Original article Strahlenther Onkol 2014 DOI 10.1007/s00066-014-0652-y Received: 13 January 2014 Accepted: 3 March 2014

Gregor Heiduschka1 · Anja Grah2 · Felicitas Oberndorfer3 · Rudolf Seemann4 · Alexander Kranz2 · Gabriela Kornek5 · Fritz Wrba3 · Dietmar Thurnher1 · Edgar Selzer2

© Springer-Verlag Berlin Heidelberg 2014

2Department of Radiotherapy, Medical University of Vienna, Vienna, Austria

1Department of Otorhinolaryngology—Head and Neck Surgery, Medical University of Vienna, Vienna, Austria

3Department of Clinical Pathology, Medical University of Vienna, Vienna, Austria 4Department of Maxillofacial Surgery, Medical University of Vienna, Vienna, Austria 5Department of Medicine I—Division of Clinical Oncology, Medical University of Vienna, Vienna, Austria

Significance of p16 expression in head and neck cancer patients treated with radiotherapy and cetuximab The increasing incidence of human papillomavirus (HPV) infection and its rising clinical significance for head and neck squamous cell carcinoma (HNSCC) has been clearly documented [7, 12, 13, 22]. In the majority of the tumors investigated, infection with HPV is associated with de novo expression of the cyclin-dependent kinase inhibitor 2A, which is often referred to as p16 [21, 31, 35]. p16, in contrast to HPV infection, can be easily detected by immunohistochemical methods. Numerous clinical studies, both retrospective and prospective in nature, as well as one meta-analysis including 34 studies with over 5,600 patients, demonstrated an improved survival in patients with HPV-positive tumors treated with radiotherapy (RT) alone or in combination with induction and/or concurrent chemotherapy [2, 28, 29, 36]. Hence, there is now robust evidence to suggest that the presence of HPV infection and/or p16 expression as a surrogate marker confers a survival advantage in patients suffering from locally advanced HNSCC treated with primary RT in combination with chemotherapy. Since the contemporary standard treatment for locally advanced HNSCC is radiochemotherapy, the vast majority of data are available for this type of treatment combination. However, the introduction of cetuximab as a novel treatment option for

patients who are not eligible for a combined radiochemotherapy now leads to the question of whether HPV positivity and/or p16 expression might also affect treatment response in this patient collective. The possible influence of epithelial growth factor receptor (EGFR) expression on treatment response in this context is also of great interest. EGFR has been implicated in a variety of publications as a modulator of radio-response—not only in vitro in preclinical models but also in vivo in head and neck cancer patients [1, 8, 9, 24, 32, 34]. Thus, the primary aim of this retrospective analysis was to determine whether p16 expression and/or HPV infection would affect the outcome of HNSCC patients who received primary RT in combination with cetuximab within a single institution (General Hospital of Vienna). In addition, we sought to analyze the expression levels of EGFR and to investigate a possible correlation between EGFR expression and p16 status with respect to treatment outcome. Finally, we sought to compare the outcome of patients treated with cetuximab and accelerated-fractionation RT with the results of patients treated with cetuximab and standard-fractionation RT as well as with a historical institutional control group treated without cetuximab.

Patients and methods Patients Between 2006 and 2012, 144 patients with HNSCC were treated consecutively with cetuximab in combination with RT at the Department of Radiotherapy at the Medical University of Vienna. Of these, 78 were eligible for further analysis. All patients were presented at the Institutional Tumor Board of the General Hospital Vienna. Patients received concurrent radiotherapy with cetuximab if they were deemed ineligible for chemotherapy due to medical reasons. For comparative analysis, a historical control group (treated between January 2001 and December 2004) of 43 consecutively treated (concomitant boost technique without systemic therapy) patients was analyzed. This patient group resembles the patient collective treated with cetuximab with respect to age, tumor characteristics, and institutional selection criteria, particularly with regard to non-eligibility for combination treatment with radiochemotherapy.

Radiotherapy Out of 78 eligible patients, 58 received standard fractionation RT, whereas 20 patients were treated with accelerated fractionation RT plus cetuximab. StandardStrahlentherapie und Onkologie X · 2014 

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Original article fractionation RT consisted of the delivery of 70 Gy in 35 fractions (2 Gy per fraction, over a 7-week period). By contrast, the accelerated-fractionation RT consisted of 72 Gy given in 42 fractions over a 6-week period plus a concomitant boost comprising twice-daily irradiation (1.8 Gy and 1.5 Gy) for 12 treatment days (as described previously)[11, 33]. The total dose delivered was either 70 Gy or 72 Gy to sites of gross disease and 50 Gy or 54 Gy to adjacent lymphatic drainage regions at risk for subclinical metastasis. The spinal cord dose was limited to a maximum of 46 Gy. The historical control group was treated with the concomitant boost protocol without cetuximab (before the approval of cetuximab for the treatment of head and neck cancer patients in 2006).

Cetuximab The administration of intravenous cetuximab (Erbitux(R)), Merck KGA, Darmstadt, Deutschland) was initiated 1 week before the start of RT at a loading dose of 400 mg/m2 of body-surface area over 120 min, followed by 60-min infusions of 250 mg/m2 for the duration of RT (one infusion per week). Furthermore, all patients received intravenously cortisone and H1 blockers (diphenhydramine hydrochloride) before the first three to four infusions of cetuximab. Dose modification options for grade 3 skin toxicity included a 1-week delay, with dose reductions in increments of 50 mg/m2 (minimum cetuximab dose 150 mg/m2).

Clinical evaluation and follow-up The entire group of patients receiving RT alone or in combination with cetuximab was monitored weekly during RT. Clinical follow-up after the end of RT was conducted both at the Department of Radiotherapy and at the Department of Otorhinolaryngology (Head and Neck Surgery) according to the institutional guidelines.

Tumor specimen and detection methods In situ hybridization as well as immunohistochemistry was performed on routinely obtained biopsy tissues, after fixa-

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tion in formalin and embedding in paraffin. Hematoxylin & eosin-stained slides were used as a control for the presence of invasive squamous cell carcinoma in each sample. For detection of HPV, a commercially available validated detection system from Ventana [INFORM Probes In Situ Hybridization (ISH) system] was used. This kit (HPV II Family 6 and HPV III Family 16) contains a cocktail of HPV genomic probes targeting the following genotypes: 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 66. The INFORM HPV probes are able to detect the presence of episomal as well of integrated HPV DNA and differentiate between high- and lowrisk HPV subtypes. Interpretation of HPV in situ hybridization staining was performed by means of the Ventana Interpretation Guide that is provided by the manufacturer (positive = 1, negative = 0) and conducted in principle as described by others as well as our group [16, 20].

p16 and EGFR immunohistochemistry EGFR and p16 immunohistochemistry was performed on a BenchMark Ultra platform (Ventana Medical Systems, Inc.), as recently described by our group [6, 25]. After pre-treatment with protease (for EGFR staining) and with heat (in EDTA pH 8.0) for p16 immunohistochemistry, slides were incubated with antibodies (EGFR-3C6; p16-E6H4). IHC slides were semiquantitatively scored by two pathologists (F.O., F.W.) for intensity of cytoplasmatic and membrane staining (EGFR) of the cellular nucleus (p16), respectively. Staining intensity was scored as 0 (none), 1 (weak), 2 (moderate), or 3 (strong). In addition, the proportion of cells stained was determined for both p16 and EGFR. EGFR expression was defined as high if the percentage of cells characterized by intense staining (score 3) was ≥ 70 %. For statistical analysis, samples were scored as p16 positive if at least 90 % of the cells (the sum of percentages) stained at least moderate and/or strong. All procedures followed in this retrospective study were in accordance with the standards of the hospital’s ethics committee (Medical University of Vienna)

and with the Helsinki Declaration of 1975 (in its most recently amended version).

Statistical analysis Rates of overall survival (OS) were estimated by means of the Kaplan–Meier method and were compared between the groups using the log-rank test. Cox proportional-hazards models were used to estimate hazard ratios. Data are presented including standard errors of the mean (SEM) and confidence intervals (CI). Unadjusted and adjusted hazard ratios were utilized to obtain estimates of the differences in survival that could be attributed to the investigated covariates. The software package SPSS Version 21 (IBM) was used for analysis.

Results Patient characteristics As of 2006, we identified 144 patients in our database suffering from HNSCC, who were treated with cetuximab in combination with RT (Department of Radiotherapy at the Medical University Vienna). Finally, 78 patients were eligible for our study; 66 patients were excluded from further analysis. Of these, 31 patients were not eligible for evaluation due to their participation in a multicentric phase II induction chemotherapy study [17]. Further exclusion criteria were unavailability of patient samples in cases where the initial biopsies had been performed at the external referring institutions (n = 10), second malignancies (n = 13), distant metastases (n = 4), previous tumor therapy (n = 5), and tumor locations other than those analyzed in this study (n = 3). Important baseline characteristics of the patients are summarized in . Table 1.

HPV, p16, and EGFR expression EGFR and p16 expression data are shown in .  Table 2. p16 positivity was found in 36.6 % of the available samples (N = 30) from the OPSCC patient group. Only one sample from a non-oropharyngeal tumor site (larynx) showed positive results for p16 or HPV positivity. High EGFR levels were detected in 82 % of the analyzed tu-

Abstract · Zusammenfassung mors (N = 55) irrespective of the localization. Nine out of 11 HPV high-risk positive tumors were detected in oropharyngeal cancer samples; 72 % of p16-positive OPSCC patients were positive for highrisk HPV. No low-risk HPV subtype was detected. In 12 (6 OPSCCs and 6 non-OPSCCs) patients, no or not enough material was available for determination of p16 and/or EGFR status. By comparison, Ang et al. [2] reported in their analysis of the RTOG 0129 study that 63 % of the OPSCC and over 25 % of the laryngeal cancer patients treated were HPV-positive.

Survival Kaplan–Meier estimates of OS for the complete RT plus cetuximab collective are shown in .  Fig. 1a. Median survival was 651 days (SEM = 47; 95 % CI = 558– 743 days). OS after 2 years was 42 % and after 3 years 27 %. There was no significant statistical difference as determined by the log-rank test (Mantel–Cox; p = 0.98) in OS between patients treated with cetuximab combined with standard fractionated RT (n = 58; median survival = 641 days, SEM = 40.6) or with cetuximab and accelerated fractionation RT (concomitant boost protocol; n = 20; median survival = 662 days, SEM = 207) (. Fig. 1b). When the entire cetuximab plus RT group was statistically analyzed according to tumor site (oropharyngeal vs. nonoropharyngeal cancer), differences in OS were either marginally significant by the log-rank test (Mantel–Cox; p = 0.06) or significant [p = 0.048 by the Breslow (generalized Wilcoxon) test; p = 0.047 by the Tarone–Ware test] (see . Fig. 1c). Median survival in the OPSCC group was estimated to be 841 days (SEM = 190) versus 583 days (SEM = 116) for the other tumor sites combined. By contrast, when OS in the historical control group (treated without cetuximab) was analyzed according to tumor site (oropharyngeal vs. non-oropharyngeal cancer), no statistical difference was observed (log-rank test, Mantel–Cox: p = 0.86; .  Fig. 1d). OS according to initial response (complete remission vs. noncomplete remission) in the RT plus cetuximab group was significantly better in the responder group (p ≤ 0.0001; log-rank test) (. Fig. 1e). OS according to N-stage

Strahlenther Onkol 2014  DOI 10.1007/s00066-014-0652-y © Springer-Verlag Berlin Heidelberg 2014 G. Heiduschka · A. Grah · F. Oberndorfer · R. Seemann · A. Kranz · G. Kornek · F. Wrba · D. Thurnher · E. Selzer

Significance of p16 expression in head and neck cancer patients treated with radiotherapy and cetuximab Abstract Background.  HPV-infection, p16 positivity, and EGFR expression have been correlated with favorable responses of head and neck cancer patients treated with radiotherapy (RT) with or without chemotherapy. However, a possible correlation of HPV/p16 and EGFR status on the effect of RT in combination with cetuximab has not been sufficiently investigated. Materials and methods.  We analyzed tumor samples for p16 and EGFR expression and correlated these variables with treatment outcome. Cox-proportional-hazard regression models were applied to compare the risk of death among patients stratified according to risk factors. Survival was estimated by the Kaplan–Meier method. Results were compared with an institutional historical control group treated without cetuximab and with published data. Results.  Expression of p16 was predominantly found in oropharyngeal squamous

cell cancer patients (OPSCC; 36.6 % positivity; 92 % of all cases), while EGFR was expressed at high levels in all tumor subsites (82 %). p16 expression was associated with improved overall survival in irradiated OPSCC patients (2-year overall survival of 80 % in p16-positive vs. 33 % overall survival in p16-negative patients). In a multivariable analysis covering all tumor sites, nodal stage (> N2a vs. ≤ N2a) and tumor site (OPSSC vs. non-OPSCC) had an impact on overall survival. Conclusion.  Our results show that p16 positivity is associated with a favorable outcome in OPSCC patients treated with RT and cetuximab. Keywords Head and neck cancer · Radiotherapy · Cetuximab · Human papilloma virus · p16 protein

Signifikanz der p16-Expression bei mit Radiotherapie und Cetuximab behandelten Patienten mit Kopf-Hals-Tumor Zusammenfassung Hintergrund.  HPV-Infektion, p16-Positivität und EGFR-Expression wurden bei Kopf-HalsTumorpatienten, die mit einer Strahlentherapie (RT) mit oder ohne Chemotherapie behandelt wurden, mit einem besseren Ergebnis in Verbindung gebracht. Bis jetzt wurde eine solche Korrelation bei Patienten, die mit einer RT in Kombination mit Cetuximab therapiert wurden, nicht untersucht. Material und Methoden.  Es wurden die p16- und die EGFR-Expression in Tumormaterial untersucht und die Daten mit dem Behandlungsergebnissen korreliert. Um die Sterberisiken zu vergleichen, wurden Cox-Regressionsmodelle angewendet und die Patienten nach Risikofaktoren stratifiziert. Das Überleben wurde nach der Kaplan-MayerMethode bewertet. Die Resultate wurden mit einer historischen Vergleichsgruppe aus unserem Institut und der Literatur verglichen. Ergebnisse.  Die p16-Expression wurde vorrangig in Oropharynxkarzinompatienten

(2b-N3 vs. N0-2a) was not significantly different (p = 0.14; log-rank test; . Fig. 1f).

gefunden (OPSCC; 36,6 % positiv; 92 % aller positiven Fälle), während EGFR in allen Tumorlokalisationen stark exprimiert war (82 %). Die p16-Expression war in OPSCC-Patienten mit einem verbesserten Gesamtüberleben (OS) assoziiert (2-Jahres-OS von 80 % in p16-positiven vs. 33 % in p16-negativen Patienten). In einer multivariablen Analyse, die sämtliche Tumorlokalisationen umfasste, zeigten der Lymphknotenstatus (> N2a vs. ≤ N2a) und die Tumorlokalisation (OPSSC vs. non-OPSCC) einen Einfluss auf das OS. Schlussfolgerung.  Unsere Resultate zeigen, dass eine p16-Expression mit einem besseren Outcome von OPSCC-Patienten, die mit einer Kombination aus RT und Cetuximab behandelt wurden, assoziiert ist. Schlüsselwörter Kopf- und Halstumore · Radiotherapie · Cetuximab · Humaner Papillomavirus · p16-Protein

Estimates of OS rates of oropharyngeal cancer patients treated with RT and cetuximab according to p16 expression are Strahlentherapie und Onkologie X · 2014 

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Original article Table 1  Baseline characteristics of the study patients Characteristic

Standard fractionated RT Concomitant-boost RT Age (median) Male: n (%) Female: n (%) Primary site: n (%) Oral cavity Oropharynx Hypopharynx Larynx Tumor stage: n (%) T1-3 T4 Nodal stage: n (%) N0-2a N2b-3 ACJCC stage: n (%) II III IV

Treatment groups RT plus cetuximab All tumor sites (n = 78) 58 (74 %) 20 (25 %) 66 60 (77 %) 18 (23 %)

Historical control group (n = 43) 0 (0 %) 43 (100 %) 60 34 (79 %) 9 (21 %)

RT plus cetuximab Oropharynx (n = 36) 27 (75 %) 9 (25 %) 68 28 (78 %) 7 (22 %)

27 (35 %) 36 (46 %) 7 (9 %) 8 (10 %)

8 (19 %) 20 (46 %) 11 (26 %) 4 (9 %)

0 (0 %) 36 (100 %) 0 (0 %) 0 (0 %)

26 (33 %) 52 (67 %)

21 (49 %) 22 (51 %)

14 (39 %) 22 (61 %)

25 (32 %) 53 (68 %)

7 (16 %) 36 (84 %)

10 (28 %) 26 (72 %)

4 (5 %) 8 (10 %) 66 (85 %)

2 (5 %) 2 (5 %) 39 (90 %)

2 (6 %) 3 (8 %) 31 (86 %)

Table 2  p16 and EGFR expression in pa-

tients treated with RT plus cetuximaba

p16 positive p16 negative EGFR high EGFR low

All sites combined 12 (18.2 %) 54 (81.8 %) 55 (82.1 %) 12 (17.9 %)

Oropharyngeal cancer 11 (36.6 %) 19 (63.4 %) 28 (90 %) 3 (10 %)

Percentages were calculated after subtracting the numbers of patients from which no tumor specimen were available for analysis

Table 3  Hazard ratios for overall survival

of RT plus cetuximab group Covariate

All patients RT plus cetuximab Hazard ratio (95 % p value CI) Tumor stage 1.3 (0.73–2.3) 0.38 T4 vs. T1-3 Nodal stage 0.4 (0.19–0.89) 0.02 Tumor site 2.4 (1.2–4.6) 0.01 Age 1.2 (0.62–2.2) 0.63 Sex 1.4 (0.7–2.8) 0.3

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shown in . Fig. 2a. OS after 2 years in the p16-positive subgroup was estimated to be 80 % as compared with 33 % in the p16negative group. Patients with p16-positive tumors had significantly better OS (logrank test; p = 0.032). When OS within the OPSCC group was analyzed according to T stage (T4 vs. T1-3; p = 0.73, log-rank test), N stage (N0-2a vs. N2b-3; p = 0.25, log-rank test), or EGFR status (EGFR high vs. low; p = 0.447, log-rank test), sex (male vs. female; p = 0.17, log-rank test), age ( 63 years; p = 0.67, log-rank test), no statistically significant differences were observed (. Fig. 2b–f). EGFR status and age had no impact on survival in the complete collective of oropharyngeal plus non-oropharyngeal cancer patients (EGFR high vs. low; p = 0.47, logrank test). In a multivariable analysis (of the complete RT plus cetuximab group), nodal stage and tumor site were significant determinants of OS (.  Table 3). Since only one tumor in the non-oropharyngeal cancer group was p16 positive, no analysis was performed with regard to p16 for the other tumor sites. EGFR status was part of a multivariable analysis within the

RT + cetuximab group but had no significant impact on survival. The hazard ratio (by univariable analysis) of the impact of p16 expression on survival in the OPSCC group was 3.7 (CI 1.04–13.5; p = 0.04). Analysis of HPV expression alone did not yield a statistical significant correlation, presumably due to the limited number of HPV-positive samples in our collective (data not shown).

Discussion In the present study, we retrospectively analyzed the treatment outcome of patients treated with RT plus cetuximab as well as with RT alone. In addition, we analyzed the expression of p16 and EGFR by immunohistochemistry, and the presence of HPV by in situ hybridization. Taken together, our data provide evidence that p16 expression might serve as a surrogate marker for a better treatment response in OPSCC patients treated with RT and cetuximab. While this result may not seem to be unexpected, we would like to stress the fact that, until now, the vast majority of data in the literature regarding the prognostic value of HPV/p16 has been obtained from studies of patients treated with chemotherapy in various combinations with RT. To our knowledge, the first published study to retrospectively investigate the relevance of HPV/p16 expression in cetuximab plus RT-treated patients was conducted by Pajares et al. [23]. Pajares et al. [23] showed that p16-positive patients treated with RT plus EGFR inhibitors had an improved OS compared with patients treated with RT plus chemotherapy. In our analysis, expression of p16 in OPSCC was found to be associated with a dramatically improved OS over p16-negative oropharyngeal tumors. OS after 2 years in the p16-positive OPSCC group was estimated to be 80 % as compared with 33 % in the p16-negative group vs. 89 % in p16-positive and 59 % in p16-negative patients as reported by Pajares et al. [23]. An indirect comparison of our results with published data from several prospective studies— ECOG [10]; TROG 2.02 [31]; RTOG 9003 and 0129 [14]; TAX324 [27]; DHANCA 5,7,10 [19]—confirms that the extent of the added benefit (2–3-year OS improve-

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Fig. 1 8 Overall survival of oropharyngeal squamous cell cancer patients (OPSCC) and non-OPSCC patients combined. Data on overall survival of all patients are shown in a. Data were stratified according to radiation protocol (b), oropharyngeal vs. other primary site in patients with cetuximab (c), oropharyngeal vs. other primary site in patients treated without cetuximab (d), according to primary treatment response (e) (complete remission (CR) vs. non-CR), and N stage (f)

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Strahlentherapie und Onkologie X · 2014 

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Original article ments up to 30 %) conferred by the presence of HPV or by p16 positivity reported is comparable to our data. Of note, in our collective, 36 % (11/36) of the OPSCCs but only 2 % (1/42) of the non-oropharyngeal tumors were p16positive. By comparison, Ang et al. [2] reported an almost twice as high prevalence of p16-positive OPSCCs (64 %) in their study population (USA), while Pajares et al. [23], on the other hand, reported HPV16 DNA in only 15 % of OPSCCs (Spain). Indirect comparisons of published data from randomized trials between intensified radiotherapeutic regimens, such as the concomitant boost protocol [11] and RT plus cetuximab [4] or RT plus cisplatin [26], suggest that these combinations are superior to a similar extent—the absolute benefit range being between 10–20 %—to conventional RT alone with respect to local control and/or OS. Cisplatin is now regarded to be equally efficient with respect to OS when given in combination with a standard-fractionation radiotherapeutic protocol as compared with a combination with intensified RT [2, 5]. While our study suffers from drawbacks generally associated with retrospective analyses, we would like to stress the fact that OS, initial response rates, as well as the rate of incidence of side effects [33] in our patient collective match the data published in the Bonner trial [4] and in the RTOG 9003 study [11] in which the same accelerated fractionation with concomitant boost protocol was investigated. We are therefore confident that the collective of patients analyzed in this study is representative with regard to key variables from several relevant publications in this field. As mentioned, expression of EGFR has been correlated in several studies with locoregional treatment outcome in head and neck cancer patients. Patients with tumors expressing high EGFR levels (EGFR index > median population value) generally had a worse outcome if treated with conventional RT, but may benefit from intensified RT with respect to locoregional tumor control [3, 9]. Evidence of an inverse correlation between HPV status and EGFR expression

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has been provided in recent studies [30]. In addition, HPV negativity in conjunction with high EGFR expression may be associated with an increase in the risk of locoregional failure as well as a reduction in survival [15, 18, 37]. In our collective, EGFR was expressed in the majority of tumors at high levels, with the median of the percentage of cells expressing high levels of EGFR being over 90 %. No correlation between EGFR expression and treatment outcome could be detected in our study.

Conclusion Taken together, our data indicate that the treatment effect of cetuximab on OS is noticeably affected by the p16 status. p16 expression as well as HPV DNA was predominantly found in OPSCC sites.

Corresponding address E. Selzer MD Department of Radiotherapy Medical University of Vienna Waehringer Guertel 18–20 1090 Vienna [email protected]

Compliance with ethical guidelines Conflict of interest.  G. Heiduschka, F. Oberndorfer, R. Seemann, A. Kranz, G. Kornek, F. Wrba, and D. Thurner state that there are no conflicts of interest. E. Selzer is a recipient of a scientific grant (for basic stem cell research) from Merck Austria. The position of A. Grah is funded by a grant from Merck Austria. All studies on humans described in the present manuscript were carried out with the approval of the responsible ethics committee and in accordance with national law and the Helsinki Declaration of 1975 (in its current, revised form). Informed consent was obtained from all patients included in studies.

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