Download IGF-1R expression is associated with HPV

Carcinogenesis, 2015, Vol. 36, No. 6, 648–655
doi:10.1093/carcin/bgv053
Advance Access publication April 20, 2015
Original Manuscript
original manuscript
IGF-1R expression is associated with HPV-negative
status and adverse survival in head and neck squamous
cell cancer
Oliver T.Dale1,2,9, Tamara Aleksic1, Ketan A.Shah3, Cheng Han4, Hisham Mehanna5,
Davy C.M.Rapozo5, Jon D.H.Sheard6, Paul Goodyear7, Navdeep S.Upile7,
Max Robinson8, Terence M.Jones7, Stuart Winter2 and Valentine M.Macaulay1,4,*
1
Department of Oncology, University of Oxford, Old Road Campus Research Building, Oxford OX3 7LJ, UK, 2The Blenheim
Head and Neck Unit, Churchill Hospital, Oxford OX3 7LE, UK, 3Department of Cellular Pathology, Oxford University Hospitals
NHS Trust, John Radcliffe Hospital, Oxford OX3 9DU, UK, 4Oxford Cancer and Haematology Centre, Oxford University
Hospitals NHS Trust, Churchill Hospital, Oxford OX3 7LE, UK, 5Institute of Head and Neck Studies and Education (INHANSE),
University of Birmingham, Birmingham B15 2TT, UK, 6Department of Cellular Pathology, Aintree University Hospitals NHS
Foundation Trust, Liverpool L9 7AL, UK, 7Department of Molecular and Clinical Cancer Medicine, University of Liverpool,
Liverpool L3 9TA, UK and 8School of Dental Sciences, Newcastle University, Framlington Place, Newcastle upon Tyne NE2
4BW, UK
9
Present address: ENT Department, University Hospital Bristol, Bristol BS2 8EG, UK
*To whom correspondence should be addressed. Tel: +44 1865 617337; Fax: +44 1865 01865 617334; Email: [email protected]
Correspondence may also be addressed to Oliver T.Dale. Tel: +44 1173 425872; Fax: +44 117 3425117; Email: [email protected]
Abstract
Head and neck squamous cell carcinomas (HNSCC) are treated with surgery, radiotherapy and cisplatin-based
chemotherapy, but survival from locally-advanced disease remains poor, particularly in patients whose tumors
are negative for Human papillomavirus (HPV). Type 1 IGF receptor (IGF-1R) is known to promote tumorigenesis and
resistance to cancer therapeutics. Here, we assessed IGF-1R immunohistochemistry on tissue microarrays containing
852 cores from 346 HNSCC patients with primary tumors in the oropharynx (n = 231), larynx (85), hypopharynx
(28), oral cavity (2). Of these, 236 (68%) were HPV-negative, 110 (32%) positive. IGF-1R was detected in the cell
membrane of 36% and cytoplasm of 92% of HNSCCs; in 64 cases with matched normal tonsillar epithelium, IGF-1R
was overexpressed in the HNSCCs (P < 0.001). Overall survival (OS) and disease-specific survival (DSS) were reduced
in patients whose tumors contained high membrane IGF-1R [OS: hazard ratio (HR) = 1.63, P = 0.006; DSS: HR = 1.63,
P = 0.016], cytoplasmic IGF-1R (OS: HR = 1.58, P = 0.009; DSS: HR = 1.58, P = 0.024) and total IGF-1R (OS: HR = 2.02,
P < 0.001; DSS: HR = 2.2, P < 0.001). High tumor IGF-1R showed significant association with high-tumor T-stage
(P < 0.001) and HPV-negativity (P < 0.001), and was associated with shorter OS when considering patients with HPVpositive (P = 0.01) and negative (P = 0.006) tumors separately. IGF-1R was independently associated with survival in
multivariate analysis including HPV, but not when lymphovascular invasion, perineural spread and T-stage were
included. Of these factors, only IGF-1R can be manipulated; the association of IGF-1R with aggressive disease supports
experimental incorporation of anti-IGF-1R agents into multimodality treatment programs for HPV-negative and high
IGF-1R HPV-positive HNSCC.
Received: December 4, 2014; Revised: March 16, 2015; Accepted: March 21, 2015
© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected].
648
O.T.Dale et al. | 649
Materials and methods
Abbreviations
CI
DSS
HNSCC
HPV
HR
IGF
IGF-1R
IHC IRS
LVI
OS
PNS
TMA
confidence intervals
disease-specific survival
head and neck squamous cell carcinoma
human papillomavirus
hazard ratio
insulin-like growth factor
type 1 IGF receptor
Immunohistochemistry
immunoreactive score
lymphovascular invasion
overall survival
perineural spread
tissue microarray
Introduction
Head and neck squamous cell carcinomas (HNSCCs) arise most
commonly from the mucosa of the oral cavity, pharynx and larynx, with approximately 600 000 new cases each year worldwide (1). Risk factors for HNSCC include smoking, alcohol and
infection with Human papillomavirus (HPV). The association
between HPV and HNSCC is particularly strong for oropharyngeal tumors (2). The incidence of HPV positivity in HNSCC
has been rising over the last 30 years and is reported to range
from 10–70% in Europe and North America (3–5). One-third of
patients with HNSCC present with small node-negative tumors
(T1-2, N0) that are treated surgically or with radiotherapy,
whereas patients with loco-regionally advanced disease require
multimodality therapy including surgery, radiation therapy and
cisplatin-containing chemotherapy (6,7). It has become apparent that HPV-positive disease is a distinct clinical entity characterized by younger age, better performance status, greater
sensitivity to chemo-radiation and better prognosis than HPVnegative HNSCC (8).
Despite recent therapeutic advances, improvements in survival have been modest, particularly in advanced disease (9).
Outcomes are particularly poor for patients with HPV-negative
tumors, with median survival for HPV-negative versus HPVpositive oropharyngeal cancer of 20 versus 131 months, prompting a search for novel treatment targets (3). Epidermal growth
factor receptor copy number gain occurs in up to 58% of HNSCCs
and is associated with adverse prognosis and radio-resistance
(10,11). Selected patients may benefit from the combination of
radiotherapy with epidermal growth factor receptor antibody
cetuximab, although recent analysis suggests that outcomes are
not as good as for chemo-radiation (12,13). Insulin-like growth
factors (IGFs) are known to influence tumor biology, via activation of type 1 IGF receptor (IGF-1Rs). The IGF signaling axis is
required for cellular transformation and promotes tumorigenesis, and low IGF bioactivity is known to protect from cancer
development (14–17). Solid tumors often overexpress IGF-1Rs,
as a result of gene amplification or, more often, loss of tumor
suppressor gene function (18–21). IGF-1R overexpression is
associated with poor prognosis in several tumors including
non-small cell lung, oesophageal, gastric, pancreatic, colorectal,
renal and ovarian cancers (22–28). Two relatively small studies
have recently reported an association between adverse survival
and IGF-1R expression in HNSCC (29,30). However, it is unclear
to what extent variation in IGF-1R expression correlates with
other prognostic factors particularly HPV. Therefore, we aimed
to explore associations between IGF-1R and other clinico-pathological variables in a large cohort of HNSCC patients of known
HPV status.
Patients and tumor tissue
Samples of formalin-fixed, paraffin-embedded HNSCC tissue were obtained
from three centers in the UK. Included were patients undergoing primary
surgery for oropharyngeal squamous cell cancer at the John Radcliffe
Hospital, Oxford, UK between 1 January 2000 and 31 December 2010 (n = 97),
patients with oropharyngeal, laryngeal, hypopharygeal or oral HNSCC
treated with primary surgery between February 5 1998 and 13 January
2009 at Aintree University Hospitals NHS Foundation Trust, Liverpool, UK
(n = 186), and patients with oropharyngeal or oral SCC treated with primary
surgery or primary radiotherapy alone or with chemotherapy between 5
May 1999 and 4 October 2011 at the University Hospital of Coventry and
Warwick, UK (n = 63). Case note review was performed for all patients, in
order to record demographic details, tumor characteristics including TNM
(tumor, nodes, metastasis) stage (Union for International Cancer Control
7th edition), treatment details, pathological findings and survival outcomes.
Overall survival (OS) and disease-specific survival (DSS) were defined as the
time from primary treatment until death or last clinical contact.
Cores of 0.6–1 mm were taken from each archival sample of primary
tumor and embedded in paraffin to create tissue microarrays (TMAs)
using TMA Grand Master 3DHISTECH (Budapest, Hungary). HPV status
was determined by dual modality testing on formalin fixed tissue using
p16 immunohistochemistry and in situ hybridization for HPV DNA as
previously described (31). Immunostaining for p16INK4A was performed
using monoclonal antibody MTM-E6H4 (MTM Laboratories, Heidelberg,
Germany), and in situ hybridization for HPV DNA used Ventana INFORM
HPV probes (Ventana Medical Systems, Tucson, AZ), which detect common high-risk HPV genotypes including HPV-16. Tumors were defined as
HPV-positive when both p16 immunostaining and in situ hybridization for
HPV DNA were positive. Human tissue samples were used under National
Research Ethics approved study 07/H0606/120.
IGF-1R immunohistochemistry (IHC)
Freshly cut 4 μm sections of each TMA were dewaxed in Citroclear (TCS
Biosciences Ltd, UK) for 16 min and serially rehydrated in ethanol (100%,
80%, 70% and 50%) for 2 min each and distilled water for 5 min. Antigen
retrieval was performed as described (32) in Tris/EDTA buffer (Trisma
base 50 nM, EDTA 2 mM, pH 9) in a decloaking chamber (DC2002, Biocare
Medical) at 125°C for 2 min and 85°C for 10 min. Endogenous peroxidase
was blocked by incubation with 3% peroxide at room temperature for
10 min, and sections were blocked with 5% goat serum/5% bovine serum
albumin in phosphate-buffered saline for 1 h. Primary IGF-1R antibody
(#9750, Cell Signaling Technology) was applied at 1:50 dilution in 5% goat
serum/5% bovine serum albumin in phosphate-buffered saline for 16 h at
4°C. Bound antibody was detected using secondary antibody (Rabbit HRPPolymer, Menarini Diagnostics) for 20 min at room temperature followed
incubation with diaminobenzidene substrate (Envision) for 5 min at room
temperature. Sections were counter-stained with Mayer’s haematoxylin (Vector Laboratories), dehydrated in serial dilutions of ethanol (50%,
70%, 80% and 100%) and then in Citroclear for 5 min, and mounted using
DePex reagent (VWR International, UK). Each staining run included controls made by formalin-fixing, paraffin-embedding and sectioning pellets
of SKUT-1 leiomyosarcoma cells that are IGF-1R deficient, and MCF-7 cells
that overexpress IGF-1R, as described (32).
Cores were scored by two observers (O.T.D., K.A.S.) as described (33)
according to the intensity (0, no staining; 1, weak; 2, moderate; 3, heavy)
and percentage (0%, nil; 1: 1–10% of tumor stained; 2: 11–50%; 3: 51–80%;
4: 81–100%) of IGF-1R signal. This generated an immunoreactive score
(IRS, range 0–12) for membrane and cytoplasmic IGF-1R. The final IRS was
calculated as the mean IRS of all cores scored for each tumor, and total
IGF-1R IRS was calculated as the sum of mean membrane and cytoplasmic
scores for each tumor. The significance of differences in IGF-1R expression between paired samples of oropharyngeal tumor and benign epithelium was tested by paired t-test (two-tailed) using Prism v6 (GraphPad,
San Diego California, USA). Mann–Whitney U-Test (Prism v6) was used to
assess the significance of variation in IGF-1R expression with HPV status
and tumor T-stage. Kaplan–Meier, univariate and multivariate analyses
used Prism v6 and Stata package release 11.0 (Stata Corporation, TX).
650 | Carcinogenesis, 2015, Vol. 36, No. 6
Results
Surgical specimens from 346 HNSCC patients were sampled for
this study; their demographic details are shown in Table 1. The
tumors were from a variety of subsites within the head and neck
including 231 (66.8%) oropharyngeal tumors, 85 (24.6%) laryngeal, 28 (8%) hypopharyngeal and 2 (0.6%) from the oral cavity.
The primary treatment modality was surgery in 305 patients
(88%) and radiotherapy or chemoradiotherapy in 41 (12%),
although the majority of patients (n = 296, 86%) received radiotherapy at some stage in their treatment. HPV status was determined by in situ hybridization for HPV DNA, revealing that 236
(68%) were HPV-negative, and 110 (32%) HPV-positive. Consistent
with published data (34), HPV positivity was detected in 50% of
oropharyngeal tumors, and only 4% and 7% of laryngeal and
hypopharyngeal tumors, respectively. In primary tumor cores,
lymphovascular invasion (LVI) was present in 79 of 270 primary
tumors (29%), and perineural spread (PNS) in 66 of 270 (24%).
From each formalin-fixed, paraffin-embedded tumor block
2–3 cores were taken, to generate 852 cores used in TMA construction. Before staining the TMAs, the specificity of IGF-1R
monoclonal antibody #9750 (Cell Signaling Technology) was
tested in IHC, using as controls formalin-fixed, paraffin-embedded sections of SKUT-1 (IGF-1R-deficient) and MCF-7 (high
IGF-1R) cells as described (35), and whole-mount sections of
tonsil SCC. This revealed very low IGF-1R immunoreactivity in
SKUT-1 cells, and moderate signal in the cell membrane and
cytoplasm of MCF-7 cells and tonsil SCC (Figure 1A). These patterns supported the specificity of this IHC protocol, as reported
(32), Aleksic et al (in preparation). The same staining protocol
was used for the HNSCC TMAs, revealing considerable variation in the intensity, subcellular distribution and percentage of
tumor tissue staining positive for IGF-1R. Representative images
of different staining patterns are shown in Figure 1B. IGF-1R was
detected in the cell membrane and cytoplasm of 36% and 92% of
HNSCCs, although in the majority of cases, cytoplasmic IGF-1R
expression was low (IRS 0–4; Figure 1C). Linear regression analysis demonstrated a significant positive association between
membrane and cytoplasmic IGF-1R (P < 0.001, Figure 1C right). In
Table 1. Demographic data. Table shows clinical characteristics and
treatment of HNSCC patients included in this study
Demographic data
Age
Gender
Primary tumor site
Tumor T-stage
HPV status
Treatment
No. patients
Mean 58.7 years
Range 19–86 years
Male
264
Female
82
Oropharynx
231
Larynx
85
Hypopharynx
28
Oral cavity
2
1
17
2
40
3
65
4
219
Negative
236
Positive
110
Primary surgery
305
Radiotherapy
296
Median dose 64 Gy, 32 fractions
Range 20–78 Gy, 5–38 fractions
Chemotherapy
49
64 cases of oropharygeal cancer, cores of matched normal epithelial tissue were available for analysis alongside the HNSCCs,
revealing that cytoplasmic IGF-1R was significantly higher in
tumor tissue than normal epithelium (P < 0.001, Figure 1D and
E). These data suggest that IGF-1R is overexpressed in HNSCC
relative to matched normal tissue from the same patients.
Having found variable IGF-1R staining between tumors,
we investigated the relationship between IGF-1R and clinical
parameters. IGF-1R IRS data were grouped into categorical variables, categorizing membrane IGF-1R as absent (IRS = 0, n = 220)
or present (IRS > 0, n = 126), and cytoplasmic IGF-1R as absent/
low (IRS < 4, n = 182), and moderate to high (IRS ≥ 4, n = 164).
We also assessed total IGF-1R as the sum of membrane and
cytoplasmic IRS, categorized as low (cIGF-1R + mIGF-1R 0–4.5)
or moderate to high (>4.5). From Kaplan–Meier analysis, it was
apparent that high IGF-1R expression was associated with
reduced survival at 5 years (Figure 2A–C). This finding held true
for IGF-1R in the membrane (P = 0.006) and cytoplasm (P < 0.001)
and for total IGF-1R (P < 0.001). When analyzing by HPV status,
high total IGF-1R was associated with adverse OS in both HPVnegative cases, with hazard ratio (HR) of 2.17 [95% confidence
intervals (CI) = 1.38–5.83, P = 0.006; Figure 2D] and HPV-positive
cohorts (HR = 2.88, CI = 1.40–10.39, P = 0.01; Figure 2E), although
there were few HPV-positive tumors in the high IGF-1R category.
We also performed univariate analysis (Table 2) to assess correlations with OS and DSS. Supporting the Kaplan–Meier analysis, IGF-1R expression was associated with an increased HR for
death, when considering high membrane IGF-1R (OS: HR = 1.63,
P = 0.006; DSS: HR = 1.63, P = 0.016), cytoplasmic IGF-1R (OS:
HR = 2.17, P < 0.001; DSS: HR = 1.91, P = 0.002) and total IGF-1R
(OS: HR = 2.00, P < 0.001; DSS: HR = 2.16, P < 0.001). Univariate
analysis also confirmed associations between adverse survival
and recognized clinical and pathological factors (36,37) (Table 2A
and B). Specifically, adverse OS and DSS were associated with
increasing age (P < 0.001), HPV-negative status (P < 0.001), LVI
(P < 0.001) and PNS (P < 0.001). Although not significantly associated with OS in this series, high-tumor T-stage and extracapsular spread did show significant association with DSS (P = 0.032
and P = 0.026 respectively). The adverse association between
clinico-pathologic factors and survival was also apparent from
Kaplan–Meier analysis (Figure 3A–D), and HPV-negative status
was significantly associated with high-tumor T-stage (P < 0.001),
as previously reported (37).
We next tested for association between these clinical variables and IGF-1R. The most noteworthy finding was the association between HPV-negative status and higher expression
of IGF-1R, whether in the cell membrane, cytoplasm, or total
IGF-1R (P < 0.001 for each association, Figure 3E). High membrane, cytoplasmic and total IGF-1R expression were also associated with increased tumor T-stage (P < 0.001, Figure 3F), but not
with PNS, LVI or extracapsular spread. Analysis of IGF-1R expression by anatomical subsite showed significantly lower IGF-1R in
oropharyngeal cancers than cancers of the larynx or hypopharynx (P < 0.001), probably reflecting the higher proportion of HPVpositive tumors in the oropharynx.
Thus we identify a significant association between high
IGF-1R expression, HPV-negative status, higher T-stage and
adverse survival. These data provide evidence for complex interaction between IGF-1R, HPV status and tumor T-stage, in which
high IGF-1R expression is associated with HPV negativity and
increased tumor T-stage, and where T-stage and HPV status are
also directly related. To further probe the relationship between
these factors, multivariate analyses were performed to determine whether variables were independently associated with OS.
O.T.Dale et al. | 651
Figure 1. Upregulation of IGF-1R in HNSCC compared with paired benign epithelium. (A) Controls for immunostaining with IGF-1R-beta antibody #9750 (Cell Signaling
Technology), showing negligible staining in IGF-1R deficient SKUT-1 cells, and moderately intense membrane and cytoplasmic staining in MCF-7 cells and tonsil SCC
tissue that was abolished by omission of primary antibody. (B) HNSCC cores stained for IGF-1R showing representative appearances of cores stained 0–3 for the intensity of membrane and cytoplasmic IGF-1R. (C) Distribution of mean IRS scores for: left, membrane; center: cytoplasmic IGF-1R, in 346 cases of HNSCC. Right panel shows
linear regression analysis of mean cytoplasmic IGF-1R IRS and mean membrane IGF-1R IRS, showing strong correlation between IGF-1R staining in the two subcellular
compartments (P < 0.001). (D) Representative oropharygeal squamous cell cancer (OPSCC) core showing more intense IGF-1R staining in malignant component. (E)
Cytoplasmic IGF-1R score in OPSCCs and normal epithelial tissue from the same cases (n = 64). Box and whiskers plot showing median, 25th and 75th percentiles and
range, showing significantly greater expression of IGF-1R in OPSCC compared with paired benign tissues (****P < 0.001).
For this purpose, total IGF-1R was used in preference to cytoplasmic or membrane IGF-1R, since it demonstrated the largest
effect size in univariate analysis (Table 2B). Initial multivariate
analysis incorporating only IGF-1R and HPV status suggested
that high IGF-1R expression and HPV-negative status were significantly and independently associated with adverse OS in
HNSCC (Table 3A). In order to control for other variables that
may independently influence survival, a second multivariate
analysis was performed (Table 3B), including the significant
pathological prognostic indicators from univariate analysis. HPV
status, LVI, PNS and tumor T-stage were all confirmed to be of
independent prognostic significance, but total IGF-1R expression appeared not to be an independent predictor of survival
in this cohort. Finally, the patients were stratified by HPV status and a subgroup analysis was performed (Table 3C and D).
While IGF-1R expression was associated with adverse survival
652 | Carcinogenesis, 2015, Vol. 36, No. 6
Figure 2. IGF-1R expression is associated with adverse OS in HNSCC. Kaplan–Meier analyses showing reduced OS in patients with (A) tumors expressing membrane
IGF-1R (mIGF-1R) compared to those with no detectable mIGF-1R; (B) tumors with moderate to high cytoplasmic IGF-1R (cIGF-1R), compared with those with low or
absent cIGF-1R; (C) tumors expressing higher total IGF-1R (tIGF-1R = mIGF-1R + cIGF-1R); (D) HPV-negative tumors containing higher total IGF-1R; (E) HPV-positive
tumors containing higher total IGF-1R.
Table 2. Univariate analysis of clinico-pathological variables in HNSCC. The effect of clinical and pathological parameters, including
IGF-1R scoring in the membrane (IRS 0 versus >0), cytoplasm (IRS <4
versus ≥4) and total IGF-1R (IRS ≤4.5 versus >4.5), on (A) OS, (B) DSS of
patients with HNSCC, showing HR for death, with 95% CI
A: OS
independently associated with survival, whereas LVI, PNS and
tumor T-stage retained independent prognostic significance in
this HPV-negative cohort (Table 3C). There were fewer patients
with HPV-positive tumors (n = 86, 15 deaths), and none of the
variables analyzed showed an independent association with OS
in this analysis (Table 3D).
Variable
HR
95% CI
P value
Discussion
Age
HPV-positive status
Tumor T-stage
LVI
Perineural invasion
Extracapsular spread
Membrane IGF-1R
Cytoplasmic IGF-1R
Total IGF-1R
1.05
0.37
1.13
2.12
2.15
1.36
1.63
2.17
2.00
1.03–1.07
0.23–0.59
0.91–1.39
1.41–3.20
1.41–3.28
0.86–2.16
1.16–2.30
1.52–3.10
1.41–2.82
<0.001
<0.001
0.274
<0.001
<0.001
0.186
0.006
<0.001
<0.001
Variable
HR
95% CI
P value
Age
HPV-positive status
Tumor T-stage
LVI
Perineural invasion
Extracapsular spread
Membrane IGF-1R
Cytoplasmic IGF-1R
Total IGF-1R
1.05
0.35
1.36
3.05
3.11
1.86
1.63
1.91
2.16
1.03–1.07
0.20–0.60
1.03–1.79
1.91–4.87
1.95–4.98
1.08–3.21
1.10–2.43
1.27–2.86
1.45–3.21
<0.001
<0.001
0.032
<0.001
<0.001
0.026
0.016
0.002
<0.001
Our data indicate that compared with paired benign oropharyngeal epithelium, IGF-1R is upregulated in HNSCC, with
prominent IGF-1R signal in the plasma membrane in 36% and
cytoplasm in 92% of tumors. While we have detected nuclear
IGF-1R in other tumor types including renal and prostate cancers, associated with adverse prognosis in renal cancer (32), we
did not detect nuclear IGF-1R immunoreactivity in any samples
of HNSCC or benign epithelia. This reinforces the idea, as we previously suggested, that the presence of nuclear IGF-1R does not
simply reflect IGF-1R over-expression (32). The results of the current study in HNSCC support and extend the findings of several
previous reports, the first of which examined a series of 57 oral
cavity SCCs and found that 56% expressed detectable IGF-1R,
although there was no comparison with normal epithelial tissue
(38). Western blotting of fresh tissue lysates was used to compare expression in 12 oral cavity SCCs, showing increased IGF-1R
expression in tumors compared with matched normal tissue
(39). More recently, a study of 131 patients with oral cavity SCC
reported that 46% exhibited moderate or high IGF-1R immunoreactivity (29), compared with 36% in the current study. IGF-1R
was also detected by immunostaining for the extracellular IGF1R-alpha subunit in 56% of 289 cases of operable laryngeal cancer, with evidence that IGF-1R-alpha expression represented an
independent predictor of OS, whereas in contrast antibody to
the transmembrane IGF-1R-beta subunit showed positive signal
in <20% (40). Finally, in a study of 59 patients with oropharyngeal
B: DSS
in both HPV-negative and -positive cohorts in Kaplan–Meier
analysis (Figure 2D and E), multivariate analysis in HPV-negative
cases (n = 176, 81 deaths), showed that total IGF-1R was not
O.T.Dale et al. | 653
Figure 3. IGF-1R expression is associated with HPV negativity and advanced tumor T-stage. Kaplan–Meier analyses showing reduced OS in patients whose tumors show
(A) PNS (P < 0.001); (B) LVI (P < 0.001); (C) HPV negativity (P < 0.001); (D) T-stage T3/T4 versus T1/T2 (P < 0.001). (E) Tumors testing negative for HPV DNA are associated with
increased membrane (mIGF-1R), cytoplasmic (cIGF-1R) and total (tIGF-1R) IGF-1R expression (****P < 0.001, Mann–Whitney U-Test). (F) Compared with T1/T2 tumors, T3/
T4 tumors contain higher levels of membrane, cytoplasmic and total IGF-1R (****P < 0.001, Mann–Whitney U-Test).
cancer, IGF-1R was detectable by IHC in ~50% and was associated with HPV negativity (30). Potential sources of variation in
these studies include inclusion of different subpopulations of
HNSCC, use of different IHC antibodies and protocols, and variation in the proportion of HPV-positive cases, not reported by
(29,38–40), and probably very low in the laryngeal cancer series
(40).
The key findings of our study are that IGF-1R overexpression
is associated with adverse survival, HPV negativity and high
tumor T-stage. These findings in a large HNSCC cohort (n = 364)
support recent data from a smaller study of 59 patients with
oropharyngeal cancer, which found a significant association
between high IGF-1R, poor survival and HPV negativity, and a
non-significant trend to association between IGF-1R and T-stage
(30). The large numbers of cases in our series enabled us to
assess the association of IGF-1R with other clinico-pathological
variables. In multivariate analysis that included only HPV and
IGF-1R expression (Table 3A), both HPV (P < 0.001) and IGF-1R
(P = 0.002) appeared to be of independent prognostic significance. We confirmed that HPV status, LVI, PNS and high-tumor
T-stage are associated with adverse prognosis (Figure 3); when
all four parameters were included in multivariate analysis, all
showed independent prognostic significance (Table 3B). HPV status is well known to be an important prognostic factor (34,37),
654 | Carcinogenesis, 2015, Vol. 36, No. 6
Table 3. Multivariate analysis of clinico-pathological parameters in
HNSCC. (A) The effect of HPV status (positive versus negative) and
total IGF-1R (IRS ≤4.5 versus >4.5) on OS in HNSCC (n = 338), showing
HR for death and 95% CI. (B) Effect of inclusion of additional clinico-pathological variables including tumor T-stage (1–2 versus 3–4)
on significance of independent predictors of OS in HNSCC (n = 262).
(C, D) Associations between clinico-pathological variables including
IGF-1R (total IRS ≤4.5 versus >4.5) and OS in (C) HPV-negative patients
(n = 176) and (D) HPV-positive patients (n = 86)
A
Variable
HR
95% CI
P value
Total IGF-1R
HPV-positive
status
1.74
0.42
1.22–2.46
0.26–0.67
0.002
<0.001
B
Variable
HR
95% CI
P value
HPV-positive status
LVI
PNS
Tumor T-stage
Total IGF-1R
0.46
1.76
1.62
1.60
1.30
0.26–0.84
1.15–2.68
1.05–2.50
1.02–2.50
0.84–2.01
0.011
0.009
0.030
0.042
0.246
Variable
HR
95% CI
P value
LVI
PNS
Tumor T-stage
Total IGF-1R
1.87
1.72
1.75
1.08
1.19 – 2.95
1.09 – 2.73
1.06 – 2.89
0.68 – 1.72
0.007
0.021
0.029
0.737
Variable
HR
95% CI
P value
LVI
PNS
Tumor T-stage
Total IGF-1R
1.65
1.11
1.11
2.59
0.48–5.69
0.21–5.79
0.31–3.93
0.85–7.87
0.428
0.901
0.872
0.093
C: HPV-negative cases
D: HPV-positive cases
first identified by Ang et al.(37) who reported the association of
HPV positivity with favorable prognostic factors including lower
tumor T-stage, non-smoking status, better performance status
and younger age at diagnosis. In our study, after controlling for
HPV status and other pathological variables, IGF-1R expression
did not appear to be independently associated with survival
outcome in HNSCC (Table 3B–D).
It is not possible to ascertain from these data whether there
is any causal link between tumor size, HPV status and IGF-1R
expression. Although not independently related to survival outcome following multivariate analysis, the association of both
membrane and cytoplasmic IGF-1R with HPV-negative disease
and higher tumor T-stage (Figure 3E and F) indicates that IGF-1R
overexpression is associated with more aggressive disease.
IGF-1R has been linked in clinical and experimental studies with
resistance to chemotherapy and radiation (41–43). These findings
raise the question as to the extent to which low IGF-1R expression contributes to the chemo- and radio-sensitive phenotype of
HPV-positive HNSCC. While clinical trials are pursuing the prospect of de-escalating treatment for good prognosis patients with
HPV-positive disease (8), there is a need for treatment intensification for the HPV-negative cohort, and also for the poor prognosis subset of HPV-positive oropharyngeal cancer patients, where
further stratification is needed. Indeed, within the HPV-positive
cohort, there was evidence of adverse prognosis in patients whose
tumors expressed relatively high levels of IGF-1R (Figure 2E). As
in other common cancers, IGF-1R inhibitory antibodies have not
shown activity as monotherapy in HNSCC (44,45), but the ability of IGF-1R targeting to enhance chemo- and radio-sensitivity
(46–49) suggests that IGF inhibitory drugs should be considered
for incorporation into multimodality treatment trials for patients
with poor prognosis high IGF-1R HNSCC.
Funding
Heads Up; the Royal College of Surgeons of England; the British
Association of Head and Neck Oncologists; The Oracle Cancer
Trust (to O.T.D.); the National Institutes of Health Research;
Oxford Biomedical Research Centre funding (to V.M.M.); Prostate
Cancer UK (grant G2012/25 to T.A.).
Acknowledgements
We are grateful to the HNSCC patients who gave permission for
use of surplus tumor tissue in research and to Drs A. Kong and
K. Shan, Department of Clinical Oncology Oxford, for comments
on the manuscript.
Conflict of Interest Statement: None declared.
References
1. Machiels, J.P. et al. (2014) Advances in the management of squamous
cell carcinoma of the head and neck. F1000Prime Rep., 6, 44.
2. Hobbs, C.G. et al. (2006) Human papillomavirus and head and neck
cancer: a systematic review and meta-analysis. Clin. Otolaryngol., 31,
259–266
3. Chaturvedi, A.K. et al. (2011) Human papillomavirus and rising oropharyngeal cancer incidence in the United States. J. Clin. Oncol., 29,
4294–4301.
4. Ribeiro, K.B. et al. (2011) Low human papillomavirus prevalence in head
and neck cancer: results from two large case-control studies in highincidence regions. Int. J. Epidemiol., 40, 489–502.
5. Mehanna, H. et al. (2013) Prevalence of human papillomavirus in oropharyngeal and nonoropharyngeal head and neck cancer-systematic
review and meta-analysis of trends by time and region. Head Neck,
35, 747–755.
6. Jemal, A. et al. (2007) Cancer statistics, 2007. CA: Cancer J Clin, 57, 43–66
7. Gregoire, V. et al. (2010) Squamous cell carcinoma of the head and neck:
EHNS-ESMO-ESTRO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann. Oncol., 21(suppl. 5), v184–186.
8. Masterson, L. et al. (2014) De-escalation treatment protocols for human
papillomavirus-associated oropharyngeal squamous cell carcinoma: a
systematic review and meta-analysis of current clinical trials. Eur J
Cancer, 50, 2636–2648.
9. Forastiere, A. et al. (2001) Head and neck cancer. N. Engl. J. Cancer, 345,
1890–1900
10.Chung, C.H. et al. (2006) Increased epidermal growth factor receptor
gene copy number is associated with poor prognosis in head and neck
squamous cell carcinomas. J. Clin. Oncol., 24, 4170–4176.
11.Bentzen, S.M. et al. (2005) Epidermal growth factor receptor expression
in pretreatment biopsies from head and neck squamous cell carcinoma as a predictive factor for a benefit from accelerated radiation
therapy in a randomized controlled trial. J. Clin. Oncol., 23, 5560–5567.
12. Bonner, J.A. et al. (2010) Radiotherapy plus cetuximab for locoregionally
advanced head and neck cancer: 5-year survival data from a phase 3
randomised trial, and relation between cetuximab-induced rash and
survival. Lancet. Oncol., 11, 21–28.
13.Petrelli, F. et al. (2014) Concomitant platinum-based chemotherapy or
cetuximab with radiotherapy for locally advanced head and neck can-
O.T.Dale et al. | 655
cer: a systematic review and meta-analysis of published studies. Oral
Oncol., 50, 1041–1048.
14.Sell, C. et al. (1994) Effect of a null mutation of the insulin-like growth
factor I receptor gene on growth and transformation of mouse embryo
fibroblasts. Mol. Cell. Biol., 14, 3604–3612.
15.Steuerman, R. et al. (2011) Congenital IGF1 deficiency tends to confer protection against post-natal development of malignancies. Eur.
J. Endocrinol., 164, 485–489.
16.Guevara-Aguirre, J. et al. (2011) Growth hormone receptor deficiency is
associated with a major reduction in pro-aging signaling, cancer, and
diabetes in humans. Sci. Transl. Med., 3, 70ra13.
17.Pollak, M. (2012) The insulin and insulin-like growth factor receptor
family in neoplasia: an update. Nat. Rev. Cancer, 12, 159–169.
18.Badzio, A. et al. (2010) Increased insulin-like growth factor 1 receptor
protein expression and gene copy number in small cell lung cancer. J.
Thorac. Oncol., 5, 1905–1911.
19.Papa, V. et al. (1997) Sporadic amplification of the insulin receptor gene
in human breast cancer. J. Endocrinol. Invest., 20, 531–536.
20.Yuen, J.S. et al. (2007) The VHL tumor suppressor inhibits expression of
the IGF1R and its loss induces IGF1R upregulation in human clear cell
renal carcinoma. Oncogene, 26, 6499–6508.
21.Werner, H. (2012) Tumor suppressors govern insulin-like growth factor signaling pathways: implications in metabolism and cancer. Oncogene, 31, 2703–2714.
22.Parker, A. et al. (2003) Expression of insulin-like growth factor I receptor and survival in patients with clear cell renal cell carcinoma. J. Urol.,
170(Pt 1), 420–424.
23.Spentzos, D. et al. (2007) IGF axis gene expression patterns are prognostic of survival in epithelial ovarian cancer. Endocr. Relat. Cancer, 14,
781–790.
24.Matsubara, J. et al. (2008) Impact of insulin-like growth factor type 1
receptor, epidermal growth factor receptor, and HER2 expressions on
outcomes of patients with gastric cancer. Clin. Cancer Res., 14, 3022–3029.
25.Takahari, D. et al. (2009) Relationships of insulin-like growth factor-1
receptor and epidermal growth factor receptor expression to clinical
outcomes in patients with colorectal cancer. Oncology, 76, 42–48.
26.Valsecchi, M.E. et al. (2012) Epidermal growth factor receptor and insulinlike growth factor 1 receptor expression predict poor survival in
pancreatic ductal adenocarcinoma. Cancer, 118, 3484–3493.
27. Donohoe, C.L. et al. (2012) Role of the insulin-like growth factor 1 axis and
visceral adiposity in oesophageal adenocarcinoma. Br. J. Surg., 99, 387–396.
28.Yamamoto, T. et al. (2012) Clinical significance of immunohistochemical expression of insulin-like growth factor-1 receptor and matrix
metalloproteinase-7 in resected non-small cell lung cancer. Exp. Ther.
Med., 3, 797–802.
29.Lara, P.C. et al. (2011) IGF-1R expression predicts clinical outcome in
patients with locally advanced oral squamous cell carcinoma. Oral
Oncol., 47, 615–619.
30.Matsumoto, F. et al. (2014) Relationship between insulin-like growth
factor-1 receptor and human papillomavirus in patients with oropharyngeal cancer. Head Neck, epub April 2.
31.Westra, W.H. (2009) The changing face of head and neck cancer in the
21st century: the impact of HPV on the epidemiology and pathology of
oral cancer. Head Neck Pathol., 3, 78–81.
32.Aleksic, T. et al. (2010) Type 1 insulin-like growth factor receptor translocates to the nucleus of human tumor cells. Cancer Res., 70, 6412–
6419.
33.Winter, S.C. et al. (2006) The relation between hypoxia-inducible factor
(HIF)-1alpha and HIF-2alpha expression with anemia and outcome in
surgically treated head and neck cancer. Cancer, 107, 757–766.
34. Leemans, C.R. et al. (2011) The molecular biology of head and neck cancer. Nat. Rev. Cancer, 11, 9–22.
35.Asmane, I. et al. (2012) Insulin-like growth factor type 1 receptor (IGF1R) exclusive nuclear staining: a predictive biomarker for IGF-1R monoclonal antibody (Ab) therapy in sarcomas. Eur. J. Cancer, 48, 3027–3035.
36.Argiris, A. et al. (2008) Head and neck cancer. Lancet, 371, 1695–1709.
37.Ang, K.K. et al. (2010) Human papillomavirus and survival of patients
with oropharyngeal cancer. N. Engl. J. Med., 363, 24–35.
38. Reszeć, J. et al. (2004) Insulin-like growth factor-I receptor in human
oral cancer. Rocz. Akad. Med. Bialymst., 49(suppl. 1), 58–60.
39.Barnes, C.J. et al. (2007) Insulin-like growth factor receptor as a therapeutic target in head and neck cancer. Clin. Cancer Res., 13, 4291–4299.
40.Mountzios, G. et al. (2013) Insulin-like growth factor 1 receptor (IGF1R)
expression and survival in operable squamous-cell laryngeal cancer.
PLoS One, 8, e54048.
41.Turner, B.C. et al. (1997) Insulin-like growth factor-I receptor overexpression mediates cellular radioresistance and local breast cancer
recurrence after lumpectomy and radiation. Cancer Res., 57, 3079–
3083.
42. Lloret, M. et al. (2007) IGF-1R expression in localized cervical carcinoma
patients treated by radiochemotherapy. Gynecol. Oncol., 106, 8–11.
43. Cosaceanu, D. et al. (2007) Ionizing radiation activates IGF-1R triggering
a cytoprotective signaling by interfering with Ku-DNA binding and by
modulating Ku86 expression via a p38 kinase-dependent mechanism.
Oncogene, 26, 2423–2434.
44.Schmitz, S. et al. (2010) Phase II study of figitumumab in patients with
recurrent and/or metastatic squamous cell carcinoma of the head and
neck: GORTEC 2008-02. J. Clin. Oncol., 23, 2153–2161
45.King, H. et al. (2014) Can we unlock the potential of IGF-1R inhibition in
cancer therapy? Cancer Treat. Rev., 40, 1096–1105.
46.Riesterer, O. et al. (2011) Combination of anti-IGF-1R antibody A12 and
ionizing radiation in upper respiratory tract cancers. Int. J. Radiat.
Oncol. Biol. Phys., 79, 1179–1187.
47.Matsumoto, F. et al. (2012) The impact of timing of EGFR and IGF-1R
inhibition for sensitizing head and neck cancer to radiation. Anticancer Res., 32, 3029–3035.
48.Chitnis, M.M. et al. (2014) IGF-1R inhibition enhances radiosensitivity
and delays double-strand break repair by both non-homologous endjoining and homologous recombination. Oncogene, 33, 5262–5273.
49.Ferté, C. et al. (2013) IGF-1R targeting increases the antitumor effects of
DNA-damaging agents in SCLC model: an opportunity to increase the
efficacy of standard therapy. Mol. Cancer Ther., 12, 1213–1222.