TISSUE MICROARRAY EVIDENCE OF ASSOCIATION BETWEEN
p16 AND PHOSPHORYLATED eIF4E IN TONSILLAR
SQUAMOUS CELL CARCINOMA
Matthew G. Fury, MD, PhD,1Marija Drobnjak, MD,2Camelia S. Sima, MD, MS,3Marina Asher, BS,2
Jatin Shah, MD, PhD(Hon),4Nancy Lee, MD,5Diane Carlson, MD,2H. Guido Wendel, PhD,6
David G. Pfister, MD1
1Head and Neck Medical Oncology Service, Memorial Sloan–Kettering Cancer Center, New York, New York 10021. E-mail:
2Department of Pathology, Memorial Sloan–Kettering Cancer Center, New York, New York 10021
3Department of Epidemiology and Biostatistics, Memorial Sloan–Kettering Cancer Center, New York, New York 10021
4Department of Surgery, Memorial Sloan–Kettering Cancer Center, New York, New York 10021
5Department of Radiation Oncology, Memorial Sloan–Kettering Cancer Center, New York, New York 10021
6Cancer Biology and Genetics Program, Memorial Sloan–Kettering Cancer Center, New York, New York 10021
Accepted 12 August 2010
Published online 10 November 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/hed.21621
human papillomavirus (HPV)-related carcinogenesis in head
and neck cancer. The purpose of this study is to determine if
p16 immunoreactivity is associated with aberrant expression of
components of the PI3 kinase pathway.
Methods. A tissue microarray (TMA) was constructed for
46 archived tonsillar squamous cell carcinoma specimens.
Clinical demographics of these patients were analyzed, and
the TMA was interrogated with antibodies directed against
p16, phosphorylated AktSer473, phosphorylated S6Ser240/244,
phosphorylated eIF4ESer209, PTEN, p21, and p53.
Results. There was a significant correlation between history
of tobacco abuse (>10 pack/years) and absence of p16
expression (p ¼ .01). Expression of p16 was significantly asso-
ciated with immunoreactivity of p21 (p ¼ .02), PTEN (p ¼ .02),
and phosphorylated eIF4E (p ¼ .03). There was no evidence
of association between p16 status and expression of phospho-
rylated S6, phosphorylated 4E-BP1, or p53.
Conclusion. p16 positive tonsillar squamous cell carcinoma
is characterized by expression of phosphorylated eIF4E that
may occur via a mammalian target of rapamycin (mTOR)-inde-
pendent mechanism. V V
Neck 33: 1340–1345, 2011
Background. Expression of p16 is a marker for
C 2010 Wiley Periodicals, Inc. Head
Keywords: HPV; tonsil; p16; tissue microarray; mTOR
Aberrant signaling in the PI3K (phosphatidylinositol
3-kinase)/Akt pathway drives the malignant pheno-
type of many solid tumors, including head and neck
squamous cell carcinoma (HNSCC).1The Head and Neck
Cancer Tissue Array Initiative found that alterations in
this pathway were present in most head and neck
tumors. Specifically, levels of phosphorylated AktSer473,
phosphorylated AktThr308, and pS6 were significantly
higher in HNSCC tumors compared with normal mu-
cosa.2Mammalian target of rapamycin (mTOR) is a ser-
ine/threonine kinase that is an important downstream
regulator of the pathway.1,3mTOR inhibitors are an
attractive option for clinical study in HNSCC, in part due
to the observation of additive or synergistic activity
against some HNSCC cell lines when mTOR inhibitors
are combined with platinum or taxane agents.4
The development of mTOR inhibitors and other new
targeted therapies in HNSCC must take into account the
fact that HNSCC comprises at least 2 distinct clinical
entities: human papillomavirus (HPV)-positive tumors
and HPV-negative tumors.5Approximately half of oro-
pharyngeal (tonsil, base of tongue) squamous cell carci-
nomas are HPV-positive, but tumors arising at other
sites in the upper aerodigestive tract (e.g., oral cavity,
hypopharynx, or larynx) generally are not associated
with HPV.5HPV-negative tumors typically occur in indi-
viduals with histories of tobacco abuse, whereas HPV-
positive HNSCC usually is not associated with tobacco
abuse.6,7In a prospective randomized clinical trial of cis-
platin plus concurrent radiation in HNSCC, HPV-posi-
tive squamous cell carcinomas arising in the oropharynx
were associated with superior overall survival compared
with HPV-negative oropharyngeal tumors.8
Potentially, differences in the molecular pathoge-
nesis mechanisms of these tumors account for their
distinct treatment responsiveness. Landmark cytoge-
netic studies of HNSCC demonstrate frequent gene
loss at the locus encoding p16 and p14 (CDKN2A),
Correspondence to: M. G. Fury
Contract grant sponsor: This work was supported by a grant from
C 2010 Wiley Periodicals, Inc.
1340Association between p16 and Phosphorylated eIF4E in Tonsillar SCC HEAD & NECK—DOI 10.1002/hedSeptember2011
and amplification at the gene locus encoding cyclin
D1.9–11Whereas p16 loss and cyclin D1 amplification
characterize HPV-negative (tobacco-related) tumors,
p16 overexpression and downregulation of cyclin D1
typify HPV-positive tumors.12The high p16/low cyclin
D1 profile in HPV-positive tumors is due to loss of
negative feedback on p16 when the E7 viral oncopro-
tein inactivates the pRB tumor suppressor protein.5,13
Immunohistochemical detection of p16 is a marker for
HPV-associated carcinogenesis in HNSCC.14–16
We now wish to explore differences in expression
of PI3K pathway proteins according to p16 expression
status. Gene expression microarray analysis of 36
patients with HNSCC specimens (28 HPV-negative, 8
HPV-positive) found overexpression of the catalytic
subunit of PI3K in HPV-positive tumors, suggesting
that this pathway may be preferentially activated in
HPV-positive tumors.17A potentially important down-
stream signaling event in the PI3K pathway is phos-
phorylation of the eukaryotic translation initiation
factor eIF4E. One mechanism by which mTOR stimu-
lates protein translation is phosphorylation of the 4E-
binding protein (4E-BP1), which releases eIF4E from
the inactive4E-BP1 complex.3
enhances translation of mRNAs with 50untranslated
regions, which include transformation-related and
survival genes.18,19In a genetically engineered mu-
rine lymphoma model, eIF4E functions as an onco-
gene and confers resistance to therapeutic mTOR
inhibition.20,21Phosphorylation of eIF4E was essen-
tial for its oncogenic activity in this model.21In
patients with head and neck cancer, expression of
eIF4E in histologically negative surgical margins has
been associated with increased risk of tumor recur-
rence.22,23Expression of total eIF4E is detectable in
virtually all HNSCC tumors,22but expression of phos-
phorylated eIF4E is significantly more common in
In this retrospective study of tonsil squamous cell
carcinomas, we use a tissue microarray (TMA) to
explore potential associations between expression of
components of the PI3K/Akt/mTOR pathway and
HPV status, using p16 expression as a marker for
HPV positivity. We interrogated expression of phos-
phorylated S6 and 4E-BP1, which are well-character-
ized downstream components of the mTOR pathway.
We also interrogated expression of phosphorylated
eIF4E, because phosphorylation of eIF4E seems to be
essential for its oncogenic activity.21,24In view of the
prior report that overexpression of PI3K may be a
characteristic of HPV-positive HNSCC,17we propose
that p16 status may impact the expression of signal-
ing proteins downstream of PI3K.
MATERIALS AND METHODS
Tumor Material and Patient Data.
retrospective study was obtained from the local Insti-
Approval for this
tutional Review Board, Privacy Board, and Human
Biospecimen Utilization Committee. Formalin-fixed,
paraffin-embedded archival biopsy and surgical resec-
tion samples of 52 primary tonsil squamous cell carci-
identified in a query of the Pathlite database of the
Department of Pathology at Memorial Sloan–Ketter-
ing Cancer Center. Clinical and demographic data
were collected, including age at diagnosis, sex, smok-
ing history, and survival status. TNM staging was
determined by review of clinical notes, surgical notes,
pathology reports, and radiology reports.
Tissue Microarray Construction and Immunohisto-
In the Core Facility of the Department of
Pathology, Dr. M. Drobnjak supervised TMA construc-
tion. Typically, 0.6 mm core punches were trans-
ferred in triplicate to a recipient TMA block, as
previously described.21,25,26Table 1 provides details
about the antibodies used to stain the TMA for the fol-
lowing proteins: p16,12,27phosphorylated AktSer473,28
phosphorylated S6Ser240/244,20phosphorylated S6Ser235/
236,29phosphorylated eIF4ESer209,21phosphorylated 4E-
BP1Thr37/46,30PTEN,31p53,31and p21.32For each anti-
body, each tumor was analyzed in triplicate in the
TMA. Both the percentage of stained tumor cells (0% to
100%) and staining intensity (-, þ, þþ, or þþþ) were
scored. For p53 and p21, only nuclear staining was
scored as positive. For p16, phosphorylated AktSer473,
phosphorylated pS6Ser240/244, and PTEN, cytoplasmic
and/or nuclear staining were scored together for analy-
sis. For eIF4E, only cytoplasmic staining was scored for
components of the PI3K/Akt/mTOR pathway and p16,
several prior studies have defined positive expression
as approximately 30% of tumor cells with positive im-
munostaining.21,27,28In the current study, criteria for
positive expression was ?30% of tumor cells with nu-
clear and/or cytoplasmic staining (þ, þþ, or þþþ) for
all antibodies, except p53 and p21. Criteria for positive
expression of p53 and p21 were >10% of tumor cells
with nuclear staining (þ, þþ, þþþ).12Some studies
have used a lower threshold to define positive tumor
staining with antibodies directed against components of
types.31,33In addition to the main analysis, we per-
formed a sensitivity analysis using ?5% positive nu-
clear and/or cytoplasmic staining as criteria for positive
staining for phosphorylated AktSer473, phosphorylated
S6Ser240/244, phosphorylated S6Ser235/236, and phospho-
rylated 4E-BP1Thr37/46; ?5% cytoplasmic staining for
phosphorylated eIF4ESer209, and ?5% positive nuclear
staining for p53 and p21.
Associations of p16 status (positive for expression
vs negative for expression) with clinical variables
(tobacco smoking history) and with expression of
For antibodies directed against
Association between p16 and Phosphorylated eIF4E in Tonsillar SCC HEAD & NECK—DOI 10.1002/hed September20111341
other molecular markers (phosphorylated AKTSer473,
phosphorylated S6Ser240/244, phosphorylated S6Ser235/
236, phosphorylated eIF4ESer209, phosphorylated 4E-
BP1Thr37/46, PTEN, p53, and p21) were analyzed by
use of the 2-sided Fisher exact test.
For the entire study population, progression-free
survival and overall survival were estimated accord-
ing to the Kaplan–Meier method.
cell carcinomas were selected for construction of the
TMA. Six samples were excluded from the analysis
due to inadequate specimens for analysis in the TMA.
Regarding the 46 patients with tonsil squamous cell
carcinoma included in the analysis, the patient char-
acteristics are shown in Table 2. Most patients were
men (80%), and the median age at the time of diagno-
sis was 52 years old. Seventeen patients were never-
smokers, and 15 patients had a greater than 20 pack-
year tobacco history. The study population included
patients with stage II to stage IVB disease, with stage
IVA being the most common stage at diagnosis in this
group (67% of patients). With a median follow-up for
Initially, 52 tonsillar squamous
all patients of 3.5 years, progression-free survival was
91% and overall survival was 92%.
Tissue Microarray Results.
with antibody against p16, as a surrogate marker for
stained with antibodies directed against signaling mole-
AktSer473, phosphorylated S6Ser240/244, phosphorylated
S6Ser235/236, phosphorylated eIF 4ESer209, phosphorylated
4E-BP1Thr37/46, and PTEN) and against the p53/p21 DNA
damage response pathway. Table 1 provides additional in-
formation regarding antibodies used in the analysis. Fig-
ure 1 provides examples of positive and negative
immunostaining of p16 and eIF4E in the TMA.
As shown in Table 3, 30 tumors (65%) were posi-
tive for p16. Expression of components of the PI3K/
Akt/mTOR pathway (phosphorylated Akt, phosphoryl-
eIF4E) was common (Table 4). Low or absent PTEN
detectability was observed in 15 samples (33%).
Regarding mediators of DNA-damage response, most
tumors expressed p21 (Table 4).
The TMA was stained
The TMA was also
Expression of p16: Clinical and Pathological Corre-
Clinical and pathologic associations with p16
status were evaluated. There was a significant associa-
tion between history of tobacco abuse (>10 pack/years)
and absence of p16 expression (p ¼ .01; Table 3). Table 4
demonstrates the associations between p16 status and
the other cell cycle markers that were analyzed in this
TMA. p16 expression was significantly associated with
expression of p21 (p ¼ .02), PTEN (p ¼ .02), and phos-
phorylated eIF4E (p ¼ .03). There was a trend toward
significance regarding the association of p16 expression
and phosphorylated Akt expression (p ¼ .12). There was
no evidence of association between p16 status and
expression of phosphorylated 4E-BP1, phosphorylated
S6Ser235/236, phosphorylated S6Ser240/244, or p53.
For most of the cores in the TMA, immunostaining
with these antibodies was either diffusely negative or
was positive in the majority of cancer cells. There were
few cores in which the percentage of positively stained
cells was >5% but ?29%. We performed a sensitivity
Table 1. Primary antibodies used to interrogate the tissue microarray.
AntigenCloneIsotype Dilution Company, catalog no.
Cell Signaling, 3787
Cell Signaling, 2215
Cell Signaling, 2211
Cell Signaling, 9741
Cell Signaling, 2855
Abbreviations: IgG, immunoglobulin G; n/a, not applicable.
Table 2. Patient characteristics (n ¼ 46).
Median age, y (range)
Tobacco, no. of patients (%)
Stage, no. of patients (%)
Abbreviations: M, male; F, female.
Note: All patients had tonsillar squamous cell carcinoma. Four patients in this analy-
sis had tonsil squamous cell carcinoma with involvement of neighboring structures,
as follows: tonsil plus soft palate (n ¼ 2), tonsil plus base of tongue (n ¼ 1), and
tonsil plus pharynx (n ¼ 1).
Percentages are rounded to the nearest whole number.
1342Association between p16 and Phosphorylated eIF4E in Tonsillar SCC HEAD & NECK—DOI 10.1002/hedSeptember2011
analysis, which investigated alternative cutpoints for
the antibodies. Cutpoints for phosphorylated AktSer473,
phosphorylated S6Ser240/244, phosphorylated S6Ser235/236,
BP1Thr37/46, PTEN, p21, and p53 were all adjusted to
5%, and all of the associations with p16 were replicated
(data not shown). This indicates that our results were
not influenced by the selected cutpoint.
In this TMA consisting of 46 tonsillar squamous cell
carcinomas, we demonstrate that downstream compo-
nents of the PI3K pathway, such as phosphorylated
Akt and S6, are expressed in the majority of the
specimens, in agreement with the results of the Head
and Neck Cancer Tissue Array initiative.2Sixty-five
percent of the tumors express p16, a marker for HPV-
driven carcinogenesis.14–16We find that p16 expres-
sion is associated with expression of phosphorylated
eIF4E. In agreement with multiple reports regarding
HPV-status and tobacco history,6,7we find a signifi-
cant correlation between history of tobacco abuse
(>10 pack/years) and absence of p16 expression (p ¼
.01). Expression of p16 also correlates with expression
of p21 and with PTEN. There is a trend toward the
association between expression of p16 and phospho-
rylated Akt, but no association between expression of
p16 and phosphorylated S6 or phosphorylated 4E-
BP1. The 3.5-year median progression-free survival
and overall survival are 91% and 92%, respectively,
and there were not enough events to explore potential
survival associations with biomarkers in the TMA.
The results in this TMA add to the growing under-
standing that HPV-positive and HPV-negative head and
neck cancers are distinct entities at the molecular pa-
thology level. There are converse molecular profiles for
FIGURE 1. Representative examples of immunostaining of tonsillar squamous cell carcinoma in tissue microarray (TMA) cores. Posi-
tive immunostaining for anti-p16 (80%, þþþ cytoplasmic) (A) and anti-eIF4E (90%, þþþ cytoplasmic) (B) for subject 49. Negative im-
munostaining for anti-p16 (0%) (C) and anti-eIF4E (0%) (D) for subject 48.
Association between p16 and Phosphorylated eIF4E in Tonsillar SCC HEAD & NECK—DOI 10.1002/hed September 20111343
HPV-positive tumors (e.g., overexpression of p14 and
p16; downregulation of cyclin D1; p53 wildtype) and
HPV-negative tumors (e.g., overexpression of cyclin D1;
downregulation p14, p16,
tant),7,12,14,27,34,35and these molecular profiles can
inform the development of clinical studies. The salient
new finding here is the apparent association between
p16 and phosphorylated eIF4E.
Yarbrough et al17
reported that HPV-positive
tumors are associated with upregulation of the cata-
lytic subunit of PI3K. The current TMA results sug-
associated with p16/HPV status, because no associa-
tions were seen between p16 expression and phospho-
rylated 4E-BP1 or phosphorylated S6. The association
between p16 and phosphorylated eIF4E in the TMA
suggests the presence of an mTOR-independent mech-
anism of eIF4E phosphorylation, which has been pre-
viously described in non-small cell lung carcinoma
cell lines.36In A549 and H157 cell lines, adenovirus-
driven expression of the catalytic subunit of PI3K
was associated with enhanced eIF4E phosphoryla-
tion.37Therefore, we propose that HPV-positive head
and neck squamous cell tumors may be characterized
by overexpression of PI3K that leads to enhanced
phosphorylation of eIF4E, and that this occurs by a
mechanism that seems to be independent of PTEN
loss or mTOR activation.
Our results suggest that HPV/p16 status is not
associated with mTOR activation, but we cannot
exclude the possibility of such an association due to
the small sample size and the inherent limitation of
TMA regarding the issue of intratumoral heterogene-
ity. For example, expression in phosphorylated S6 in
HNSCC seems to be strongest at the invasive borders
of the tumor,2and our TMA analysis did explicitly
restrict analysis to the invasive borders of the tumors.
Similarly, it is unclear to what extent the immunohis-
tochemical detection of PTEN represents the func-
tional status of this molecule.
Our results are in agreement with the immuno-
histochemistry study of Hafkamp et al,12demonstrat-
immunopositivity in tonsillar squamous cell carci-
is unlikelyto be
p16 and p21
noma. The association between p16 and p21 expres-
sion may have implications for clinical research
because the mTOR inhibitor everolimus sensitizes tu-
mor cells to cisplatin by a mechanism that involves
downregulation of p21.38This observation led to the
initiation of a phase I clinical study of everolimus
plus low-dose cisplatin.39
The association between p16 and phosphorylated
eIF4E may reflect interaction between virus and cellu-
lar translational apparatus. In cells infected with her-
pes simplex virus-1 or vaccinia virus, phosphorylation
of eIF4E by mnk-1 is critical for viral protein synthesis
and replication.40,41It is not known if similar mecha-
nism occurs in HPV-positive HNSCC, but HPV infec-
tion has been associated with increased cap-dependent
translation in differentiating cervical cancer cell
lines.42A topic for further research in HNSCC is the
potential interplay between HPV and cap-dependent
mRNA translational machinery that might be amena-
ble to targeted therapeutic intervention.
The TMA provides strong evidence that p16/HPV
positivity is associated with expression of phosphoryl-
ated eIF4E and p21, but provides no evidence of pref-
HNSCC. The gene expression data of Yarbrough et
al17demonstrated that PI3K overexpression is com-
mon in HPV-positive HNSCC, and PI3K overexpres-
expression of phosphorylated eIF4E.37Future studies
Table 3. Associations between smoking history and anti-p16
p16 expression status
p value* PositiveNegative
Positive for tobacco
abuse, >10 pack-years
>0 but ?10 pack-years
9 11 .01
30 (65%)16 (35%)
*p value determined by Fisher exact test.
Table 4. Associations between anti-p16 and 6 other antibodies.
Abbreviations: Pos, positive; Neg, negative.
*p values determined by Fisher exact test.
Note: For the following antibodies, tumor sample was missing from the TMA for one
patient each: p21, p53, phosphorylated S6Ser240/240. Tumor sample was missing
from the TMA for 2 patients each for: phosphorylated 4E-BP1Thr37/46and phospho-
1344 Association between p16 and Phosphorylated eIF4E in Tonsillar SCC HEAD & NECK—DOI 10.1002/hedSeptember 2011
should address the mechanism that leads to eIF4E Download full-text
phosphorylation in p16-positive HNSCC. We feel that
inhibitors of PI3K and/or eIF4E are appropriate can-
didates for the next generation of clinical studies in
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