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Original Article |

Frequent Mutations in TP53 and CDKN2A Found by Next-Generation Sequencing of Head and Neck Cancer Cell Lines

Anthony C. Nichols, MD; John Yoo, MD; David A. Palma, MD, PhD; Kevin Fung, MD; Jason H. Franklin, MD; James Koropatnick, PhD; Joe S. Mymryk, PhD; Nizar N. Batada, PhD; John W. Barrett, PhD
Arch Otolaryngol Head Neck Surg. 2012;138(8):732-739. doi:10.1001/archoto.2012.1558.
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Objective  To conduct high-throughput mutational analysis in 6 commonly used head and neck cancer cell lines. Comprehensive mutation analysis of primary head and neck squamous cell carcinoma (HNSCC) tumors has recently been reported, and mutations in the NOTCH receptors, TP53 and CDKN2A, were key findings. Established cell lines are valuable tools to study cancer in vitro. Similar high-throughput mutational analysis of head and neck cancer cell lines is necessary to confirm their mutational profile.

Design  DNA was extracted from American Type Culture Collection (ATCC) cell lines Cal27, Detroit562, FaDu, SCC4, SCC15, and SCC25. Cell line identity was confirmed by short tandem repeat (STR) analysis, and human papillomavirus (HPV) infection status was assessed by real-time polymerase chain reaction. A total of 535 cancer-associated genes were sequenced through a limited exome capture on the Illumina HiSeq system.

Setting  London Regional Cancer Program.

Results  The identity of the 6 cell lines was confirmed by STR analysis, and all lines tested negative for HPV infection. We achieved an average of 129-fold coverage with paired-end 100 base-pair reads. Sequencing revealed an average of 38 damaging mutations in each cell line (range, 30-45). The TP53 mutations, predicted to confer loss of function, were noted in all cell lines, and damaging CDKN2A mutations were found in all lines except SCC15.

Conclusions  High-throughput sequencing of head and neck cancer cell lines revealed similar mutations to those observed in primary tumors. Thus, these lines reflect the tumor biology of HNSCC and can serve as valuable models to study HNSCC in vitro.

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Figures

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Grahic Jump Location

Figure. Schematic diagrams of mutations in selected head and neck squamous cell carcinoma genes. The features illustrated were identified using the Conserved Domain Architecture Retrieval Tool (CDART) and the Conserved Domain Database (CDD) at the National Center for Biotechnology Information (NCBI). A, Multiple mutations were observed in NOTCH2 and NOTCH3 in numerous cell lines. In contrast, only a single cell line exhibited a damaging single-nucleotide polymorphism (SNP) in PIK3CA. Epidermal growth factor (EGF) repeats, Lin-12/Notch repeats (LNR), and ankyrin (ANK) repeats are found in multiple copies within the NOTCH proteins. NOD and NODP are members of a superfamily found in most NOTCH proteins. The region rich in proline (P), glutamic acid (E), serine (S), and threonine (T) makes up the PEST domain. The p85-binding domain (p85), ras-binding domain (RBD) and catalytic domain (Kinase) of PIK3CA are indicated. The C2 and helical domains of PIK3CA are superfamily members with structural functions. B, Cell lines exhibited SNP mutations in TP53 and CDKN2A that were predicted to be damaging by altering the protein structure. Arrowheads indicate position of observed point mutations. Numbers indicate length in amino acids. The transcription activation domain (TAD) and tetramerization (TETRA) domain of p53 are illustrated.

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