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Keywords:

  • oropharyngeal carcinoma;
  • HPV;
  • p16;
  • radiotherapy;
  • tissue microarray

Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References

The incidence of human papilloma virus (HPV) induced oropharyngeal squamous cell carcinoma (OPSCC) increases in the western countries. These OPSCC show distinct molecular characteristics and are characterized by an overexpression of p16, considered a surrogate marker for HPV infection. When compared to patients with p16 negative OPSCC, patients with HPV induced p16 positive OPSCC show a significantly better prognosis, which is reported to be caused by increased radiosensitivity. The objective of the present study was to analyze the impact of p16 expression status on the prognosis of OPSCC treated by either radiotherapy (RT) or primary surgery. Results are based upon a tissue microarray (TMA) of 365 head neck squamous cell carcinomas (HNSCC) including 85 OPSCC with clinico-pathological and follow-up data. p16 positivity correlated significantly with oropharyngeal tumor localization (p < 0.001). Patients with p16 positive OPSCC exhibited a significantly better overall survival than those with p16 negative tumors (p = 0.007). In a multivariate analysis, survival benefit of patients with p16 positive OPSCC was independent of clinico-pathological parameters such as cT and cN classification and treatment modality. The improved prognosis of p16 positive OPSCC is found after RT as well as after surgery.

Although incidence of head and neck squamous cell carcinoma (HNSCC) in general has decreased progressively during the last two decades,1 an increased incidence of oral and oropharyngeal squamous cell carcinoma (OPSCC) was reported in the United States as well as in Europe.2–4 As already suggested in 1983, these OPSCC are characterized by human papilloma virus (HPV) infection.5 Viral DNA of high risk HPV 16 can be detected in nuclei of tonsillar cancer cells,6 and is responsible for the vast majority of HPV positive tumors.7 These tumors arise mainly in the lingual and palatal tonsils and are characterized as being a type of HNSCC with different distinct epidemiological, molecular, and clinical characteristics.8 The main risk factor for these HPV positive OPSCC is sexual behavior with a high number of vaginal or oral sex partners.8, 9 HPV-induced OPSCC show distinct molecular characteristics suggesting a different pathway in carcinogenesis compared to HPV negative HNSCC.10 One particular molecular difference among others concerns p16 expression. p16 functions as a cell-cycle checkpoint regulator, a tumor suppressor gene located on chromosome 9p21. In HPV negative HNSCC p16 is frequently inactivated by deletions, mutations or promoter methylation.11 In HPV positive OPSCC, overexpressed viral oncoprotein E7 degrades pRb,12 which otherwise inhibits p16 transcription.13 The resulting nuclear and cytoplasmic p16 overexpression correlates precisely to HPV positivity and is suggested to be specific for HPV positive OPSCC.14, 15 As p16 overexpression is very rarely seen in HPV negative HNSCC it is considered a surrogate marker for HPV positive OPSCC. Direct diagnosis of HPV in HNSCC is only reliably possible by in situ hybridization, an expensive and elaborate test available only at a limited number of centers.16, 17

In the majority of publications, patients with HPV positive OPSCC show an improved prognosis when compared to patients with HPV negative tumors.18–23 Various, yet unclear possible factors include immune surveillance to viral antigens or absence of field cancerization but also an improved response to RT is discussed. HPV positive tumors treated by RT show a better prognosis than HPV negative OPSCC,21, 24, 25 which leads to the hypothesis of increased radiosensitivity of these cancers. Other authors reported an improved prognosis also in surgically treated HPV positive OPSCC.26 The question arises as to whether these OPSCC benefit from a treatment by RT rather than of primary surgery or whether improved prognosis is independent of the chosen treatment modality. In our study, we compare survival time in a collective of OPSCC p16 positive and negative tumors after primary surgical treatment or primary RT. Treatment modality was chosen for each patient individually in a multidisciplinary pretreatment setting.

Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References

Patients

All patients with HNSCC treated from 1988 to 2003 at the ENT department of the University Hospital Basel were collected. From an initial collective of 696 patients, 365 untreated primary HNSCC specimens with clinico-pathological data were included into the tissue microarray (TMA). The remaining patients were excluded for the following reasons: histology other than squamous cell carcinoma (N = 47), nasopharyngeal cancer (N = 31), second primary cancer after initial curative therapy (N = 30), no histological material at our institute of pathology (N = 61), biopsy too small or frozen section material only (N = 162).

This collective of HNSCC includes curatively treated patients only. Treatment modality was chosen for each patient individually in a multidisciplinary setting based on the official published guidelines (Clinical practice guidelines in head and neck cancers V.I. 2006; http://www.nccn.org). Primary RT or radio-chemotherapy (RCT) was given mainly with a hyperfractionated regimen and 64–74 Gy with or without a concomitant platinum-based chemotherapy, adjuvant RT was given with normal fractionation and 54–64 Gy.

Tissue microarray

A TMA of these 365 unselected and untreated patients was constructed as previously described.27 Formalin-fixed paraffin-embedded pre-treatment biopsies and resected HNSCC were collected from the archives of the Institute of Pathology of the University Hospital Basel. One tissue cylinder with a diameter of 0.6 mm was punched from morphologically representative tissue areas of each donor tissue block and brought into a recipient paraffin block using a homemade semi-automated tissue arrayer.

Immunohistochemical p16 analysis

Four micrometer sections of TMA blocks were transferred to an adhesive coated slide system (Instrumedics, Hackensack, New Jersey) supporting the cohesion of 0.6 mm array elements on glass. Standard indirect immunoperoxidase procedures were used for immunohistochemistry (ABC-Elite, Vector Laboratories, Burlingame, CA) on an automated stainer (Bond® System, Menarini Diagnostics). A monoclonal antibody was used for p16 detection (Clone E6h4, MTM Laboratories AG, Heidelberg, Germany). Optimal staining could be achieved after pretreatment with microwave oven (100°C 20 min. Bond® puffer, dilution 1/400). Nuclei were counterstained with haematoxylin. The primary antibody was omitted as a negative control (Fig. 1).

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Figure 1. Representative immunostaining of p16 in oropharyngeal squamous cell carcinoma. (a) Negative p16 expression (<5% stained tumor cells) and (b) positive p16 expression (>90% stained tumor cells) (Magnification ×20).

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p16 protein expression was scored semi-quantitatively by two independent observers (L.T., E.G.) blinded to clinical parameters. Scores were given by evaluating the proportion of nuclear immunoreactive tumor cells over total number of tumor cells/nuclei ranging from 0% to 100% and the scores of both observers were averaged. Staining intensity was not assessed. Failure of analysis (about 21% of all cases) was related to TMA technology, including a fraction of missing samples (empty spot) and those containing no or only a few tumor cells.

Outcome definitions

The time point of loco-regional recurrence was defined as the date of the histologically/cytologically proven tumor recurrence, either at the site of the initial primary tumor or at the neck.

Overall survival was defined as the total amount of time that a patient survived after initial treatment, including relapses, until the day of death by any reason, confirmed by a legal medical professional.

Statistical analysis section

The association of p16 expression (negative <5%; positive ≥5% immunoreactivity in tumor cells) with clinico-pathological features was performed by the Chi-Square test, Fisher's Exact test and Student's t-test, where appropriate. Univariate tumor-specific survival and recurrence-free survival were evaluated by the Kaplan–Meier method and log-rank test. Considering the number of deaths in the OPSCC subgroup of patients (N = 53), only 3 well-established prognostic factors along with p16 expression and treatment modality could be adequately analyzed in multivariable setting. Therefore, the independent prognostic effect of p16 on outcome in multivariable analysis was determined using Cox regression analysis after adjustment for cT stage, cN stage and treatment modality. The hazard ratio (HR) and 95% confidence intervals (CI) were obtained to determine the effect of each variable on outcome. The assumption of proportional hazards was first verified by analyzing the correlation of Schoenfeld residuals and the ranks of individual failure times. p-values < 0.05 were considered statistically significant. All analyses were performed using SAS (Version 9, Cary, NC).

Results

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References

p16 expression and clinico-pathologic features

Of the 365 patients considered for analysis, p16 expression was evaluable in 288 pretreatment HNSCC biopsies with clinico-pathological and follow-up data. Samples were originally localized, 117 in the oral cavity, 85 in the oropharynx, 48 in the larynx and 38 in the hypopharynx. A statistically significant difference was found in the oropharynx, as this localization was correlated with significantly more p16 positive tumors (57.5%) compared to any of the other tumor sites (5.0–27.5%) (p < 0.001) (Table 1). No differences in p16 expression were found by cT and cN classification or tumor grading, neither was 16 expression associated with loco-regional recurrence. Of the 85 analyzed OPSCC, most tumors were localized in palatal tonsils (N = 40) or base of the tongue (N = 27). p16 positivity did not vary significantly among the four oropharyngeal localizations (palatal tonsil, base of the tongue, soft palate, back wall of the oropharynx) nor among other clinico-pathological parameters such as cT and cN classification or tumor grading.

Table 1. Association of p16 expression with clinico-pathological parameters of HNSCC (N = 288)
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Impact of p16 expression on survival (univariate analysis)

Overall survival analysis was possible for 267 patients with complete clinico-pathological data for multivariable analysis. The median survival time of the entire cohort was 74.0 (95% CI 52–145) months. A trend toward improved 5-year overall survival time with p16 positivity was observed (p = 0.097). 5-year survival rate for p16 negative and positive patients were 39.2% (95% CI: 32–46) and 59.2% (95% CI: 44–72), respectively (Fig. 2a). Similar results were observed in patients with OPSCC; namely prognosis for patients with p16-positive cancers was significantly more favourable compared to p16-negative cases (p = 0.007). Specifically, 5-year survival rates for p16-negative and -positive patients were 26.8% (95% CI: 15–41) and 57.1% (95% CI: 37–73), respectively (Fig. 2b). p16 status did not demonstrate an influence on survival time in patients with tumors in other localizations, such as oral cavity (p = 0.171), larynx (p = 0.519) and hypopharynx (p = 0.593).

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Figure 2. Kaplan–Meier survival curve demonstrating differences in survival time for patients with positive (≥5%) and negative (<5%) expression of p16 in (a) all head neck squamous cell carcinomas of different localizations, (b) oropharyngeal squamous cell carcinoma only. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

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The impact of p16 expression on outcome of OPSCC after treatment by either surgery or surgery with adjuvant RT and RT with or without concomitant platinum-based chemotherapy was subsequently evaluated. In the group of 57 patients with survival information undergoing surgery with or without adjuvant RT, a favourable effect of p16-positivity on outcome was observed (Fig. 3a). Twenty-six patients were positive for p16 of whom 10 died of disease (5-year survival rate (95% CI): 76.7% (55–89)). Twenty-one of 31 patients with p16-negative tumors died from OPSCC (5-year survival rate (95% CI) = 41.5% (23–59)). This difference was determined to be marginally significant (p = 0.067), with a trend toward improved outcome in patients with p16-positive cancers.

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Figure 3. Kaplan–Meier survival curves demonstrating significant differences in survival time for patients with positive (≥5%) and negative (<5%) expression of p16 in oropharyngeal squamous cell carcinoma, treated by (a) surgery with or without adjuvant radiotherapy and (b) primary radiotherapy or radiochemotherapy. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

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Of the 25 patients treated with RT and RT/CT, only 8 cases were positive, including 6 patients who died of disease. In contrast, of the 17 patients with p16-negative tumors, 16 patients died of disease prior to or at 5-year follow-up. This difference in survival time again exhibited a trend toward improved prognosis (p = 0.096) in the RT or RCT arm in patients with p16-positivity (Fig. 3b).

Independent prognostic effect of p16 in HNSCC and OPSCC

Multivariable survival analysis was carried out to determine whether the effect of p16 protein expression on prognosis was independent of other well-established prognostic features. Taking into consideration the sample size of OPSCC and the total number of deaths in this subgroup of patients, the effect of p16 was determined after adjustment for cT classification, cN classification, and the treatment modality. In the entire group of HNSCC, only cT and cN classification had an independent effect on prognosis, while p16 status was not found to contribute additional prognostic information. However, in the subgroup of patients with OPSCC, p16 positivity was determined to have a significant beneficial effect on outcome (p = 0.01; HR (95%CI): 0.47 (0.3–0.9), maintained after considering cT and cN classification and treatment modality (Table 2).

Table 2. Multivariable survival analysis demonstrating the prognostic impact of p16 expression in HNSCC and OPSCC after adjustment for CT and CN classification and treatment modality
  1. HR, hazard ratio; CI, confidence interval; RT, radiotherapy; RCT, radiochemotherapy.

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Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References

Among the whole group of HNSCC we can differentiate a distinct subgroup of OPSCC, which are induced by HPV infection rather than by smoking and alcohol consumption. HPV positive OPSCC show a different expression of various molecular tumor markers, such as p16. p16 is over-expressed in most HPV positive OPSCC and considered a surrogate marker for HPV infection in these carcinomas.28 We investigated the effect of p16 expression on prognosis of HNSCC in general and OPSCC in particular. The novel findings in our study show that p16-positive protein expression in OPSCC is an independent and favorable prognostic factor in these patients, not only after RT or RCT but equally after surgery with or without adjuvant RT.

p16 positivity corresponded significantly with tumor localization in the oropharynx, the tumor site in the upper aerodigestive tract most often affected by HPV infection.29 Unlike HPV infection, carcinogenesis induced by alcohol and nicotine consumption leads to a defect of the p16 gene, being the most frequent genetic alteration in these carcinomas.30 In the whole group of HNSCC, p16 expression did not correlate with cT and cN classification although others found significantly more p16 negative tumors with advanced T stages.31 These contrary results must be compared cautiously, as Yuen et al. analyzed only 5% (12 out of 225) OPSCC compared to 28% (52 out of 186) in our TMA. As in other publications, tumor grading of HNSCC did not correlate to p16 expression.31, 32

p16 expression did not influence loco-regional tumor control but prognosis in our heterogeneous collective of HNSCC samples. The impact of the survival benefit for patients with p16 positive tumors relied on the highly significant correlation to improved survival in the included 28% of OSCC. This could also be the reason for the similar results reported by Karsai et al. presenting a collective of 670 HNSCC including 26% OSCC.33 In other localizations, no relevant prognostic difference by p16 expression was found. This corresponds to the vast majority of publications, confirming significantly improved overall survival of HPV induced p16 positive OPSCC only.15, 28, 31, 32, 34–36

Focusing on OSCC, most tumors in our TMA were localized in the palatal tonsils or in the base of the tongue. As reported by others, p16 expression did not differ between the four different localizations nor did it correlate with clinico-pathological parameters cT and cN classification or tumor grade.15, 35 Additional prognostic parameters such as anemia or performance status could not be included in this retrospective analysis do to lack of this data in a majority of patients. Furthermore, loco-regional tumor relapse was independent of p16 expression in OPSCC. A significant correlation was found between p16 expression and improved overall survival, with p16 negative patients demonstrating more than a 2-fold greater risk of death compared to p16-positive patients. A majority of investigators analyzing prognostic relevance of HPV infection and p16 expression in OPSCC confirmed our results.18, 19, 22, 28, 35 Although p16 is considered a biomarker for HPV infection in OPSCC and prognostic information of both markers is reported to match independently in the above mentioned studies, Li et al. reported prognostic relevance of HPV status only but not of p16 expression alone.15

After analyzing the whole collective of OPSCC differentiated by p16 expression only, we compared overall survival in the two main treatment groups, surgery alone or with adjuvant RT with primary RT or combined RCT. p16 positive OPSCC treated by primary RT or RCT, exhibited a clear trend to better prognosis compared to p16 negative OSCC. Similar results were published by others.25, 37 Jayasurya et al. further differentiated oral squamous cell carcinoma treated by normal from altered fractionation. Loss of p16 expression combined with cyclin D1 overexpression correlated to worse prognosis in the 90 tumors treated by altered fractionation only. As we apply primary radiotherapy (RT) generally in a hyperfractionated regimen, their results confirm our findings. Whether the type of fractionation plays a significant role in treatment of p16 positive OPSCC will have to be part of prospective randomized trials. Various authors mention, the improved overall survival after RT or RCT in p16 positive OSCC to relay on increased radiosensitivity of these particular carcinomas. We consider this conclusion to be drawn cautiously, as some publications summarized various RT modalities as primary RT, RCT and adjuvant RT after radical surgery in one and the same group.15, 24, 34, 35, 38, 39 A conclusion regarding radiosensitivity should be based on results of tumors treated with primary RT or RCT only, which are directed against the tumor volume as a whole. Primary RT normally differs from adjuvant RT in decreased dosage and single fractionation directed against possible microscopic tumor cell remains, utilized after primary radical surgery.

One of the limitations of our study is the small sample size of OPSCC, which first may explain the non-significant difference in survival time stratifying patients by treatment modality and moreover, played a role in restricting our multivariable analysis of the independent effect of p16 on outcome to only the most relevant prognostic factors, namely cT and cN classification and treatment modality. Our study represents a retrospective analysis of follow-up data of a head and neck cancer team, which evaluates treatment choice for each patient individually in a multidisciplinary setting. The aim of this conjoint evaluation is optimal oncological outcome combined with best function and least long-term side effects by the chosen treatment modality, therefore treatment groups include tumor samples not randomized by stage and treatment modality. As a consequence we can not compare outcome of surgically treated tumors with those treated by RT/RCT, as small tumors were treated more frequently surgically, advanced stage tumors requiring extensive, more debilitating surgery were more often treated by organ sparing RT or RCT. Nevertheless, we can compare the influence of p16 expression on overall survival in both treatment groups. Reports on prognosis of HPV induced p16 positive OPSCC including tumor collectives treated by different treatment modalities such as surgery,26, 37 RT,37 RCT25, 40 or OPSCC treated by various, but summarized modalities.15, 20, 21, 24, 34, 35, 38, 39 In addition, our single-institutional study requires validation in large prospective trials prior to implementation of p16 as a potential prognostic marker in OPSCC.

Underlying mechanisms for proposed increased radiosensitivity of p16 expressing OSCC are unclear although different hypotheses are discussed. p53 degradation related to the viral oncoprotein E6 in HPV positive tumors might not be functionally as relevant compared to functional loss of p53 depending pathways after p53 mutations found in HPV negative HNSCC.41, 42

Our results suggest that p16 expression is of considerable prognostic value, particularly in patients with OPSCC. p16 positivity confers a survival benefit in these patients which is independent of clinico-pathological parameters such as cT and cN classification. HPV induced p16 positive OPSCC are a distinct type of HNSCC with a generally better outcome than p16 negative carcinomas, which may be independent of the treatment modality chosen.

References

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References
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