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, 122 (5), 1189-94

Novel Mutant-Enriched Sequencing Identified High Frequency of PIK3CA Mutations in Pharyngeal Cancer

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Novel Mutant-Enriched Sequencing Identified High Frequency of PIK3CA Mutations in Pharyngeal Cancer

Wanglong Qiu et al. Int J Cancer.

Abstract

We previously reported 4 PIK3CA mutations in 38 head and neck cancer samples, 3 of which were identified in 6 pharyngeal cancer samples. To determine the mutation frequency of PIK3CA in pharyngeal cancer, we studied 24 additional cases of pharyngeal squamous cell carcinoma in this study. Using both direct genomic DNA sequencing and novel mutant-enriched sequencing methods developed specifically for the 3 hot-spot mutations (H1047R, E545K and E452K) of PIK3CA, we detected 5 mutations of PIK3CA in the 24 pharyngeal cancers (20.8%). Three of the 5 mutations had been missed by the conventional sequencing method and were subsequently detected by novel mutant-enriched sequencing methods. We showed that the mutant-enriched sequencing method for the H1047R hot-spot mutation can identify the mutation in a mixed population of mutant and wild-type DNA sequences at 1:360 ratios. These novel mutant-enriched sequencing methods allow the detection of the PIK3CA hot-spot mutations in clinical specimens which often contain limited tumor tissues (i.e., biopsy specimens). The data further support that oncogenic PIK3CA may play a critical role in pharyngeal carcinogenesis, and the mutant-enriched sequencing methods for PIK3CA are sensitive and reliable ways to detect PIK3CA mutations in clinical samples. Because PIK3CA and its pathway are potential targets for chemotherapy and radiation therapy, and frequent somatic mutation of PIK3CA has been identified in many human cancer types (e.g., breast cancer, colorectal cancer), the abilities to detect PIK3CA mutations with enhanced sensitivities have great potential impacts on target therapies for many cancer types.

Figures

Fig. 1
Fig. 1. The schematic of the mutant-enriched sequencing methods for detecting PIK3CA mutations, H1047R, E545K and E542K
A. The protocol for detectingPIK3CA mutation H1047R. Enzyme BsaBI specifically cuts the wild-type sequences of the exon 20, but not the mutant copies with A3140G nucleotide alteration. After digesting the first PCR product with the enzyme BsaBI, the second PCR selectively amplifies the mutant copies. B. A unique restriction enzyme site Hpy188I was introduced by mismatch PCR for detecting PIK3CA mutation E545K. The mismatch primer (PIK-E9MF) has two A→ T nucleotide substitutions in the forward primer to create a unique enzyme site Hpy188I in the wild-type sequences of the PIK3CA exon 9, but not the mutant sequences. C. The mismatch primer PIK-2E9MR was designed to create a unique restriction enzyme site EcoRI for enriching PIK3CA mutation E542K (G1624A) and E542G (A1625G) with the similar strategy for the hot-spot mutation E545K.
Fig. 2
Fig. 2. Two mutations of PIK3CA were identified in 24 pharyngeal cancer samples using conventional DNA sequencing
A. Hot-spot mutation G1633A (E545K) of PIK3CA gene causes a change in amino acid codon from 545 GAG (glutamic acid) to AAG (lysine). B. A point mutation at PIK3CA exon 20 nt 3127A→G leads to codon substitution from 1043 ATG (Met) to GTG (Val).
Fig. 3
Fig. 3. The detection of PIK3CA hot-spot mutations by mutant-enriched sequencing
A1–2. The sensitivity of the mutant-enriched sequencing protocol for the exon 20 H1047R mutation was investigated in head and neck cell line Detroit 562, whose genome had been reported to harbor a H1047R mutation . A1. Both wild-type A and mutant G peaks were detected at 1:1 ratio as expected by conventional genomic sequencing of the cell line Detroit 562 DNA (non-diluted). A2. When the ratio of mutant and wild-type DNA reached 1: 360, the mutant G peak was still the only peak detected in the cell line Detroit 562 DNA by mutant-enriched sequencing. B1–2. A patient sample with a known E545K mutation was used to test the mutant-enriched sequencing protocol for the exon 9 E545K mutation. B1. Antisense sequencing of the mutated site by the conventional genomic sequencing (marked by the black arrow) detected both the wild-type and the mutant alleles. B2. The peak representing the wild-type allele in the same sample disappeared when the mutant-enriched sequencing method was applied (indicated by the black arrow). The red arrow marks the nucleotide A1630T change that was introduced by mismatch primer (PIK-E9MF) in order to generate the restriction enzyme Hpy188I (TCNGA) site. C1–4. The mutant-enriched sequencing method identified an undetected PIK3CA hotspot mutation E542K (G1624A) by the conventional sequencing. C1. Forward sequencing of a clinical sample with a known E542K mutation by the conventional genomic sequencing method (marked by the black arrow) displayed a dominant presence of the wild-type allele over the mutant allele. C2. The wild-type allele in the same sample disappeared when the mutant-enriched sequencing method was applied (indicated by the black arrow). The red arrow marks the nucleotide A1627T change that was introduced by mismatch primer (PIK-2E9MF) to generate a unique restriction enzyme EcoRI (GAATTC) site. The case of pharyngeal cancer that had been tested negative for a mutation by the conventional genomic sequencing method (C3), but was subsequently identified with a PIK3CA E542K mutation using the mutant-enriched sequencing method (C4).

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