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Salas et al. J Cancer Metastasis Treat 2018;4:15 I http://dx.doi.org/10.20517/2394-4722.2017.66 Page 3 of 13

transferred to an empty paraffin recipient block in order to construct tissue microarrays (TMA). FFPEs
samples were fixed for 6 to 8 h in 10% neutral buffered formalin and routinely processed with standard
methodologies.

In total, 41 tissue cores were distributed into ten TMA blocks. Tissue sections were cut at 3 mm and float-
mounted on adhesive (silanized) glass slides. Immunohistochemistry (IHC) for p16 status was performed
using the DAKO EnVision™ FLEX+ detection system together with the Autostainer Link instrument (DAKO
Corp, Carpentaria, California) on FFPE tissue. Antigen was retrieved using EnVision™ FLEX Target Retrieval
Solution, High pH, and p16 was detected using p16 mouse monoclonal antibody (clone 16p04, JC2, BSB
5828, prediluted, Bio SB, Santa Barbara, California). The EnVision™ FLEX+, Mouse, High pH, (LINK) Kit was
used to perform the assay according to the manufacturer’s instructions. It contains the substrate chromogen
3-3’-diaminobenzidine (DAB), which, on staining, results in a brown-colored precipitate at the antigen site.

Positive p16 expression was defined as a strong and diffuse nuclear and cytoplasmic staining in at least 70%
of the tumor cells.

Immunohistochemical evaluation was carried out by three pathologists in independent readings (LC, HG,
and SC). Reports that varied among readers were reevaluated to determine a consensus.

Statistical analysis
The log-rank statistic was used for univariate analysis, and Cox proportional hazards regression was used for
multivariable analysis. Categorical comparisons were carried out using the chi-square statistic or Fisher exact
test. In all cases, the level of alpha was set at 0.05 a priori. Survival analysis was calculated using the Kaplan-
Meier method. All analyses were performed in SPSS version 17.0 (SPSS, Chicago, IL).

RESULTS
Tumor primary location for this cohort was distributed in 5 patients for hard palate and 73 for upper alveolar
ridge. Mean age was 64.47 years old and 55.1% were female. There were tabaquismo and alcoholism history
in 10.3% and 6.4% cases, respectively. Two (40%) hard palate and 52 (71.2%) upper alveolar ridge tumors
were clinically classified cT4, and 3 (60%) hard palate and 21 (28.8%) upper alveolar ridge tumors were
clinically node-positive at presentation. Clinical stages I-IV of upper alveolar ridge SCC were found in 1
(1.4%), 13 (17.8%), 5 (6.8%) and 54 (74%) of cases, respectively. Clinical stages II-IV of hard palate SCC were
found in 1, 1 and 3 cases, respectively [Table 1].

The primary tumor was resected in all cases (n = 78). Neck dissections were initially performed in 24 cases (21
in clinically node-positive and 3 in node-negative). Nineteen (79.16%) of cases who went to neck dissections
had confirmed nodal metastases on pathological examination (including the 3 clinically node-negative
cases). Cervical metastases in these 19 node-positive cases were distributed between levels I (94.7%), II
(73.7%), and III (26.3%). Extracapsular extension at presentation was noted in 7 specimens of upper alveolar
ridge tumors.

In no instances were age (P = 0.329), location (P = 0.590), cT (P = 0.629), histological grade (P = 0.361), PNI (P
= 0.825), or LVI (P = 0.080) associated with cervical metastases [Table 2].

Neck recurrences
Altogether, 18 patients (75%) developed cervical recurrences, and 8 (44.4%) of them went to neck dissection
rescue (3 of them with additional radiation). Altogether, 18 patients (75%) developed cervical recurrences
and 8 (44.4%) of them went to neck dissection rescue (3 of them with additional radiation). Ten patients
did not go to surgery and treatment for them were: radiation alone (n = 2), radiation and chemotherapy