Abstract
The aim of this study was to evaluate the clinicopathological features and immunohistochemical expression of proliferation markers in oral tongue squamous cell carcinomas (OTSCC). Sixty-three patients without previous treatment or distant metastases were selected. Clinical information was retrieved from medical charts, histopathological analysis was performed and expression of proliferation markers (Ki-67, Mcm-2 and geminin) was evaluated. Most patients were men (81%) (male:female ratio 4.25:1). The age range was 31–92 years (mean 57.6 ± 11.81 years). A high Anneroth score was associated with tumour size ( p = 0.05), tumoural embolization of the blood vessels ( p = 0.003), nodal metastasis ( p = 0.05), nodal capsule rupture ( p = 0.016) and distant metastasis ( p = 0.002). Elevated Bryne scores were significantly associated with nodal capsule rupture ( p = 0.02), distant metastasis ( p = 0.002), shorter overall survival (OS) ( p = 0.03) and disease-free survival (DFS) ( p = 0.05) compared with patients with lower score. Elevated Ki-67+ cells ( p = 0.05) and Mcm-2+ cells ( p = 0.008) were associated with nodal metastasis and tumours with a high geminin score demonstrated a significant tendency for neural invasion ( p = 0.05). Anneroth and Bryne score in association with biomarkers of proliferation can be useful for evaluating the biological behaviour of OTSCC.
Oral tongue squamous cell carcinoma (OTSCC) is more commonly observed in male patients in the sixth and seventh decades of life and is associated with heavy smoking and alcohol consumption . It has been suggested that these tumours have distinct epidemiological and biological features, since they are also observed in women and young patients without any association with tobacco or alcohol . Generally, OTSCC has a worse prognosis compared with other oral sites. Initial OTSCC may have similar survival rates to stage III and IV disease from other oral sites . OTSCC thickness is related to a high rate of nodal metastasis, supporting multimodal treatment . The risk of nodal metastasis seems to increase in an antero-posterior fashion, which may occur in a tumour size-independent manner . Tumour grading and the pattern of invasion seem to be useful predictors of tumour behaviour, especially when the invasive front is analyzed . Grading the invasive front seems to correlate better with prognosis and tumour behaviour, possibly because of a unique cellular microenvironment in the deep portion of the tumour, characterized by higher immunoexpression of proliferation markers and oncoproteins .
Cell cycle regulatory proteins, such as minichromosome maintenance proteins (Mcm) and geminin, are altered in many malignant lesions from distinct origins . Mcm proteins act in DNA replication and cell cycle initiation, and are expressed throughout the cell cycle including at the G 0 –G 1 checkpoint. Notably, Mcm complexes warrant a replication license and act as helicase unwinding the double helix . Geminin regulates cell cycle initiation by impeding the assembly of a second replication complex after the beginning of replication, and has also demonstrated altered expression in cancer cells . Mcm-2 and geminin are potential proliferation markers capable of predicting the prognosis of human malignancies . Previous studies have shown promising results regarding the combined use of Mcm-2 and geminin on evaluating the prognosis of malignant tumours of distinctive origins, including oral cancer .
The purpose of this study was to evaluate the histopathological grading systems and the immunohistochemical expression of the proliferation markers Ki-67, Mcm-2 and geminin in a sample of OTOSCC followed for 10 years to establish their prognostic significance.
Material and methods
63 patients with OTSCC treated at the Department of Otolaryngology and Head and Neck Surgery of the A.C. Camargo Cancer Hospital, São Paulo, Brazil, were selected. Criteria for inclusion were biopsy-proven OTSCC, no previous tumour treatment, absence of distant metastasis (stage M0), surgery as the first treatment used and, at least 5 years of follow-up. The Ethics Committee of the Piracicaba Dental School approved the research protocol (no. 62/2006) according to Brazilian law and the Helsinki Statement.
A central portion of the tumoural mass was collected, fixed in 10% neutral-buffered formalin and processed for routine light microscopy and immunohistochemical analysis. Histopathological grading of the tumours was performed according to the criteria of A nneroth et al. and B ryne et al. Anneroth’s score evaluates keratinization, nuclear pleomorphism, mitosis, pattern of invasion, stage of invasion and inflammatory infiltrate. Bryne’s scores were determined at the tumour front and do not consider the number of mitotic cells or stage of invasion. Blood and lymphatic vessel embolization, perineural invasion, tumour thickness and nodal metastases were evaluated. Observers were calibrated by evaluating 20 cases of OSCC not included in the sample together. After calibration, the intra-observer agreement rate was evaluated by determining the kappa value.
Immunohistochemical reactions were performed using the streptavidin-biotin peroxidase complex method with the following primary antibodies: anti-Ki-67 (MIB-1, Dako A/S, 1:200), anti-Mcm-2 (NCL-MCM2, Novocastra, 1:50) and anti-geminin (NCL-geminin, Novocastra, 1:50). The evaluation was performed under high power magnification (400x) on a Kontron KS400 (Karl Zeiss, Germany). All marked cells were considered positive when nuclear staining was observed, regardless of staining intensity, and the labelling index (LI) was determined by counting the labelled nuclei of 1000 cells. The slides were independently analyzed by two of the authors (LAG and MAL). The labelling indices of biomarkers were considered as the mean value of both observers’ scores.
For frequency analysis in contingency tables, statistical analyses of associations between variables were performed using Fisher’s exact test and the non-parametric Mann–Whitney U -test for continuous variables with the aid of NCSS 2000 software (NCSS, Kayville, USA). The overall survival (OS) was defined as the interval between the diagnosis and the date of death or the last information for censored observations. The disease-free interval was measured from the date of the treatment (surgery) to the date when recurrence was diagnosed. OS and disease-free survival (DFS) probabilities were estimated by the Kaplan–Meier method, and the log-rank test was applied to assess the significance. Statistical significance was defined as p ≤ 0.05.
Results
A strong male prevalence was observed, and the majority of patients were in the sixth decade of life. Most of the patients reported smoking (54 patients, 86%) and drinking alcohol (52 patients, 83%). The association of both habits was distributed as follows: 50 patients (78%) were smokers and drinkers, 4 patients (6%) were smokers only, 7 patients (11%) were nonsmokers/nondrinkers and 3 patients (5%) did not inform the researchers about these habits. Regarding tumour stages, almost half of the patients (49%) were classified as T3 and T4 and 37% presented positive lymph nodes at diagnosis. 60% of the patients had advanced clinical stages of the disease (stages III and IV). All patients were surgically treated, 31 (49%) exclusively with surgery, 31 (49%) with a combination of surgery and radiation therapy (RT) and 1 patient (2%) with a combination of surgery, RT and chemotherapy. The majority of patients (32 cases, 51%) presented recurrence. Of these, 11 patients presented exclusively local recurrence, five presented only nodal metastases, seven presented distant metastases, and nine presented more than one site of recurrence ( Fig. 1 ). The 5-year survival rate was 48% and the 10-year survival rate was 14%. Clinical data are summarized in Table 1 .
| Variable | Patients | % |
|---|---|---|
| Age | ||
| Range | 31–92 | |
| Mean | 57.6 | |
| Median | 58 | |
| Gender | ||
| Male | 51 | 80.95 |
| Female | 12 | 19.05 |
| Smoking | ||
| No | 7 | 11.11 |
| Yes | 54 | 85.72 |
| Not informed | 2 | 3.17 |
| Drinking | ||
| No | 10 | 15.87 |
| Yes | 52 | 82.54 |
| Not informed | 1 | 1.59 |
| Tumour size | ||
| T1 | 11 | 17.46 |
| T2 | 21 | 33.33 |
| T3 | 23 | 36.51 |
| T4 | 8 | 12.70 |
| Nodal status | ||
| N0 | 40 | 63.49 |
| N1 | 17 | 26.98 |
| N2 | 5 | 7.94 |
| N3 | 1 | 1.59 |
| Clinical stage | ||
| Stage I | 10 | 15.87 |
| Stage II | 15 | 23.81 |
| Stage III | 26 | 41.27 |
| Stage IV | 12 | 19.05 |
| Treatment | ||
| Surgery | 31 | 49.20 |
| Surgery + RT | 30 | 47.62 |
| Surgery + RT + CT | 1 | 1.59 |
| RT + surgery | 1 | 1.59 |
| Recurrence | ||
| No | 31 | 49.21 |
| Yes | 32 | 50.79 |
| Local | 20 | 31.75 |
| Regional metastasis | 12 | 19.05 |
| Distant metastasis | 13 | 20.63 |
| Status | ||
| Alive | 25 | 39.68 |
| Dead | 38 | 60.32 |
| Survival | ||
| 5 years | 30 | 47.62 |
| 10 years | 9 | 14.29 |
Kappa values for Anneroth’s and Bryne’s score systems were 0.86 and 0.91, respectively. Although the individual scores from Anneroth’s system were often higher than those from Bryne’s system, a significant concordance in all individual components of both systems was observed. Comparing the two scores, a high concordance was observed ( r = 0.82). The clinicopathological correlations with the histopathological scores of Anneroth’s and Bryne’s systems are listed in Table 2 . The score values of Anneroth’s (score 15) and Bryne’s (score 12) systems were used to classify the tumours into two groups, below and above each score, which were used for statistical analysis. A high Anneroth’s score was associated with the presence of vascular invasion ( p < 0.01), nodal metastasis ( p = 0.05), nodal capsule rupture ( p = 0.01), advanced T stage ( p = 0.05) and distant metastasis ( p < 0.01). Tumours with a Bryne’s score above 12 demonstrated significantly more nodal capsule rupture ( p = 0.02) and distant recurrence ( p < 0.01). Higher Bryne scores were related to shorter OS rate ( p = 0.03) and DFS rate ( p = 0.05) ( Figs 2 and 3 ).
| Clinicopathological features | Anneroth | Bryne |
|---|---|---|
| Age | 0.78 | 0.19 |
| Gender | 0.19 | 0.35 |
| Symptoms at diagnosis | 0.34 | 0.53 |
| Tumour size | 0.05 * | 0.29 |
| Margin status | 0.73 | 0.28 |
| Tumoural embolization of lymphatic vessels | 0.17 | 0.32 |
| Tumoural embolization of blood vessels | 0.003 * | 0.11 |
| Neural invasion | 0.89 | 0.98 |
| Tumour thickness | 0.50 | 0.69 |
| Nodal metastasis | 0.05 * | 0.23 |
| Nodal capsule rupture | 0.016 * | 0.02 * |
| Local recurrence | 0.71 | 0.72 |
| Nodal recurrence | 0.60 | 0.35 |
| Distant metastasis | 0.002 * | 0.002 * |
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