Transoral Robotic Surgery and Adjuvant Therapy for Oropharyngeal Carcinomas and the Influence of p16 INK4a on Treatment Outcomes

The Laryngoscope VC 2013 The American Laryngological, Rhinological and Otological Society, Inc. Transoral Robotic Surgery and Adjuvant Therapy for Oropharyngeal Carcinomas and the Influence of p16 INK4a on Treatment Outcomes Harry Quon, MD; Marc A. Cohen, MD; Kathleen T. Montone, MD; Amy F. Ziober, JD; Li Ping Wang, MD; Gregory S. Weinstein, MD; Bert W. O Malley, Jr., MD Objectives/Hypothesis: To determine the prognostic influence of p16 INK4a immunohistochemistry on the survival of resectable oropharyngeal carcinomas (OPSCC). Study Design: Retrospective pathologic evaluation of a prospective single-arm cohort study at a tertiary referral center. Methods: There were 48 patients with resectable OPSCC who consented for transoral robotic surgery (TORS) and banked tissue specimen for assessment. TORS was with or without adjuvant radiation or chemoradiation. Main outcome measures were p16 INK4a status, human papillomavirus status, local-regional disease control, and overall, disease-specific, and disease-free survival. Results: p16 INK4a and HPV positivity were identified in 73% and 74% of patients respectively. With a median follow-up of 38.8 months (2.5 63.3 months), only one local-regional relapse has occurred in both the p16 INK4a -positive and p16 INK4a - negative cohorts. No disease-specific, disease-free, and overall survival differences were observed between p16 INK4a -positive and p16 INK4a -negative patients (P ¼.446, P ¼.277, P ¼.643, respectively). Conclusions: p16 INK4a was not prognostic in resectable OPSCC when treated with an initial TORS approach. Key Words: p16 INK4a, prognosis, oropharyngeal carcinoma, human papilloma virus, transoral robotic surgery. Level of Evidence: 2b. Laryngoscope, 123:635 640, 2013 INTRODUCTION A significant advancement in the management of oropharyngeal squamous cell carcinomas has been the consistent demonstration that human papillomavirus (HPV)-associated oropharyngeal carcinomas (HPV- OPSCC) have a favorable prognosis. 1,2 This appears to be independent of the treatment approach, be it radiation therapy, 3 chemoradiation, 2,4 or surgery. 5 The mechanism for this is unclear, but evidence suggests that the favorable prognosis may in part be dependent on an intact and functional p16 INK4a protein expression. 5,6 Recently, our group demonstrated that the survival outcome for HPV-negative OPSCC when treated surgically with the transoral robotic surgical (TORS) technique was not statistically different from HPV-positive OPSCC. 7 This observation is contrary to other reports where From the Department of Radiation Oncology (H.Q.), Department of Pathology and Laboratory Medicine (K.T.M., A.F.Z., L.P.W.), Department of Otorhinolaryngology Head and Neck Surgery (H.Q., M.A.C., G.S.W., B.W.O.), Abramson Cancer Center (H.Q., K.T.M., G.S.W., B.W.O.), University of Pennsylvania (H.Q., K.T.M., G.S.W., B.W.O.), Philadelphia, Pennsylvania, U.S.A. Editor s Note: This Manuscript was accepted for publication April 18, 2011. The authors have no funding, financial relationships, or conflicts of interest to disclose. Harry Quon, MD, Gregory S. Weinstein, MD, and Bert W. O Malley, Jr., MD, contributed equally to this work as first authors. Send correspondence to Harry Quon, MD, MS, Johns Hopkins University, Department of Radiation Oncology and Molecular Radiation Sciences, 410 North Broadway, Suite 1440, Baltimore, MD 21231-2410. E-mail: hquon2@jhmi.edu DOI: 10.1002/lary.22172 OPSCC and head and neck squamous cell carcinoma (HNSCC) have been treated nonsurgically. 2 As daily fractionated radiotherapy is an oxygen-dependent process for there to be effective local-regional control (LRC) of HNSCC, 8 we hypothesized that the absence of survival difference between HPV-positive and HPV-negative OPSCC may be attributed to the beneficial effects of adding TORS surgery in combination with radiation and chemotherapy as pathologically indicated. As such, we sought to analyze the outcomes of p16 INK4a -positive and p16 INK4a -negative OPSCC patients treated with an upfront TORS approach. MATERIALS AND METHODS Patients analyzed were prospectively enrolled on a clinical trial evaluating the safety and efficacy of TORS. The primary aim of this study was to evaluate the influence of p16 INK4a in newly diagnosed oropharyngeal carcinomas treated initially with TORS. The outcome data for this trial was collected prospectively from a pool of 162 patients enrolled in a phase I protocol approved by the institutional review board at the Hospital of the University of Pennsylvania (Philadelphia, PA). The inclusion and exclusion criteria have previously been reported. 9 Of the 162 patients enrolled in the TORS protocol, nine patients did not receive surgery at all. Of the 153 patients receiving some type of surgery, three patients were excluded from TORS during a preoperative endoscopy that demonstrated that exposure for TORS was suboptimal. Of these final 150 patients who received TORS, there were 79 patients identified with previously untreated, biopsy-proven, squamous cell carcinoma of the oropharynx. Of these 79 patients, there were tumor-banked specimens for 48 patients for p16 INK4a 635

Patient Characteristics TABLE I. Summary of Patient Characteristics. No. of Patients (N¼48) T1-2 37 T3-4 11 N0-1 29 N2-3 19 Adjuvant radiotherapy, no (%) 12 (25) Adjuvant chemoradiotherapy, no (%) 25 (52 HPV positive:hpv negative, no (%) 37 (74):13 (26) p16 INK4a positive:p16 INK4a negative, no (%) 35 (73):13 (27) HPV positive/p16 INK4a positive, no (%) 32 (66) HPV positive/p16 INK4a negative, no (%) 4 (8) HPV negative/p16 INK4a positive, no (%) 3 (6) HPV negative/p16 INK4a negative, no (%) 9 (19) HPV ¼ human papillomavirus. assessment. These patients underwent TORS from May 24, 2005 to November 5, 2007. Clinical data, including age, gender, tumor, and American Joint Committee on Cancer stage, histopathological findings, resection margins, recurrence, speech and swallowing outcomes, and mortality were obtained prospectively with the last data collection performed in February 2009. The procedures for TORS of the oropharynx have been described in detail previously. 10,11 The surgical sites of 47 of the 48 patients were allowed to heal by secondary intention, with one early patient undergoing free tissue transfer. TORS was performed with the da Vinci surgical system (Intuitive Surgical, Inc., Sunnyvale, CA) in all cases. A thorough description of the pathologic assessment of tumor specimens has been described previously. 11 The indications for adjuvant therapy with intensity modulated radiation therapy with or without concurrent cisplatin have been described previously. 11 Briefly, indications for postoperative concurrent chemotherapy (typically, cisplatin) included the presence of nodal extracapsular extension and/or positive surgical margins. Relative indications for concurrent chemotherapy included multiple positive lymph nodes. Absolute indications for postoperative radiation without chemotherapy included the presence of two or more positive lymph nodes or positive surgical margins. We defined close or positive margins at the University of Pennsylvania as <2 mm. Relative indications for adjuvant radiation included T4 lesions with infiltrative growth patterns, perineural invasion, lymphovascular invasion, and the presence of one pathologically positive lymph node. In the early years as experience with the TORS technique was developing, there was a tendency to favor postoperative radiotherapy due to the uncertain negative predictive value of a negative margin (designated as 2 mm or greater) when evaluating a new surgical technique. p16 INK4a Immunohistochemistry Paraffin-embedded formalin-fixed tissue specimens of the primary oropharyngeal tumor were available for analysis. A total of 48 patients had sufficient material for analysis. There were 5-lm sections of formalin-fixed and paraffin-embedded biopsy samples processed by the CINtec Histology Kit (MTM Laboratories, Inc., Westborough, MA). Sections were de-waxed by passage through xylene and rehydrated by a graded series of ethanol, followed by boiling in 10 mmol/l of citrate buffer (ph 6.0) for 15 minutes. Endogenous peroxidase was inactivated by 3% H 2 O 2 in phosphate-buffered saline (PBS) (ph 7.4) for 10 minutes. After rinsing with PBS, sections were incubated at room temperature for 45 minutes with anti-p16 INK4a mouse monoclonal antibody (clone E6H4 TM ; MTM Laboratories, Inc.). After rinsing thoroughly with PBS, the slides were incubated with Visualization Reagent (MTM Laboratories, Inc.) for 30 minutes. After washing with PBS, visualization was performed with diaminobenzidine tetrahydrochloride (MTM Laboratories, Inc.), and sections were counterstained with hematoxylin. Strong nuclear staining as well as strong cytoplasmic staining were considered positive for p16 INK4a expression. Immunostaining was graded and scored on whole sections according to Klaes et al. 12 as follows: negative (<1% of the cells were positive), sporadic (isolated cells were positive, but <5%), focal (small cell clusters, but <25% of the cells were positive), and diffuse positive (>25% of the cells were stained). For survival analyses, immunoreactivity of >25% of the tumor cells were scored as p16 INK4a positive, <75% as p16 INK4a negative. HPV Polymerase Chain Reaction HPV type was assessed with the AttoSense HPV test (Sensi- Gen, Ann Arbor, MI) in an identifier blinded fashion by Sensigen. The AttoSense HPV test is a commercially available assay that uses a competitive real-time quantitative polymerase chain reaction technique in addition to flight mass spectroscopy, the details of which have been previously published. 13 Primers for serotype specific E6 allowed for detection of high-risk HPV types. These include HPV-16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68. 13 Standards allowed for quantification of each individual HPV serotype copies per cellular genome copy, and 10% percent of all samples were tested in duplicate to assure accuracy. Statistical Analysis Statistical analyses were computed using Statistical Package for Social Sciences version 17.0 (SPSS, Inc., Chicago, IL). Nonparametric statistics were performed using Pearson s v 2 and the Fisher exact test. Overall survival was defined as the time from TORS to the date of death. Disease-specific survival was defined as the time from TORS to the date of death secondary to OPSCC. Death without evidence of disease involved censorship at the patient s date of death. Disease-free survival (DFS) was defined as the time from TORS until disease recurrence or the date of death. Patients who were lost to follow-up were censored as per the disease status at the last clinic visit. Two patients total were lost to follow-up prior to 18 months follow-up with phone number changed and no response to letters. These two patients were p16ink4a-negative patients, one lost at 2.5 months without evidence of disease and one at 6.0 months following regional and distant disease recurrence. At last contact with the patient with disease recurrence, the patient was living with disease. The Kaplan-Meier method was used for analysis of survival data, and survival plots were compared using the logrank test. All statistics were two-tailed, and items were considered statistically significant with a P value <.05. RESULTS Patients analyzed were accrued and treated with TORS and adjuvant therapy as pathologically indicated from May 24, 2005 to November 5, 2007. Patient characteristics are summarized in Tables I and II. The majority of the patients were both HPV positive and p16 INK4a positive. Only 6% were HPV negative but p16 INK4a positive that would be expected to favorably influence the outcome of the HPV-negative cohort. There was a high degree of 636

TABLE II. Summary of T and N Stages for Patients by p16 INK4a Status. T1 T2 T3 T4 Total (%) p16 INK4a negative N0 1 2 0 1 4 (31) N1 2 2 0 0 4 (31) N2 3 1 0 1 5 (38) Total (%) 6 (46) 5 (39) 0 (0) 2 (15) 13 p16 INK4a positive N0 2 2 1 0 5 (14) N1 4 10 1 1 16 (46) N2 2 6 6 0 14 (40) Total (%) 8 (23) 18 (51) 8 (23) 1 (3) 35 concordance between the HPV and p16 INK4a status in 85% of the patients. Adjuvant therapy was administered in 85.7% of p16 INK4a -positive and 69.2% in p16 INK4a -negative patients. Of these, concurrent chemoradiotherapy was administered in 51.4% and 53.8%, respectively. Positive surgical margins were seen in 0% of p16 INK4a -positive and 7.7% in p16 INK4a -negative patients. The survival outcomes by HPV status have been previously been reported. 7 Table II summarizes the T and N stage by p16 INK4a status. Although there was more T1 disease (46% vs. 23%) and combined more T1 and T2 disease (85% vs. 74%) in the p16 INK4a -negative cohort compared to the p16 INK4a -positive cohort, there was comparable N2 disease in both study cohorts. Comparisons between the p16 INK4a -negative and the p16 INK4a -positive cohorts demonstrated a trend toward higher T-stages in the p16 INK4a -positive cohort (P ¼.06). Comparisons of nodal stages revealed a nonsignificant difference between the two p16 INK4a cohorts (P ¼.389). With a median follow-up of 38.8 months (mean, 36.5 months; range, 2.5 63.3 months), only one local-regional relapse has occurred in both the p16 INK4a -positive and p16 INK4a -negative cohorts. As such, Figure 1, which demonstrates the overall actuarial local-regional disease control rates, was not separated by p16 INK4a status. Fig. 2. Actuarial disease-specific survival by p16ink4a status. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] Figure 2 demonstrates the actuarial disease-specific survival rates by p16 INK4a status. No survival differences were observed (P ¼.446). The relapse pattern is summarized in Table III, which demonstrates that distant relapses appear to be the dominant pattern of relapse. Figure 3 demonstrates the actuarial DFS rates by p16 INK4a status. No significant differences were observed (P ¼.277). The DFS rate for the p16 INK4a -negative cohort was influenced by one patient with relapse (Table III), who died from other causes following successful surgical salvage, and two patients who died due to other causes. Figure 4 demonstrates the overall survival rates. This also demonstrated no statistically significant difference between the p16 INK4a -positive and p16 INK4a -negative cohorts (P ¼.643). When analyzed by both HPV and p16 INK4a status, the survival analysis is limited by the small patient numbers for the HPV-positive/p16 INK4a -negative and HPV-negative/p16 INK4a -positive cohorts. All patients in these discordant cohorts are free of recurrent disease. DISCUSSION The results reported suggest that p16 INK4a may not have a significant influence on the treatment outcomes of HPV-OPSCC when treated with an initial surgical approach using the TORS technique followed by adjuvant radiotherapy as pathologically indicated. Cohen TABLE III. Summary of Relapse Patterns by p16 INK4a Status. Fig. 1. Actuarial local-regional disease control rates. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] Relapse Pattern No. of p16 INK4a - Positive Patients No. of p16 INK4a - Negative Patients Local alone 0 0 Regional alone 0 0 Local and regional 1 0 Regional and distant relapse 0 1 Distant relapse alone 2 0 637

Fig. 3. Actuarial disease-free survival by p16ink4a status. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] Fig. 4. Actuarial overall survival by p16ink4a status. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] Reference TABLE IV. Summary of Prognostic Studies for Survival of p16 INK4a Protein Expression Evaluated by Immunohistochemistry. p16 INK4a Prognostic Significance HPV Prognostic Significance Patient Population p16 INK4a -Monoclonal Antibody p16 INK4a -Positive Definition Primary surgical therapy Licitra et al. 5 Negative Positive Oropharynx NS* NS Fischer et al. 20 Positive NA All sites E6h4 a Rich et al. 21 Positive Negative Oropharynx NS b Klussman et al. 19 Positive NA Oropharynx 16p04 and 16p07* c Current series Negative Negative Oropharynx E6h4 d Primary radiation therapy Lassen et al. 3,17 Positive NA All sites JC8 e Rischin et al. 18 Positive NA Oropharynx NS* f Kumar et al. 22 Positive Positive Oropharynx 16p04 k g Higuchi et al. 23 Positive NA All sites Ab-7* h Shi et al. 24 Positive Positive Oropharynx E6h4 i Fischer et al. 20 Positive NA All sites E6h4 a Mixture of treatment approaches Smith et al. 6 Positive Positive All sites E6h4 j Li et al. 25 Positive Positive Oropharynx Ab-4/16p04 clone* k HPV ¼ human papillomavirus; NS ¼ not specified; NA ¼ not available; a ¼ nuclear accounting only for the proportion of cells and not staining intensity with <5% staining negative and >5% staining positive; b ¼ intracellular location not specified accounting only for the proportion of cells and not staining intensity with no staining negative and >75% staining positive; c ¼ nuclear and cytoplasmic with semiquantitative scores accounting only for the proportion of cells and not staining intensity: <25% staining negative and >25% staining positive; d ¼ nuclear and cytoplasmic with semiquantitative scores accounting only for the proportion of cells and not staining intensity: <25% staining negative and >25% staining positive; e ¼ nuclear and cytoplasmic with semiquantitative scores accounting only for the proportion of cells and not staining intensity: <10% staining negative and >10% staining positive; f ¼ nuclear and cytoplasmic with semiquantitative scores accounting only for the staining intensity: 0 (none), 1 (weak), 2 (moderate), or 3 (strong), with 0 or 1 scores defined as negative and 2 or 3 defined as positive; g ¼ intracellular location not specified accounting only for the proportion of cells and not staining intensity: 1 was <5%, 2 was 5% to 20%, 3 was 21% to 50%, and 4 was 51% to 100% tumor staining with definition of p16 positivity not specified; h ¼ nuclear staining accounting only for the proportion of cells staining with >1% staining positive; i ¼ nuclear and cytoplasmic with positivity defined as staining observed in both the nucleus and cytoplasm; j ¼ nuclear and cytoplasmic with semiquantitative scores accounting only for the proportion of cells and not staining intensity: 1 is negative for staining, 2 is slightly positive for staining, 3 is 10% to 80% positive, 4 is >80% positive; 5 is every tumor cell positive (slides in category 1 were defined as negative and 2 to 5 were positive; k ¼ intracellular location not specified accounting only for the proportion of cells and not staining intensity with <5% staining negative and >5% staining positive. *Neomarkers, Fremont, CA. MTM Laboratories AG, Heidelberg, Germany. MTM Laboratories CINtec, Westborough, MA. Santa Cruz Biotechnology, Inc., Santa Cruz, CA. k Lab-Vision, Fremont, CA. 80% of treated patients received radiotherapy. 638

et al. had reported similar nonsignificant results seen when analyzed by HPV status. 7 As Smith et al. have recently observed that p16 INK4a protein expression could influence the survival outcome of HPV-positive HNSCC patients, 6 we had speculated if the favorable survival seen in our HPV-negative patients may be due to a favorable biologic influence of an intact p16 INK4a protein expression. This analysis demonstrated a high concordance between p16 INK4a and HPV status and suggests that our TORS approach may possibly be improving the outcome of HPV-negative p16 INK4a -negative patients. Only 6% of our study patients were HPV negative/ p16 INK4a positive. The favorable prognosis associated with HPV oncogenesis for oropharyngeal carcinomas is now well recognized. 2,4,5,14 Although the biologic explanation for this is unclear at this time, several reports have suggested that this may be due to an increased radiosensitivity associated with HPV oncogenesis. 15,16 Gupta et al. demonstrated a significant difference in clonogenic radiosensitivities between two HPV-16 naturally infected head and neck cell lines compared to the HPVnegative SQ-20B head and neck cell line. 16 The degree of sensitivity correlated with the activation of the downstream Akt protein, and further radiosensitization could be achieved with Akt inhibition with the HIV protease inhibitor, nelfinavir. 16 Other potential mechanisms, including the influence of the tumor microenvironment on tumor radiosensitivity, have also been suggested. It is important to note that there are clear limitations in attributing a biologic explanation to the lack of prognostic impact of p16 INK4a on survival in our study population. The most relevant is the influence of selection bias that can occur with any institutional clinical series with a modest study population size and the low event rate that has been observed to date despite a median follow-up of 38.8 months. The findings reported should be more appropriately regarded as hypothesis generating and require further validation. With this in mind, several recent investigations provide a framework on which future investigations may be directed to improve the understanding of how p16 INK4a may be influencing the prognosis of OPSCC. Recent investigations suggest that tumor hypoxia may be more clinically important in the radiosensitivity of p16 INK4a - negative HNSCC cells based on the retrospective analysis of two large randomized trials that studied the oncologic outcome of hypoxic radiosensitization. 17,18 In the Danish Head and Neck Cancer Group (DAHANCA) 5 trial, Lassen et al. demonstrated that only in the p16 INK4a -negative cohort was there a statistically significant difference in LRC rates between patients randomized to radiotherapy and those receiving radiotherapy, and the concurrent hypoxic radiosensitizer nimorazole (5-year LRC, 20% vs. 37% respectively; 95% confidence interval [CI], 0.50 0.95). 17 In the p16 INK4a - positive cohort, the 5-year LRC rate was almost identical (59% with nimorazole, 58% in the control group [hazard ratio (HR) ¼ 0.93; 95% CI, 0.45 1.91]). In the Trans-Tasman Radiation Oncology Group (TROG) 02.02 trial, Rischin et al. demonstrated a trend favoring the tirapazamine, cisplatin, and radiotherapy arm compared to the cisplatin and radiotherapy arm for improved localregional disease control only in the p16 INK4a -negative patients (HR ¼ 0.33; 95% CI, 0.09 1.24; P ¼.13). The high LRC rates observed in our study population (Fig. 1) may be consistent with the hypothesis that surgical resection may be more effective in p16 INK4a - negative OPSCC. However, we note that only one localregional event occurred in each of the p16 INK4a -positive and p16 INK4a -negative cohorts, and thus may be influenced by the relatively modest size of the study population. In the p16 INK4a -negative OPSCC group, no local relapses have been observed to date. If p16 INK4a is influencing its prognostic significance through its relationship with the tumor oxygenation status, one might speculate that the impact of surgery on its prognostic significance may possibly be related to the burden of any residual microscopic disease requiring postoperative radiotherapy. In contrast, p16 INK4a -negative patients treated definitively with radiotherapy alone may show a more consistent detrimental prognostic effect. Table IV summarizes the studies that have reported on the prognostic impact of p16 INK4a protein immunohistochemistry (IHC). The most noteworthy observation is a consistent prognostic influence of p16 INK4a IHC in reports that have treated HNSCC with fractionated radiotherapy. Table IV also demonstrates that the adverse prognosis occurred despite the use of variable monoclonal antibodies and definitions for a positive p16 INK4a tumor. To date, no other TORS series has analyzed the impact of HPV or p16 INK4a IHC on the outcome of an upfront TORS approach. However, Rich et al. did analyze their results using a transoral laser microsurgical approach, which serially resects portions of the oropharyngeal tumor until a tumor normal-tissue interface appears to have been reached. Thus, the confidence one may have in the margin status after a complete surgical resection can be limited. The study population analyzed by Klussman et al. noted a positive margin rate of 11%. 19 The positive margin rate was not reported in the series by Fischer et al. 20 or the proportion of patients needing postoperative radiotherapy. CONCLUSION The results reported suggest that p16 INK4a may not have a significant influence on the survival rates of oropharyngeal carcinomas treated with an upfront TORS approach. Further validation of these results is required. 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