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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. 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