2.1

Patients

The medical records of all patients with head and neck cancer who presented to a single institution from 2009 to 2014 were retrospectively reviewed in an attempt to identify cases of LSCC and HSCC. Only patients with known p16 status were included in this analysis. Approval was obtained from the institutional human investigations committee prior to collection of all patient information. All patients were treated definitively with either primary surgery or intensity-modulated radiation therapy. Pre-treatment workup consisted of standard history and physical examination including direct laryngoscopy as well as positron emission tomography (PET). Patients who presented for palliative treatment and/or with known distant metastases were excluded. Thirty-one patients: 23 LSCC and 8 HSCC patients satisfied all inclusion criteria and continued on for further chart analysis.

Among all 31 cases, 22 patients were treated with primary radiation therapy (13 of whom received concurrent chemotherapy, typically using a platinum-based regimen), and 9 patients were treated by primary surgery. Of the 9 patients treated with primary surgery, 8 (89%) received adjuvant radiotherapy and 4 (44%) received adjuvant chemoradiation, typically utilizing single-agent cisplatin. The median radiation dose was 66 Gy (range, 60–70 Gy) using conventional fractionation. Doses to prophylactically-treated nodal regions ranged from 54 Gy to 63 Gy. Treatment is further summarized in Table 1 .

2.2

Data collection

Clinical characteristics noted included age, race, gender, tumor site, tobacco use, alcohol use, ECOG performance status, tumor location, T-stage, N-stage, and other oncologic history. Age was examined as a continuous variable. Tobacco exposure included cigarettes, cigars, pipes, chewing tobacco, and snuff. Tobacco use included the patient’s lifetime history of current, former, or never users. For former smokers, years since cessation were also recorded. Tobacco cumulative exposure was recorded in pack–years and grouped by exposure into two categories of <10 pack–years and ≥10 pack–years . Due to the small sample size, the continuous variable of pack–year history was stratified into these three categories. Alcohol exposure included beer, wine, or liquor (One drink is equivalent to a 12 oz beer, 4 oz. wine, or 1.5 oz of liquor). The patient’s lifetime history reflected current, former, or never users of alcohol >7 drinks/week. Alcohol cumulative exposure was positive for moderate use if >7 drinks/week or heavy use if >14 drinks/week.

2.3

Laboratory studies

Standard patient care at our institution has included testing for p16 expression through immunohistochemistry (IHC) or HPV infection through in situ hybridization (ISH) in HNSCC. All patients included in this study were previously evaluated for p16 expression from a tumor biopsy or surgical resection. The positivity of p16 and HPV status was recorded based on the clinical pathology report.

Immunohistochemistry testing for p16 expression followed the protocol supplied by the Ventana CINtec p16INK4a Histology Kit #9517. Immunohistochemistry was performed on the Leica Bond III autostainer using Leica ancillary reagents. Anti-P16 monoclonal antibody clone E6H4 immunostaining involved heat-induced epitope retrieval in High pH-EDTA buffer for 20 min, primary antibody incubation for 15 min, and application of Leica Refine (DAB) detection kit. Slides were counterstained with Refine hematoxylin. The p16 immunohistochemistry stain was considered positive if nuclear and cytoplasmic staining was greater than 70% of the cells .

HPV in situ hybridization was performed on the Ventana Ultra autostainer using DNA probes and ancillary reagents from Ventana Medical Systems up until July 9, 2014. Subsequent HPV ISH cases were sent to Integrated Oncology using the same Ventana method. The Ventana INFORM® HPV III probe set was used including the low-risk genotypes 6 and 11 and high-risk genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 66. A DNA probe was also run to assess integrity of tissue DNA. In situ hybridization for high-risk and low-risk HPV genotypes was considered positive when nuclear-specific staining was detected with all others were recorded as negative.

2.4

Statistical analysis

The following definitions were used for statistical analyses: overall survival was the time between initial treatment and the date of death from any cause. Locoregional failure was the time between initial treatment and the date of first local or regional recurrence as detected by radiologic imaging or clinical examination. Distant failure was the time between initial treatment and the date of first local or regional recurrence as detected by radiologic imaging or biopsy. Patients who were living at the last date of follow-up without recurrence were censored at the last date of follow-up. The Kaplan–Meier method was used to estimate overall survival (OS) and locoregional control (LRC) through a 48 month study period. The overall survival and locoregional failure-free survival estimates were reported for 12 and 24 months with 95% confidence intervals for all patients together and stratified by p16 status. The log rank test was used to determine predictors for locoregional failure.

2.1

Patients

The medical records of all patients with head and neck cancer who presented to a single institution from 2009 to 2014 were retrospectively reviewed in an attempt to identify cases of LSCC and HSCC. Only patients with known p16 status were included in this analysis. Approval was obtained from the institutional human investigations committee prior to collection of all patient information. All patients were treated definitively with either primary surgery or intensity-modulated radiation therapy. Pre-treatment workup consisted of standard history and physical examination including direct laryngoscopy as well as positron emission tomography (PET). Patients who presented for palliative treatment and/or with known distant metastases were excluded. Thirty-one patients: 23 LSCC and 8 HSCC patients satisfied all inclusion criteria and continued on for further chart analysis.

Among all 31 cases, 22 patients were treated with primary radiation therapy (13 of whom received concurrent chemotherapy, typically using a platinum-based regimen), and 9 patients were treated by primary surgery. Of the 9 patients treated with primary surgery, 8 (89%) received adjuvant radiotherapy and 4 (44%) received adjuvant chemoradiation, typically utilizing single-agent cisplatin. The median radiation dose was 66 Gy (range, 60–70 Gy) using conventional fractionation. Doses to prophylactically-treated nodal regions ranged from 54 Gy to 63 Gy. Treatment is further summarized in Table 1 .

2.2

Data collection

Clinical characteristics noted included age, race, gender, tumor site, tobacco use, alcohol use, ECOG performance status, tumor location, T-stage, N-stage, and other oncologic history. Age was examined as a continuous variable. Tobacco exposure included cigarettes, cigars, pipes, chewing tobacco, and snuff. Tobacco use included the patient’s lifetime history of current, former, or never users. For former smokers, years since cessation were also recorded. Tobacco cumulative exposure was recorded in pack–years and grouped by exposure into two categories of <10 pack–years and ≥10 pack–years . Due to the small sample size, the continuous variable of pack–year history was stratified into these three categories. Alcohol exposure included beer, wine, or liquor (One drink is equivalent to a 12 oz beer, 4 oz. wine, or 1.5 oz of liquor). The patient’s lifetime history reflected current, former, or never users of alcohol >7 drinks/week. Alcohol cumulative exposure was positive for moderate use if >7 drinks/week or heavy use if >14 drinks/week.

2.3

Laboratory studies

Standard patient care at our institution has included testing for p16 expression through immunohistochemistry (IHC) or HPV infection through in situ hybridization (ISH) in HNSCC. All patients included in this study were previously evaluated for p16 expression from a tumor biopsy or surgical resection. The positivity of p16 and HPV status was recorded based on the clinical pathology report.

Immunohistochemistry testing for p16 expression followed the protocol supplied by the Ventana CINtec p16INK4a Histology Kit #9517. Immunohistochemistry was performed on the Leica Bond III autostainer using Leica ancillary reagents. Anti-P16 monoclonal antibody clone E6H4 immunostaining involved heat-induced epitope retrieval in High pH-EDTA buffer for 20 min, primary antibody incubation for 15 min, and application of Leica Refine (DAB) detection kit. Slides were counterstained with Refine hematoxylin. The p16 immunohistochemistry stain was considered positive if nuclear and cytoplasmic staining was greater than 70% of the cells .

HPV in situ hybridization was performed on the Ventana Ultra autostainer using DNA probes and ancillary reagents from Ventana Medical Systems up until July 9, 2014. Subsequent HPV ISH cases were sent to Integrated Oncology using the same Ventana method. The Ventana INFORM® HPV III probe set was used including the low-risk genotypes 6 and 11 and high-risk genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 66. A DNA probe was also run to assess integrity of tissue DNA. In situ hybridization for high-risk and low-risk HPV genotypes was considered positive when nuclear-specific staining was detected with all others were recorded as negative.

2.4

Statistical analysis

The following definitions were used for statistical analyses: overall survival was the time between initial treatment and the date of death from any cause. Locoregional failure was the time between initial treatment and the date of first local or regional recurrence as detected by radiologic imaging or clinical examination. Distant failure was the time between initial treatment and the date of first local or regional recurrence as detected by radiologic imaging or biopsy. Patients who were living at the last date of follow-up without recurrence were censored at the last date of follow-up. The Kaplan–Meier method was used to estimate overall survival (OS) and locoregional control (LRC) through a 48 month study period. The overall survival and locoregional failure-free survival estimates were reported for 12 and 24 months with 95% confidence intervals for all patients together and stratified by p16 status. The log rank test was used to determine predictors for locoregional failure.