA from paraffin-embedded tissue. The primers sequences are as follows: 20a (F: 50-ACTCCAAAGCCTCTTGCTCA-30; R: 50-TTTGCTCCAAACTGACCAAAC-30), 20b (F: 50-CAGAGTGAGCTTTCATTT TCTCA-30; R: 50-ACATTCGAAAGACCCTAGCC-30), 20c (F: 50-ATGCTGTTCAT GGATTGTGC-30; R: 50-GGACAACAAAAATGGATTGGA-30) and 20b (F: 50-TTT GGACTTAAGGCATAACATGAA-30; R: 50ACTCTCAGCAGGCAAAGACC-30). The PCR products were purified and sequenced by using the BigDye terminator method (Applied Biosystems; Foster City, CA) on the ABI PRISM 3100 autosequencer. By direct DNA sequencing, we have found 2 candidate base substitutions at exon 13 (G2047C and C2164G) locating at the helical domain of PIK3CA in all 6 NPC cell lines and xenografts. These 2 candidate base substitutions were not found in published human single nucleotide polymorphism (SNP) database. To confirm whether these sequence changes are common somatic mutations or SNPs of PIK3CA, we have also examined the sequence of exon 13 in the normal nasopharyngeal epithelial outgrowths and 4 normal peripheral blood samples by DNA sequencing (Fig. 1a). However, the 2 candidate base substitutions were detected in all nasopharyngeal and blood samples. The results imply that the base substitutions are not sporadic mutations and also not likely to be common SNPs. By blasting the sequence of exon 13 of PIK3CA with the human genome through NCBI (http://www.ncbi.nlm.nih.gov/BLAST/), we found that the 172 bp sequence of exon 13 of PIK3CA shows high homology (98%) with a genomic fragment in the Cat Eye Syndrome region, clone: KB67B5 (AP000547) at chromosome 22q11.2. It is interesting that only 2 base pair differences were detected between the sequences in exon 13 of PIK3CA and that in the Cat Eye Syndrome region, clone KB67B5 (Fig. 1b). The nucleotide differences were located at position 2047 (G) and 2164 (C) in exon 13 of PIK3CA. The sequences of corresponding positions in the Cat Eye Syndrome region, clone KB67B5 are 86710 (C) and 86827 (G), respectively. Further, comparing the genomic sequences of PIK3CA with that of Cat Eye Syndrome region, we found that a 6868 bp DNA fragment in the clone KB67B5 (AP000547) at 22q11.2 was almost identical (94% homology) to the genomic sequence from intron 8 to intron 13 of PIK3CA gene. The findings imply that the base substitutions identified in our NPC samples were only generated from the sequence of putative PIK3CA pseudogene at chromosome 22q11.2 during PCR amplification. Since the homology to the Cat Eye Syndrome region harboring the intron 8 to intron 13 of PIK3CA gene, it is difficult to avoid detecting the pseudogene by primer design. Similar observation has only been reported recently. The problems did not affect the results of the studies in which single strand conformation polymorphisms (SSCP) and other mutation screening methods were used for PIK3CA mutations screening. So, particular attention should be paid when investigating these regions by direct DNA sequencing. Further, confirmation of the mutations in mRNA samples of the tumors is recommended. Nevertheless, exon 13 mutation is rare in human cancers, according to recent reports. The only mutation H701P of exon 13 was found in 6.7% and 3.8% of glioblastoma