Abstract
The tumour subtype, TNM classification, and histopathological data are sometimes not sufficient for understanding and assessing the behaviour of oral cancers. In an attempt to find additional markers of tumour biology and behaviour, this study sought to determine the incidence and consequently the relevance of c-erb-B2, c-Myc, and H-ras gene alterations in tumour-free margins of oral squamous cell carcinoma (OSCC). Fifty samples of OSCC were analyzed for c-erb-B2 and c-Myc amplification by real-time polymerase chain reaction and for H-ras point mutations by sequencing. A relatively high incidence of genetic lesions was detected: 22% of cases had c-erb-B2 and 30% had c-Myc amplification, whilst only 12% harboured H-ras mutations. Kaplan–Meier analysis and the log-rank test showed statistically significant differences in 5-year survival rates and relapse between patients with tumour margins positive for c-erb-B2 amplification and those with margins that were negative ( P = 0.002). H-ras and c-Myc alterations could not be associated with tumour behaviour. Molecular analysis of margins, targeting cancer genes, could identify additional, independent predictors of risk and outcome in OSCC.
Oral cancers consistently rank among the top 10 cancers worldwide. More than 90% of malignant neoplasms of the oral cavity are squamous cell carcinomas. The three most common localizations of oral cancers are the tongue, floor of the mouth, and lower lip. They can develop from potentially pre-malignant lesions, such as leukoplakia, erythroplakia, and lichen planus, or from apparently normal epithelium. Oral squamous cell carcinoma (OSCC) has a propensity to early and extensive lymph node metastasis. Traditionally, surgeons plan the treatment of OSCC according to the tumour subtype, TNM classification, and histopathological grading. The same parameters are also used to predict the outcome of the treatment and offer a prognosis to the patient. However, these parameters are sometimes not sufficient to understand the behaviour of oral cancers and cannot provide a reliable estimation of the patient’s outcome. Despite significant improvements in local and regional disease control by surgery and radiotherapy over the last few decades, 5-year survival rates of OSCC patients have improved only moderately.
Approximately 10–30% of patients with OSCC develop local recurrences in spite of seemingly adequate tumour resection (surgical margins are confirmed as tumour-free), and the incidence of metastasis is high as well. One critical factor affecting the rates of recurrence and metastasis could be the molecular status of the histologically tumour-free surgical margins, i.e. an unfavourable disease course could be the consequence of residual tumour cells in the surgical margins that cannot be detected by conventional diagnostic methods.
Oral carcinogenesis is a complex multi-step process that is driven by the accumulation of genetic and epigenetic alterations leading to the activation of oncogenes and inactivation of tumour suppressor genes. The detection and quantitation of certain gene alterations in oral cancers have been shown to be of prognostic importance.
Members of the c-erb-B2/c-Myc/H-ras signalling cascade are known to be involved in the pathogenesis of OSCC. c-erb-B2 (HER-2, NEU) is one of the four genes of the epidermal growth factor receptor (EGFR) family, which encodes a p185 transmembrane glycoprotein receptor with tyrosine kinase activity. c-erb-B2 amplification, resulting in oncoprotein overexpression, has been shown to play an important role in the pathogenesis of various solid tumours including OSCCs.
c-Myc, a member of the Myc oncogene family, encodes a transcription factor involved in cell proliferation, differentiation, and apoptosis. c-Myc expression can be enhanced through several mechanisms, the best known being chromosomal translocation and gene amplification. Myc amplification has also been reported in numerous solid tumours, including OSCC.
Some studies have shown that members of the ras oncogene family, which encode GTPases implicated in cellular signal transduction, are overexpressed in oral cancer too. The H-ras oncogene is usually activated by point mutations in codons 12, 13, and 61.
Interestingly, although studies have dealt with mutation screening in OSCC, findings on genetic alterations in tumour margins after surgical excision are relatively scarce and are mainly related to TP53 mutations. Indeed, mutations in TP53 are the hallmark of many cancers and have been found at high percentages in OSCC. Nevertheless, other genes in surgical margins also deserve to be studied, and these might provide some indication of the reasons for therapeutic failure.
The aim of this study was to detect alterations of c-erb-B2, c-Myc, and H-ras in histologically free tumour margins of OSCC and to try to establish a relationship between molecular changes and clinical/histopathological parameters, including the 5-year survival rate and recurrence rate.
Materials and methods
Patients and tumour samples
A cohort of 50 patients (36 male and 14 female, aged 62.74 ± 10.95 years) with a primary tumour of the oral cavity, who underwent surgery during the period January 2007 to December 2008, were included in the study. Tissue samples originated from patients with tumours of different localization, histological grade, clinical stage, and depth. They were all squamous cell carcinomas. The histopathological diagnosis was established in accordance with the World Health Organization (WHO) guidelines, using haematoxylin and eosin (H&E)-stained tissue sections, and the tumours were staged using the TNM classification.
Margin samples were taken from the edges of the surgical defect after excision of the primary tumour. Fifty consecutive samples that fulfilled the inclusion criterion of tumour-free resection margins, i.e. samples distant at least 5 mm from the tumour edge and histologically without neoplastic cells (H&E), were used in the analysis. The samples came from 25 patients with tumours of the tongue and floor of the mouth, five with buccal mucosa tumours, four with maxillary mucosa tumours, two with mandibular mucosa tumours, and 14 with lip tumours. Three patients were lost during the course of the study, i.e. could not be traced for the collection of survival/relapse data. Parameters such as tumour localization, depth of invasion, presence of tumour cells in the lymph nodes, etc., were not selection criteria.
This study was performed according to the ethical principles governing medical research and human subjects as laid down in the Declaration of Helsinki (2002 version, http://www.wma.net/e/policy/b3.htm ), and with the approval of the ethics committee of the study institution in Belgrade. All patients were informed of all procedures and signed a written informed consent form.
DNA extraction for molecular analysis was done from tumour margins that had previously been confirmed as histologically cancer-free by a pathologist. DNA was obtained after proteinase K digestion and phenol–chloroform extraction.
Molecular genetic analysis
A quantitative real-time polymerase chain reaction (qPCR) and comparative cycle threshold (Ct) method of c-erb-B2 and c-Myc quantitation (ΔΔCt) was performed. The manufacturers’ recommendations were followed for reaction mixes and profiles: total volume of 25 μl, 1× Maxim SYBR Green/ROX qPCR Master Mix (Fermentas Life Sciences, Vilnius, Lithuania) 0.35 μM of each primer (Metabion, GmbH, Munich, Germany), 20 ng template DNA, and nuclease-free water. A single-copy gene encoding the dopamine D2 receptor (D2R) was used as the reference gene. Primer sequences and annealing temperatures for c-erb-B2, c-Myc, and D2R are given in Table 1 . PCRs for all genes and for each sample were carried out in duplicate. To confirm the specificity of the amplified products, a melting curve analysis was performed in each case. The Ct values for c-erb-B2 and c-Myc were normalized against D2R as the reference gene. The ΔΔCt value was calculated for each sample and the amplification levels were calculated as 2 −ΔΔCt . A gene dose greater than 2.5 was considered as amplification.
| Gene | Primer sequence | Length (bp) | Annealing temperature (°C) |
|---|---|---|---|
| c-erb-B2 | 5′-CCTCTGACGTCCATCATCT-3′ | 98 | 55 |
| 5′-ATCTTCTGCTGCCGTCGTT-3′ | |||
| c-Myc | 5′-GCTCCAAGACGTTGTGTGTTCG-3′ | 158 | 55 |
| 5′-GGAAGGACTATCCTGCTGCCAA-3′ | |||
| D2R | 5′-CCACTGAATCTGTCCTGGTATG-3′ | 112 | 55 |
| 5′-GTGTGGCATAGTAGTTGTAGTGG-3′ | |||
| H-ras | 5′-ATGACGGAATATAAGCTGGT-3′ | 123 | 50 |
| 5′-ATATCTCCACTCGGACCGC-3′ | |||
H-ras codon 12/13 mutations were screened by PCR–single-strand conformation polymorphism (SSCP) and confirmed by sequencing. The PCR reaction was performed in a volume of 25 μl reaction mixture containing 300 ng of genomic DNA and 200 nM of each primer. Primer sequences and annealing temperatures are given in Table 1 . After amplification, the PCR products were denatured by heating at 95 °C for 5 min, separated by electrophoresis at 4 °C on an 8% and 10% non-denaturing polyacrylamide gel (LKB, Pharmacia, Sweden), and stained with silver nitrate. The samples that showed changes in band migration pattern were further subjected to commercial sequencing.
Statistical analysis
Fisher’s exact test and the χ 2 test were performed in association studies. The 5-year survival estimation was carried out by Kaplan–Meier analysis. Differences in the curves were evaluated by log-rank test. Statistical significance was set at P < 0.05. Software package SPSS v. 17 was used for the analysis (SPSS Inc., Chicago, IL, USA).
Results
A high incidence of genetic lesions was detected in seemingly cancer-free margins of OSCC: 11 (22%) had c-erb-B2 and 15 (30%) had c-Myc amplification. H-ras analysis was successful in 42 patients only, and mutations were detected in five patients (12%). The distribution of gene alterations in relation to clinical and histopathological parameters is given in Table 2 .
| c-erb-B2 | c-Myc | H-ras | ||||
|---|---|---|---|---|---|---|
| Total | Mutation | Total | Mutation | Total | Mutation | |
| No. of patients | 50 | 11 | 50 | 15 | 42 | 5 |
| Histological grade | ||||||
| 1 | 13 | 2 | 13 | 5 | 12 | 1 |
| 2 | 31 | 7 | 31 | 9 | 24 | 3 |
| 3 | 6 | 2 | 6 | 1 | 6 | 1 |
| P -value | 0.625 | 0.617 | 0.868 | |||
| Stage | ||||||
| 1 | 14 | 4 | 14 | 4 | 11 | 1 |
| 2 | 11 | 2 | 11 | 2 | 11 | 2 |
| 3 | 15 | 1 | 15 | 6 | 11 | 2 |
| 4 | 10 | 4 | 10 | 3 | 9 | 0 |
| P -value | 0.222 | 0.692 | 0.547 | |||
| Lymph node | ||||||
| Negative, N− | 29 | 3 | 29 | 9 | 25 | 4 |
| Positive, N+ | 21 | 8 | 21 | 6 | 17 | 1 |
| P -value | 0.023 * | 0.552 | 0.315 | |||
| Smoking | ||||||
| Yes | 28 | 7 | 28 | 9 | 22 | 1 |
| No | 22 | 4 | 22 | 6 | 20 | 4 |
| P -value | 0.411 | 0.477 | 0.144 | |||
| Alcohol consumption | ||||||
| Yes | 15 | 2 | 15 | 7 | 13 | 2 |
| No | 35 | 9 | 35 | 8 | 29 | 3 |
| P -value | 0.283 | 0.091 | 0.497 | |||
Remarkably, patients with c-erb-B2 amplification in cancer-free margins showed a higher mortality rate than patients without the amplification ( P = 0.033) ( Table 3 ). Using Kaplan–Meier analysis and the log-rank test for the evaluation of 5-year survival rates, a significant difference was found between c-erb-B2-positive and c-erb-B2-negative patients ( P = 0.002) ( Table 4 , Fig. 1 ). Finally, patients who were positive for c-erb-B2 amplification experienced relapses more frequently than patients who were negative ( P = 0.017) ( Table 5 ).
| Variables | Alive | Dead | P -value | |
|---|---|---|---|---|
| Family cancer history | Positive | 3 | 1 | 0.128 |
| Negative | 14 | 29 | ||
| Personal cancer history | Positive | 0 | 3 | 0.250 |
| Negative | 17 | 27 | ||
| c-erb-B2 mutation | No | 16 | 20 | 0.033 * |
| Yes | 1 | 10 | ||
| c-Myc mutation | No | 12 | 20 | 0.782 |
| Yes | 5 | 10 | ||
| H-ras mutation | No | 13 | 21 | 0.559 |
| Yes | 1 | 4 | ||
| Presence of lymph node metastasis | Negative, N− | 13 | 14 | |
| Positive, N+ | 4 | 16 | 0.045 * | |
| Stage | 1 | 7 | 7 | |
| 2 | 4 | 5 | 0.475 | |
| 3 | 4 | 10 | ||
| 4 | 2 | 8 | ||
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