Targeted next-generation sequencing of TP53 in oral tongue carcinoma from non-smokers

Abstract

Background
Little is known regarding the etiology and genomic underpinnings of Oral Tongue Squamous Cell Carcinoma (OTSCC) in patients who lack traditional risk factors, yet the incidence is increasing. In particular, the rate, and role, of TP53 mutations in this cohort has been heavily debated in the literature. Methods
Tumor DNA from forty-three non-smokers with OTSCC underwent next generation sequencing of TP53. ResultsSixty percent of samples contained a TP53 mutation. The G > T transversion rate was 5.7 %. TP53 status did not differ by age. ConclusionsOTSCC in non-smokers have TP53 mutation rates similar to other Head and Neck cancers yet these mutations do not appear related to carcinogen exposure based on the mutational spectrum and clinical history. The mechanisms driving tumorigenesis in this cohort, including mutations in TP53, remain elusive and further studies are needed.

Full text

S H O R T R E P O R T Open Access
Targeted next-generation sequencing of
TP53 in oral tongue carcinoma from non-
smokers
Daniel L. Faden
1*
, Sarah T. Arron
2
, Chase M. Heaton
1
, Joseph DeRisi
3
, Andrew P. South
4
and Steven J. Wang
1
Abstract
Background: Little is known regarding the etiology and genomic underpinnings of Oral Tongue Squamous Cell
Carcinoma (OTSCC) in patients who lack traditional risk factors, yet the incidence is increasing. In particular, the rate,
and role, of TP53 mutations in this cohort has been heavily debated in the literature.
Methods: Tumor DNA from forty-three non-smokers with OTSCC underwent next generation sequencing of TP53.
Results: Sixty percent of samples contained a TP53 mutation. The G > T transversion rate was 5.7 %. TP53 status did
not differ by age.
Conclusions: OTSCC in non-smokers have TP53 mutation rates similar to other Head and Neck cancers yet these
mutations do not appear related to carcinogen exposure based on the mutational spectrum and clinical history.
The mechanisms driving tumorigenesis in this cohort, including mutations in TP53, remain elusive and further
studies are needed.
Keywords: Oral tongue carcinoma, TP53
Introduction
Oral Tongue Squamous Cell Carcinoma (OTSCC) most
commonly occurs in older patients who smoke and
drink. The incidence of OTSCC is decreasing in the
United States as the rates of tobacco use in the popula-
tion decrease. However, OTSCC in young patients (<45)
is increasing in incidence in the United States and does
not appear to be associated with the known risk factors
for head and neck squamous cell carcinoma (HNSCC)
including tobacco/alcohol abuse, infection with human
papilloma virus (HPV), or any other virus [1, 2]. Little is
known regarding the genomic underpinnings of OTSCC
in patients who lack traditional risk factors. It remains
unclear if this cohort is genomically and etiologically dis-
tinct from traditional OTSCC.
TP53 (p53) mutations are the most common muta-
tions in HNSCC and appear to play a pivotal role in
tumor initiation. The rate of p53 mutations in non-
smokers, particularly OTSCC in young patients, is highly
variable in the literature [1, 3, 4]. In order to help shed
light on this important question, we performed targeted
Next Generation Sequencing (NGS) on OTSCC samples
from forty-three non-smokers.
Methods
After histologic assessment, tumor DNA was extracted
from FFPE tissue sections (QIAamp DNA FFPE Tissue
Kit, Qiagen, Hilden, Germany) from forty-three patients
with surgically treated OTSCC. All patients were never
smokers and had no history of radiation exposure or
chemotherapy. 454 Pyrosequencing of p53 was per-
formed using the GS Junior system (Roche/454 Life
Sciences, Branford, CT) and Fluidigm (Fluidigm Corpor-
ation, San Francisco, CA) PCR amplicon libraries as
template according to recommended guidelines (Roche,
Mannheim, Germany). 454 variant detection required a
minimum of four supporting reads and a minimum vari-
ant allele frequency threshold of 0.1. Samples with
coverage less than 20 % at 10x were filtered out to en-
sure accurate mutation frequency calculations.
* Correspondence: Daniel.Faden@ucsf.edu
1
Department of Otolaryngology-Head and Neck Surgery, University of
California, 2380 Sutter St First Floor, San Francisco, CA 94115, USA
Full list of author information is available at the end of the article
© 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Faden et al. Journal of Otolaryngology - Head and Neck Surgery (2016) 45:47
DOI 10.1186/s40463-016-0160-4

Results
Thirty-six samples had adequate sequencing coverage
for inclusion (Additional file 1: Table S1). The mean age
was 58 years old. Eight patients were under 45. Forty-
five variants in p53 were identified in 22 different sam-
ples (60 % of samples) (Additional file 1: Table S2),
including the common polymorphism Pro72Arg. Thirty-
four of these variants (53 % of samples) were known or
predicted to be deleterious. The rate of deleterious mu-
tations in patients <45 and >45 was 50 % and 58 %, re-
spectively. The mutational spectrum in order of
frequency was the following: G > A 45.7 %, T > G 20.0 %,
T > C14.2 %, G > C 11.4 %, G > T 5.7 %, T > A 2.8 %. Six
samples contained a missense mutation at codon 382
(Lys382Asn).
Discussion
OTSCC in non-smokers, particularly young patients, is
an intriguing cohort as they do not have the traditional
HNSCC risk factors, and are increasing in incidence in
the United States [1, 2]. Many of these tumors also
appear to lack key driver mutations present in other
HNSCC, suggesting they are more than just epidemio-
logically distinct. Little is known regarding the genomic
and etiologic underpinnings of these tumors. Highlight-
ing this paucity of knowledge is the considerable vari-
ability in reported mutation rates of known cancer
causing genes, particularly p53.
p53 functions as a powerful regulator of the cell cycle
and apoptosis and is the most commonly mutated gene
in HNSCC (47–62 %) [5, 6]. In OTSCC, reported p53
mutation rates are highly variable ranging from 20–94 %
[1, 3, 4]. Some of this variability may be related to muta-
tion identification techniques that either under call
(Sanger Sequencing of limited high yield exons) or over
call (WES without mutation validation) mutations.
Other reasons include small cohort sizes, imprecise pa-
tient stratification and heterogeneity within the cohorts.
Our results suggest that the rate of p53 mutations in
OTSCC from non-smokers is similar to other HNSCC,
including OTSCC from smokers, with 53 % of samples
having a deleterious mutation. If anything, our data may
under call the true mutation rate as some select exons
had sub-optimal sequencing coverage due to poor DNA
quality. Interestingly, six samples contained a common
mutation (Lys382Asn). Evidence suggests that acetyl-
ation of Lys 382 is required for P53 activation and tran-
scriptional activity [7].
p53 mutations in most tobacco/alcohol associated
HNSCC are assumed to be directly related to DNA
damage from carcinogen exposure. Examination of the
mutational spectrum of a tumor can help elucidate the
etiology of mutations. Smoking associated mutations
tend to have a preference for G > T (C > A) transversions
[8]. Our data show a low (6 %) G > T transversion rate,
consistent with other non-smoking related cancers out-
side the head and neck, and with findings in other oral
tongue cohorts [4, 8]. One caveat to consider when
assessing the mutational spectrum is that DNA extracted
from FFPE can be susceptible to cytosine deamination to
uracil as well as 5 methyl cytosine deamination to thy-
mine, which can lead to artefactual C:G > T:A mutations
during PCR amplification. Therefore, the percentage of
C > T changes could reflect some degree of artifact and
should be interpreted knowing this. None-the-less, our
data suggests that while p53 mutations in non-smokers
with OTSCC are as prevalent as other HNSCC, and
likely vital to tumorigenesis, the etiology of these muta-
tions remains unclear.
Conclusion
While the overall rate of HNSCC and OTSCC is de-
creasing in the United States, the rate of OTSCC in
young patients who do not smoke is increasing. Whether
these cancers represent simply an epidemiologically dis-
tinct cohort or if they represent a genomically and etio-
logically distinct cohort remains to be determined. Our
data suggests OTSCC in non-smokers have rates of p53
mutations similar to other HNSCC (~55 %) yet these
mutations do not appear related to carcinogen exposure
based on the mutational spectrum and clinical history.
The mechanisms driving tumorigenesis in this cohort,
including mutations in p53, remain elusive. Further
studies are needed to elucidate the processes driving
tumor development.
Additional file
Additional file 1: Table S1. Demographic data for patient samples
included in analysis (coverage >20 % at 10x). Table S2. TP53 Mutations.
(DOCX 293 kb)
Abbreviations
OTSCC: Oral tongue squamous cell carcinoma; HNSCC: Head and neck
squamous cell carcinoma; HPV: Human papilloma virus; NGS: Next
generation sequencing
Acknowledgments
Not applicable.
Funding
Funding for this manuscript is from the personal research fund of SJW. There
is no external grant support.
Authors’contributions
Conception and design of study: DLF, STA, CMH, AS, JD, SJW. Acquisition of
data, or analysis and interpretation of data: DLF, STA, CMH, AS. Drafting or
revision of manuscript: DLF, STA, CMH, AS, JD, SJW. All authors read and
approved the final manuscript.
Faden et al. Journal of Otolaryngology - Head and Neck Surgery (2016) 45:47 Page 2 of 3

Availability of data and materials
All raw data is stored in a repository at UCSF and is available by request to
the corresponding author, DLF. Processed mutation information and patient
demographics are available in the Supplemental Information.
Competing interest
None of the authors have any financial or personal relationships with other
people or organizations that could inappropriately influence (bias) the
authors’actions.
Consent for publication
Not applicable.
Ethics approval and consent to participate
Approval for this study and for sample collection was provided by the
Institutional Review Board of the University of California, San Francisco
(IRB# 11-05565).
Author details
1
Department of Otolaryngology-Head and Neck Surgery, University of
California, 2380 Sutter St First Floor, San Francisco, CA 94115, USA.
2
Department of Dermatology, University of California, San Francisco, CA, USA.
3
Department of Biochemistry and Biophysics, University of California, San
Francisco, CA, USA.
4
Department of Dermatology and Cutaneous Biology,
Thomas Jefferson University, Philadelphia, PA, USA.
Received: 9 August 2016 Accepted: 5 September 2016
References
1. Li R, Faden DL, Fakhry C, et al. Clinical, genomic, and metagenomic
characterization of oral tongue squamous cell carcinoma in patients who
do not smoke. Head Neck. 2015;37:1642–9.
2. Patel SC, Carpenter WR, Tyree S, et al. Increasing incidence of oral tongue
squamous cell carcinoma in young white women, age 18 to 44 years.
J Clin Oncol. 2011;29:1488–94.
3. Heaton CM, Durr ML, Tetsu O, van Zante A, Wang SJ. TP53 and CDKN2a
mutations in never-smoker oral tongue squamous cell carcinoma.
Laryngoscope. 2014;124:E267–273.
4. Pickering CR, Zhang J, Neskey DM, et al. Squamous cell carcinoma of the
oral tongue in young non-smokers is genomically similar to tumors in older
smokers. Clin Cancer Res. 2014;20:3842–8.
5. Agrawal N, Frederick MJ, Pickering CR, et al. Exome sequencing of head and
neck squamous cell carcinoma reveals inactivating mutations in NOTCH1.
Science (New York, NY). 2011;333:1154–7.
6. Stransky N, Egloff AM, Tward AD, et al. The mutational landscape of head and
neck squamous cell carcinoma. Science (New York, NY). 2011;333:1157–60.
7. Gu W, Roeder RG. Activation of p53 sequence-specific DNA binding by
acetylation of the p53 C-terminal domain. Cell. 1997;90:595–606.
8. Pfeifer GP, Denissenko MF, Olivier M, Tretyakova N, Hecht SS, Hainaut P.
Tobacco smoke carcinogens, DNA damage and p53 mutations in smoking-
associated cancers. Oncogene. 2002;21:7435–51.
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