The Expression of SIRT6 Is Associated With Treatment Outcome in Elder Patients With Oral Cancer

Materials and Methods

Patients and clinicopathological data. This study enrolled 78 patients (56 men and 22 women) with OSCC, with median age of 65 years (age range=33-89 years), who underwent treatment between April 2012 and March 2019 at the Department of Oral and Maxillofacial Surgery, Yokohama City University Hospital. All the tissue samples were harvested from the patients who did not have preoperative adjuvant therapy. The OSCC patients had undergone surgery or chemoradiotherapy (51 surgery and 27 chemoradiotherapy). Samples were also obtained from 10 patients with oral potentially malignant disorders (OPMD): 8 men and 2 women, with median age of 68 years (age range=43-82 years). Biopsied or surgical samples were used for the study. We obtained 21 noncancerous tissues as normal tissues from the adjacent cancerous tissues after tumor resection. Following personal information protection laws, patients were given an opportunity to opt-out of this study through public announcements published by the institution. The protocol of this study was approved by the Institutional Ethical Committee of Yokohama City University in accordance with the principles of Declaration of Helsinki (approval # B210200001). Pathological tumor-node-metastasis (pTNM) stages were determined according to the Union for International Cancer Control (UICC) 7^th edition. Pathological types were determined by two experienced pathologists, according to the World Health Organization.

Immunohistochemical staining. Immunohistochemistry was performed as described in previous reports (8). Briefly, paraffin-embedded specimens were rehydrated by gradients of xylene and ethanol, followed by antigen retrieval via heating in Tris-EDTA. Endogenous peroxidase blocking was performed with the addition of 3% H₂O₂. The samples were then incubated with anti-SIRT6 rabbit monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA, 1:100) or CINtec^® p16 Histology (Roche, Basel, Switzerland) (9) at 4°C for overnight. Samples were incubated with secondary antibody (anti-Rabbit HRP, for SIRT6 expression or anti-Mouse HRP, for p16 expression, Agilent, Santa Clara, CA, USA). DAB (Agilent was employed according to the manufacturer’s instructions, and samples were stained using Mayer’s hematoxylin. The sections were mounted, cleared, cover slipped, and examined using a Leica Application Suite microscope (Leica, Wetzlar, Germany).

Analysis of expression and localization of SIRT6 in IHC. Three investigators (H.Y., M.K., and I.K.) independently assessed the stained sections. The positive signals for SIRT6 expression were semi-quantitatively scored according to the following criteria: 0, no staining; 1, weak to normal staining; 2, moderate to strong staining. We used the highest score for each sample. We also analyzed density of SIRT6-positive cells in each tissue, in which five different sites were randomly selected at high power field, and the ratio of stained tumor cells and total tumor cells was calculated for each site. The average of the scores was indicated as percent positive of SIRT6.

Quantitative real-time RT-PCR analysis of SIRT6 genes. Total RNA was extracted and purified from 41 paraffin-embedded cancerous and 12 noncancerous specimens by using High Pure FFPE RNA isolation kit (Roche). cDNA was generated with Super Script IV VILO Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacture’s protocols. The expression of SIRT6 gene was analyzed using TaqMan^® system. SIRT6 gene information is written as below, GenBank accession number: NM_001193285.2, amplicon size: 79, assay location: 131, and assay ID: Hs00213036_m1, size: FAM-MGB 250rxns (10). The expression of human housekeeping gene, GAPDH, was used for normalization. GAPDH gene information is as below, GenBank accession number: NM_001256799.2, amplicon size: 58, assay location: 1274, and assay ID: Hs03929097_g1, size: PL VIC-MGB 360rxns. qRT-PCR was performed in an ABI 7900HT Real-Time System (Thermo Fischer Scientific) calculated to obtain the cycle of threshold (ΔCt). The relative expression levels between OSCC and noncancerous parts were calculated by the comparative Ct (ΔΔCt) method, and the relative expression folds (2–ΔΔCt) were calculated using RQ Manager. The value of mRNA expression in healthy individuals is designated 1 while the level of mRNA expression in patients with OSCC was normalized to obtain the fold change in patients with OSCC.

RT-PCR analysis. DNA was extracted and purified from paraffin-embedded 35 cancerous specimens (10 μm) by using High Pure FFPE DNA isolation kit (Roche). DNA was submitted to PCR using PGMY09/11 primers (11) to amplify a sequence of 450 base pairs from the L1 region of most HPV types. The conditions of the thermocycling reaction using the PGMY09/11 primer set were: denaturing at 95°C for 5 min, followed by 40 cycles at 95°C for 1 min, 55°C for 1 min, and 72°C for 1 min. This was followed by 10 min final extension period at 72°C. Negative control comprised a tube with water, instead of DNA. As a positive control, DNA isolated from He-La and Si-Ha cell was used. Polymerase chain reaction products were analyzed by electrophoresis on a 2% agarose gel stained with Midori Green Direct (Nippon Genetics, Tokyo, Japan) and observed under UV light.

Microarray analysis. Among the samples mentioned before, we used 6 OSCC surgical paraffin embedded samples (4 men and 2 women). Total RNAs of OSCC and normal tissues were extracted with RNeasy (QIAGEN, Valencia, CA, USA) according to the manufacturer’s instructions. The gene expression profiles were analyzed by Macrogen Inc. (Tokyo, Japan). cDNA synthesis, RNA amplification, ss-cDNA synthesis, fragmentation, and labeling and hybridization were performed with the Gene Chip WT PLUS Kit (Thermo Fisher). The gasket slides were incubated in the Gene Chip^® hybridization oven, stained using the Gene Chip^® Fluidics Station (Thermo Fisher), and scanned using the Gene Chip^® Scanner (Thermo Fisher). Array data export processing and analysis were performed using the Affimetrix Expression Console Software and TAC (Thermo Fisher) software.

The data were summarized and normalized using the SST-RMA method implemented in Affymetrix^® Power Tools. We exported the result with gene level SST-RMA analysis and performed the differentially expressed gene (DEG) analysis. Statistical significance of gene expression was determined using fold change. For a DEG set, Hierarchical cluster analysis was performed using complete linkage and Euclidean distance as a measure of similarity. Gene-Enrichment and Functional Annotation analysis for significant probe list was performed using Gene Ontology and Kyoto Encyclopedia of Genes and Genomics (KEGG) in gene set enrichment analysis (GSEA).

Statistical analysis. Statistical analysis was performed using EZR ver.1.42 (Easy R, Jichi Medical University, Tochigi, Japan). Clinicopathological parameters and the SIRT6 expression or localization results were analyzed using t-test, Mann–Whitney U-test, Kruskal–Wallis test, and Spearman’s rank correlation efficient test. Clinicopathological parameters included age at diagnosis (<65 vs. ≥65 years of age), sex, smoking history, nodal metastasis, pTNM stages (stages I-II vs. stages III-IV), pathological type (well- vs. moderately vs. poorly differentiated), and tumor region. When the incidence was less than 5, χ² tests with Fisher’s exact correction were applied. Two-sided probability values less than 0.05 were considered statistically significant. Kaplan–Meier survival curves were plotted to evaluate the association of SIRT6 expression with 5-year overall survival rates (OS) and progression free survival rates (PFS). The curves were analyzed using the log-rank test. Cox regression analysis was used to identify factors affecting 5-year survival rates.