Abstract
The purpose of this study was to evaluate the expression of the enzymes involved in the biotransformation of tobacco and alcohol. A study group of 41 young patients (≤40 years old) with oral squamous cell carcinoma (OSCC) was compared to 59 control subjects (≥50 years old) with tumours of similar clinical stages and topographies. The immunohistochemical expression of CYP1A1, CYP1B1, ALDH1A1, and ALDH2 was evaluated using the tissue microarray technique. There was a predominance of males, smokers, and alcohol drinkers in both groups. Most tumours were located in the tongue (43.9% vs. 50.8%), were well-differentiated (63.4% vs. 56.6%), and were in clinical stages III or IV (80.5% vs. 78.0%). No difference was observed in the expression of CYP1A1, ALDH1A1, or ALDH2 between the two groups. CYP1A1 and ALDH2 protein expression had no influence on the prognosis. The immunoexpression of CYP1B1 was significantly higher in the control group than in the young group ( P < 0.001). The 5-year relapse-free survival was better in patients with CYP1B1 overexpression vs. protein underexpression (64% vs. 25%; P < 0.05), regardless of age. ALDH1A1 expression improved relapse-free survival in young patients. These results suggest a lower risk of recurrence with increased metabolism of carcinogens by CYP1B1. Further studies involving other genes and proteins are necessary to complement the results of this research.
Oral squamous cell carcinoma (OSCC) mainly affects men between the fifth and sixth decades of life and is uncommon in young patients (age ≤40 years). However, the incidence in this young age group has increased in several countries over the last two decades. In older patients, the dominant risk factors for OSCC are tobacco and alcohol abuse, which are strongly synergistic. Nevertheless, there remains doubt regarding the role of these factors in young people due to the short time of exposure.
Cigarettes contain potentially carcinogenic substances such as heterocyclic amines, N -nitrosamines, and polycyclic aromatic hydrocarbons (PAH). The metabolic activation of PAH into epoxide intermediates is catalyzed by the P450 cytochrome (CYP) family. CYPs and microsomal epoxide hydrolases act together in the metabolic activation of benzo[a]pyrene (BaP), a typical promutagenic PAH found in cigarette smoke. BaP generates a final product that can degrade different molecules, including DNA. CYP1A1 and CYP1B1 are the two main enzymes involved in the activation of carcinogenic PAH into electrophilic reagents that trigger the transformation of cells. CYP1A1 is considered a classical isoform in the activation of PAH; however, recent studies have indicated that the expression of CYP1B1 plays a more important role in carcinogenesis through PAH metabolism than CYP1A1.
The penetration of carcinogens through the mucosa is facilitated by the ingestion of ethanol, which serves as a solvent. Furthermore, ethanol induces CYP2E1, an enzyme that generates reactive oxygen species (ROS). The alcohol is metabolized by alcohol dehydrogenase, which oxidizes ethanol to acetaldehyde, a highly reactive compound that can form DNA adducts. The oxidation of acetaldehyde to acetate is catalyzed by aldehyde dehydrogenases (ALDH), especially ALDH1A1 and ALDH2. A reduction in the activity of these enzymes in tissues leads to elevated levels of acetaldehyde, increasing the risk of developing head and neck cancer.
There appear to be no studies focusing on the protein expression of tobacco- and alcohol-metabolizing enzymes in young patients with oral cancer in the English language literature. If differences exist between the young group and subjects older than 50 years of age, treatment strategies may be individualized. Therefore, the aim of the present study was to evaluate the expression of enzymes involved in the biotransformation of tobacco and alcohol in patients aged ≤40 years (young group) and to compare the results with those obtained from patients aged ≥50 years (control group). The hypothesis was that the expression of these proteins would be similar in the young and control groups.
Materials and methods
A retrospective study was conducted on 100 patients with OSCC treated from 1970 to 2004 at the department of head and neck surgery and otorhinolaryngology of AC Camargo Cancer Center in São Paulo, Brazil. Demographic (age, sex, and race), lifestyle (smoking habit and alcohol consumption), clinical (tumour site and clinical stage), treatment, and pathological (histological grade) factors were analyzed. The clinical characteristics of the patients were obtained from their medical records. The tumours were restaged according to the 2002 version of the American Joint Committee on Cancer (TNM) classification and classified as early clinical stage (clinical stage I–II) or advanced clinical stage (clinical stage III–IV). The cases studied were followed up after treatment. All cases with loco-regional recurrence were confirmed microscopically. The histopathological diagnoses were reviewed and the histological grade was determined on the basis of the World Health Organization classification as well-differentiated (grade I), moderately differentiated (grade II), or poorly differentiated (grade III). Patients with squamous cell carcinoma (SCC) of the lip and oropharynx and those who had received any previous treatment were excluded from the study. The number of cases was determined by the number of young patients diagnosed and treated in this service. The control group was matched to the young group by tumour topography and clinical stage. Only cases with complete clinical information and paraffin blocks available were included in the study.
The study was approved by the necessary human research ethics committee.
Immunohistochemistry
Construction of the tissue microarray has been described previously by Kaminagakura et al. The sections were pre-heated for 12 h at 60 °C, deparaffinized in xylene, and hydrated in decreasing alcohol solutions. For each antibody, the slides were subjected to antigen retrieval with citrate buffer, pH 6.0, in a Pascal pressure cooker (DakoCytomation, Dako, Carpinteria, CA, USA). Endogenous peroxidase was blocked by incubating the slides in a solution of 3% hydrogen peroxide (Merck, Brazil). The slides were then incubated with the primary antibodies ( Table 1 ) for 18 h at 4 °C. All antibodies were diluted in phosphate-buffered saline containing 1% bovine serum albumin (Sigma–Aldrich, Saint Louis, MO, USA) and 0.1% sodium azide. The reactions were detected using the streptavidin–biotin–peroxidase system in accordance with the manufacturer’s specifications (LSAB, DakoCytomation). Diaminobenzidine (DAB) was used as the chromogenic substrate (DakoCytomation). The primary antibody was omitted from the reactions in the negative control. All experiments were performed in duplicate.
| Antibody a | Clone | Dilution | Positive control |
|---|---|---|---|
| CYP1A1 | 6G5 | 1:75 | Ovarian carcinoma |
| CYP1B1 | Polyclonal | 1:600 | Brain |
| ALDH1A1 | EP1933Y | 1:400 | Liver |
| ALDH2 | EPR4493 | 1:200 | Colon |
Immunohistochemistry
Construction of the tissue microarray has been described previously by Kaminagakura et al. The sections were pre-heated for 12 h at 60 °C, deparaffinized in xylene, and hydrated in decreasing alcohol solutions. For each antibody, the slides were subjected to antigen retrieval with citrate buffer, pH 6.0, in a Pascal pressure cooker (DakoCytomation, Dako, Carpinteria, CA, USA). Endogenous peroxidase was blocked by incubating the slides in a solution of 3% hydrogen peroxide (Merck, Brazil). The slides were then incubated with the primary antibodies ( Table 1 ) for 18 h at 4 °C. All antibodies were diluted in phosphate-buffered saline containing 1% bovine serum albumin (Sigma–Aldrich, Saint Louis, MO, USA) and 0.1% sodium azide. The reactions were detected using the streptavidin–biotin–peroxidase system in accordance with the manufacturer’s specifications (LSAB, DakoCytomation). Diaminobenzidine (DAB) was used as the chromogenic substrate (DakoCytomation). The primary antibody was omitted from the reactions in the negative control. All experiments were performed in duplicate.
| Antibody a | Clone | Dilution | Positive control |
|---|---|---|---|
| CYP1A1 | 6G5 | 1:75 | Ovarian carcinoma |
| CYP1B1 | Polyclonal | 1:600 | Brain |
| ALDH1A1 | EP1933Y | 1:400 | Liver |
| ALDH2 | EPR4493 | 1:200 | Colon |
Statistical analysis
The sections on the slides were scanned with the ScanScope GL System (Leica Biosystems, Nussloch, Germany). The scanned images were analyzed with Aperio ImageScope Viewer software using the Positive Pixel Count algorithm (version 9). Strong and medium-brown pixels were considered as positive staining, while weak-brown pixels and negative pixels were considered as negative staining. Only epithelial malignant neoplastic cells were counted. The percentage of positive staining, expressed as the ratio between positive and total (positive + negative) staining, was considered for statistical analysis.
The χ 2 test was used to verify the homogeneity of the groups in terms of clinical and pathological variables. The cytoplasmic expression of CYP1A1, CYP1B1, ALDH1A1, and ALDH2 proteins was compared between the groups using the non-parametric Mann–Whitney test. For the survival analysis, protein expression was dichotomized according to the median. The follow-up time was defined as the interval between the beginning of treatment and the date of death or the last information for censored observations. The disease-free interval was measured from the date of treatment to the date when the first recurrence was diagnosed. Disease-free survival and overall survival probabilities were estimated by Kaplan–Meier method and the log-rank test was applied to assess the significance of differences among actuarial survival curves. The statistical analysis was performed using R version 2.13 (R Development Core Team (2010), Vienna, Austria; www.R-project.org ).
Results
One hundred specimens were available for histopathological and immunohistochemical analysis: 41 for the young group and 59 for the control group. The mean age of the young patients was 34.7 (range 19–40) years and of control patients was 62.6 (range 50–90) years. The male to female ratio was 1:1.9 for the young patients and 1:3.9 for the control patients. In both groups, the tongue was the most frequently involved site (43.9% vs. 50.8%), followed by the floor of the mouth (29.3% vs. 22.0%). Thirty-one of the young patients had tobacco exposure (75.6%), while 45 of those in the control group were smokers (76.3%) ( P = 0.63). Alcohol consumption was similar in the two groups ( P = 0.96). An advanced clinical stage was observed in 33 patients in the young group (80.5%) and 46 patients in the control group (78.0%) (P = 0.76). There was no statistically significant difference between the young and the control groups regarding the treatment approach ( P = 0.15). The demographic and clinical data are summarized in Table 2 .
| Characteristics | Young (≤40 years), n (%) |
Control (≥50 years), n (%) |
P -value |
|---|---|---|---|
| 41 (100) | 59 (100) | ||
| Age, years | |||
| Mean | 34.7 | 62.6 | |
| Range | 19–40 | 50–90 | |
| Sex | 0.12 | ||
| Male | 27 (65.9) | 47 (79.7) | |
| Female | 14 (34.1) | 12 (20.3) | |
| Race | 0.53 | ||
| White | 32 (78.0) | 49 (83.1) | |
| Other | 9 (22.0) | 10 (16.9) | |
| Tobacco use | 0.63 | ||
| Tobacco exposure | 31 (75.6) | 45 (76.3) | |
| No tobacco exposure | 8 (19.5) | 9 (15.2) | |
| Not mentioned | 2 (4.9) | 5 (8.5) | |
| Alcohol use | 0.96 | ||
| Alcohol exposure | 24 (58.5) | 33 (55.9) | |
| No alcohol exposure | 15 (36.6) | 21 (35.6) | |
| Not mentioned | 2 (4.9) | 5 (8.5) | |
| Sub-site | ND | ||
| Tongue | 18 (43.9) | 30 (50.8) | |
| Floor of the mouth | 12 (29.3) | 13 (22.0) | |
| Retromolar area | 5 (12.2) | 10 (17.0) | |
| Other | 6 (14.6) | 6 (10.2) | |
| T classification | 0.25 | ||
| T1–T2 | 13 (31.7) | 25 (42.4) | |
| T3–T4 | 28 (68.3) | 34 (57.6) | |
| N classification | |||
| N0 | 18 (43.9) | 25 (42.4) | 0.87 |
| >N0 | 23 (56.1) | 34 (57.6) | |
| M classification | NA | ||
| M0 | 41 (100) | 59 (100) | |
| Clinical stage | 0.76 | ||
| I–II | 8 (19.5) | 13 (22.0) | |
| III–IV | 33 (80.5) | 46 (78.0) | |
| Histological grade a | 0.02 | ||
| Well-differentiated | 26 (63.4) | 30 (56.6) | |
| Moderately differentiated | 7 (17.1) | 20 (37.7) | |
| Poorly differentiated | 8 (19.5) | 3 (5.7) | |
| Treatment a | 0.15 | ||
| Surgery | 15 (36.6) | 22 (37.9) | |
| Surgery + RxT | 23 (56.1) | 24 (41.4) | |
| RxT alone | 1 (2.4) | 9 (15.5) | |
| Surgery + RxT + ChT | 2 (4.9) | 3 (5.2) |
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