Specific TP53 mutations predict aggressive phenotype in head and neck squamous cell carcinoma: a retrospective archival study

Head and neck squamous cell carcinoma (HNSCC) is one of the 10 most frequent malignancy in the world and more than 500 000 new cases are reported annually [1]. Despite the intense research in the area of squamous cell carcinomas of head and neck (HNSCC), the long-term survival rate has not changed significantly in this malignancy during recent decades [2]. The initial treatment approaches for a patient with HNSCC include most often radiotherapy added to primary surgery one way or another or as a definitive treatment of inoperable disease [3]. In the past decade, the role of organ-preservation protocols, with combined chemoradiation and surgery for salvage, has increased. These protocols are particularly effective for patients with moderately advanced cancers of the larynx and pharynx who are less than 70 years old and have a good performance status [2]. Also technical improvements have decreased late radiotherapy side-effects. Recently Nutting and co-workers (2011) compared conventional radiotherapy to parotid-sparing intensity-modulated radiotherapy (IMRT) in patients with pharyngeal squamous cell carcinoma and noticed significantly lower late side-effect rate but did not report any survival advantage [4]. Anti-EGFR mAb cetuximab has shown promising antitumour activity with tolerable toxicity profile but the optimal combinations and schedule is still to be found [5].

The abrogation of p53 function through the mutation of its gene, TP53 [6], the loss of heterozygosity of TP53 [7] or interaction with viral proteins [8], is one of the most common molecular alterations in squamous-cell carcinoma of the head and neck [9]. The TP53 tumour suppressor gene in chromosome 17p13.1 encodes the p53 protein, which functions primarily as a multi-target transcription factor. The p53 protein is known to be involved in various cellular functions including cell-cycle regulation, senescence, apoptosis, repair of DNA damage caused by genotoxic agents, angiogenesis, and regulation of oxidative stress and glucose metabolism [10–13]. Loss of the p53 function allows proliferation of the cells with a DNA-damage and promotes neoplasia in transgenic p53 null mice [14]. TP53 gene alterations are commonly found in head and neck cancers, and most of the published mutations affect the p53-DNA interactions, resulting in a partial or complete loss of transactivation functions [15]. TP53 differs from other tumour suppressor genes in its mode of inactivation. While most tumour suppressor genes are inactivated by mutations leading to absence of protein synthesis or production of a truncated protein, more than 80% of TP53 alterations are missense mutations that lead to the synthesis of a stable full-length protein [15]. The location of the resulting amino-acid substitution is usually within the central DNA-binding domain of the p53, resulting in a loss of DNA-binding activity with consequent failure to transcriptionally activate target genes [16].

In the past 10 years, systematic data from the functional assays have been generated and integrated in the TP53 database managed by the International Agency for Research on Cancer (IARC) (http://www-p53.iarc.fr/) [17]. Most mutant p53 proteins have lost their DNA-binding activity, leading to a loss of their growth inhibiting and apoptotic properties. The role of p53 as a prognostic marker of squamous cell carcinoma of the head and neck is controversial. The possible reasons for this include small numbers of patients studied, insufficient clinical follow-up, variable laboratory techniques used, or analysis of TP53 data based on insufficient mutation characterization [15, 18].

In the present study, we pursue the clinical importance of TP53 mutation types in head and neck cancer. We analyzed TP53 mutations in 46 patients with head and neck squamous cell carcinomas (HNSCC) by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and sequencing as earlier described [19]. The aim of this study was to examine whether specific TP53 mutation types were associated with the clinical outcome of head and neck cancer, such as lymph node metastasis, prognosis and therapy of the patients.

In the present study, we have evaluated the prognostic value of TP53 gene aberrations in a series of 46 patients with primary head and neck squamous cell carcinoma. Analysis of TP53 mutations is being studied as a clinical marker [27]. When correlating results from mutation analysis to the outcome in HNSCC, so far restricted to exons 5-8 or 5-9, contradicting results have been published (for reviews see [28, 29]). We found that tumours containing TP53 mutations in DNA-binding surface regions (L2, L3 + LSH) are more aggressive than tumours with mutations outside of those regions. Interestingly, patients affected by tumours with TP53 mutations in L3 and LSH have poorer survival, and in case of tumours with TP53 mutations in domains L2, L3 + LSH there were more relapses and shorter disease free time than with other tumours implicating a more aggressive phenotype. Similar findings have already been reported in gastric cancer [30], and colorectal carcinoma [31]. Bazan and co-workers (2005) reported that in sporadic colorectal carcinoma tumours with TP53 mutations in L3 domain are associated with a worse prognosis (as judged by disease free time only) than other tumours [31]. Furthermore, several studies have revealed a strong association between the mutations in the L2/L3 loop and a shorter survival or poorer response to treatment in breast cancer [32, 33] and in oesophageal cancer [34]. Oral squamous cell cancers with TP53 mutations in DNA-binding surface regions (L2, L3 and the LSH motif) and conserved regions (II-IV) were also associated with a significantly poorer prognosis than tumours with mutations outside of those regions [35]. In a large multi-centre study of squamous cell head and neck cancer, Poeta and co-workers (2007) found that mutations affecting the protein structure and DNA-binding capacity of TP53 were associated with shortened disease-specific and overall survival [36]. In our study, most (78%) patients that had TP53 mutations in L2, L3 and LSH motifs died of cancer. Only 42% of those patients with a wild type TP53 or 12.5% in those with TP53 mutations outside the L2/L3/LSH domain died of cancer. Interestingly, the patients with mutations outside the DNA-binding domains of p53 had a better survival than the patients with a wild-type p53. The association between TP53 mutation types and outcome presented here is also in agreement with the largest study on the TP53 mutation types and association with clinical phenotypes carried out in 630 patients screened for mutations in breast cancer and published by Alsner and co-workers (2008) [37].

When we compared the disease-free survival, disease-specific or overall survival in HNSCC patients with and without TP53 mutations, no difference was seen between the two groups. Similarly, some researchers have failed to find such a correlation [38, 39]. On the other hand, positive association between the TP53 mutations and poor prognosis have been published [36, 40, 41]. Patients with TP53 mutations have been shown to have a significantly shorter survival time than those without any TP53 mutations [42]. Also the loco-regional control rate and disease-free survival rate have been shown to be inferior in the patient group with a TP53 mutation when compared to the patients with a wild-type TP53 [43]. We show here that TP53 mutations in exons 6 and 8 correlate with poorer overall survival. Russo and co-workers (2006) have noticed that TP53 mutations within exons 5 and 8 are strong prognostic indicators of both disease recurrence and survival in patients with locally advanced laryngeal squamous cell carcinoma [41]. Similarly, Huang and co-workers (1998) have studied 204 cases of non-small cell lung carcinoma, and patients with mutations in exons 7 and 8 have a significantly shorter survival compared with patients with other mutations or no mutation [44]. Our findings together with the published data suggest that the nature and location of the mutation are connected to tumour aggressiveness and prognosis and it is not enough to analyze merely the presence or absence of the TP53 mutations. Thus, the usefulness of TP53 mutations in human tumours for clinical purposes requires a much more detailed analysis.

We found here that patients with TP53 mutations in L2, L3 or LSH motif received significantly less often combined treatment (curative intent) and had significantly lower disease-free time and cancer specific-survival. Therefore, specific TP53 mutations present in L2, L3 and LSH motifs could lead to inherently more aggressive tumours than mutations outside of these regions. The age of the patients or other diseases did not affect the treatment modalities. Mechanism of action of a mutant p53 appears to be complex [45, 46]. Many studies have shown that although mutant p53 proteins often lose the ability to activate the expression of genes that are responsive to wild-type p53, they may not be inert when it comes to the regulation of transcription [16]. Indeed, a mutant p53 can acquire the ability to both positively and negatively regulate gene expression, which clearly contributes to some of the pro-tumorigenic functions of a mutant p53, such as enhanced survival and resistance to cancer therapy [16, 47].

There have been a limited number of inconclusive studies on TP53 mutations in patients treated with primary radiotherapy (for a review, see [48]). We evaluated the relationship between TP53 mutations and radiosensitivity in 36 patients with HNSCC who underwent primary radiotherapy. Tumours containing TP53 mutations in DNA-binding surface region had significantly poorer responses to radiation than other tumours. Eriksen and co-workers (2005) have investigated the importance of TP53 mutations for the overall treatment time of radiotherapy in HNSCC patients [39]. They noticed that patients with carcinomas containing a wild-type p53 did not benefit as much as the patients with a mutated TP53 from an increase in the number of weekly fractions (i.e. a reduction in the overall treatment time), as judged by local control at T-site, disease-specific or crude survival [39]. This may be due to the decreased ability of some mutant p53 proteins to initiate apoptosis [49]. Consequently, there may be less delay in G1 resulting in increased progress through the cell cycle and increased uncontrolled proliferation [39]. Moreover, Alsner and co-workers (2001b) noticed that majority of head and neck of tumours with a mutated TP53 had a tumour potential doubling time below that in wild type-tumours [50]. Carcinomas with a mutated TP53 had higher cell proliferation potential compared to wild type-tumours. TP53 mutation status may thus identify patients where the dominating factor associated with outcome is an increased intrinsic radioresistance.

In conclusion, this study demonstrates that the clinical outcome for head and neck cancer patients is associated with the type of TP53 mutations. Mutations present in DNA-binding regions were associated with poorer prognosis and clinical response to radiotherapy. Especially, TP53 mutations in L2 and L3 + LSH motif may be usable as a marker for selecting the treatment option and predicting prognosis among HNSCC. Predictive factors become increasingly important, in particular because new treatment modalities e.g. radiotherapy combined with EGFR generally yield more treatment-related toxicity. However, due to the small number of patients in this study further confirmation is required before reliable clinical application is possible. Studies are needed to evaluate whether specific TP53 mutations influence prognosis in patients treated with conventional chemoradiotherapy or radiotherapy combined with anti-EGFR treatment.