Introduction
Esophageal cancer (EC) remains the sixth most common cause of cancer related death in the world despite recent progress in multimodal therapy concepts involving neoadjuvant therapy and standardized surgical approaches (1). EC is associated with high malignant potential for local invasion and early dissemination resulting in low overall-survival (OS) rates in patients even after curatively intended surgery (2).
There are two major histological types of EC that account for >90% of all malignant neoplasms in the esophagus: squamous cell carcinoma and adenocarcinoma. The pathogenesis in adenocarcinoma is generally considered to be driven by epithelial metaplasia, often caused by acid reflux. In squamous cell carcinoma, malignant transformation is predominantly associated with alcohol intake and smoking (3). The incidence of the particular subtypes has changed in the past years with increase of adenocarcinoma and decrease of squamous cell carcinoma in Western countries, mainly due to life style changes (2). Due to the limited prognosis of the majority of patients, identification of new biomarkers predicting the individual prognosis and response to therapy are of imperative need for allocation of individualized therapy strategies.
The TP53 gene has been known as one of the most important tumor suppressor genes, located on human chromosome 17p13.1. p53 plays a major role in tumorigenesis as it controls cell growth, apoptosis and regulation of angiogenesis (4). Mutations in TP53 were found in >50% of human cancers, which makes it one of the most mutated genes in tumors (5). In esophageal cancer, mutations have been described in frequencies between 40–70%, depending on the underlying cell type (4,6–8). As shown for several tumor entities, inactivation of the TP53 tumor suppressor gene leads to the development of malignant cell clones and accelerates the carcinogenesis (9). Different mechanisms of functional p53 inactivation have been described including functionally relevant point mutations and gross chromosomal alterations, mostly driven by chromosome 17p deletions.
Previous studies reported inconclusive results on whether p53 accumulation has a functional impact on progression of EC (10–19). While some studies found associations between the p53 expression level and tumor progression or overall-survival, other studies were not able to confirm these findings. Data on TP53 deletion status is available from only one report including 40 patients suffering from esophageal squamous cell carcinoma (20).
To further elucidate the clinical relevance of TP53 mutations and p53 expression as prognostic biomarkers in esophageal cancer we took advantage of our preexisting tissue microarray (TMA) containing nearly 700 esophageal cancer specimens with attached clinical follow-up and clinico-pathological data.
Our findings demonstrate that strong p53 immunostaining is correlated with unfavorable prognosis in esophageal cancer, while homozygous TP53 deletions represent a catastrophic event leading to cell death.
Discussion
Our study shows that p53 overexpression is linked to shortened overall survival in patients suffering from EC. To our knowledge, this is the study includes the largest number of cases for EC and SCC with corresponding survival data. Furthermore, we correlated p53 IHC and TP53 FISH analysis. As follows in the discussion, previous studies either present inconclusive results or include only few patients.
In our analysis p53 alterations are present in 40–45% of esophageal cancer, irrespective of the underlying histological type. Genome studies conducted either with array comparative genome hybridization (CGH) or whole genome sequencing repeatedly revealed TP53 mutations as the most common gene mutation in EC with reported mutation rates between 40–70% (4,7,24). The variance of the reported mutation frequencies is in concordance to immunohistochemical confirmation studies for SSC and the results revealed from our study (19,25–28). In contrast, data from large patient cohorts concerning p53 expression in esophageal adenocarcinoma are hardly available. A recent review article by Chen and colleagues analyzed biomarkers in esophageal cancer and found only 2 studies focusing on p53 expression in esophageal adenocarcinoma with 97 patients included in the meta-analysis (14). These studies also showed p53 protein alterations in 40–65%, although the case load is too small to draw general conclusions. Our group has previously shown that the applied IHC protocols, for prostate cancer, reveal high concordance of IHC data with sequencing results, arguing for a strong validity of our IHC protocol (22).
p53 is physiologically expressed by numerous cell types and plays a major role in cell cycle control. It is considered one of the most important tumor suppressor genes (9). p53 mutations have been found in multiple cancers with large variances in the mutation frequency, depending on the observed tumor entity (29).
In our study heterozygous TP53 deletions were found in 41% of AC and 19% of SCC. Only little data on TP53 gene deletions have yet been reported. Recently, a study on 40 patients suffering from esophageal squamous cell carcinoma revealed heterozygous deletion of TP53 in 22 patients (55%). A correlation with clinical data could not be established in this study (20). The deletion rate is somewhat higher than it is in our data, which is partially caused by our stringent criteria for defining TP53 deletions. These were applied to avoid false deletion calling due to truncation of the nuclei during tissue sectioning. Most samples with TP53 deletion had >80% cells with fewer TP53 fluorescence signals than centromere 17 signals. In contrast to other studies, we did not use automated systems for detection of particular gene deletions which is another possibility for deviating deletion rates compared to previous published reports (20). As already shown in prostate cancer on a TMA with >11,000 patient samples, 100% concordance between array-CGH detected deletions and FISH could be achieved for PTEN and TP53 deletions using these criteria (22,23,30). Therefore, differences in the deletion rate are explained by different protocols calling more deletions as present within one patient sample. The deletion rate of 19% supports the assumption that TP53 deletions have only minor significance as a pathway for p53 inactivation in esophageal SCC. In line with the results revealed by our study, the impact of p53 mutations is of higher importance with respect to tumor progression than the heterozygous gene deletion of chromosome 17p13. These findings also match a Northern blot analysis on expression of the p53 gene in esophageal tumorigenesis. RNA was extracted from tumor, Barrett's epithelium, and histologically normal esophageal mucosa. p53 was found to be overexpressed in cancerous or metaplastic tissue in comparison to normal tissue. Thus, the authors concluded that p53 is implicated in the progression of Barrett's epithelium to invasive cancer (31).
By analyzing p53 immunostaining and TP53 deletions, we were able to correlate p53 IHC and TP53 deletion status with corresponding clinical data of our patient cohort. Patients with p53 overexpression in IHC presented with shortened OS and unfavorable clinic-pathological data, meaning that p53 mutations must have an influence on tumor biology. This effect is also paralleled by a significant correlation between advanced tumor stages and high p53 expression levels in AC (Table I: Tumor stage (P=0.019), UICC stage (P=0.004), grading (P=0.027) and surgical resection margin status (P=0.006)). Furthermore, the multivariate analysis of p53 immunostaining in a setting with various established prognostic factors for esophageal cancer, such as UICC stage, distant metastasis, resection margin and grading revealed that p53 accumulation is an independent prognostic marker in SCC. In contrast, in our analysis, heterozygous TP53 deletions do not affect the OS, neither in AC nor in SCC. These results confirm the findings of former studies in EC and other tumor entities that mono-allelic protein expression is sufficient for regular cell cycle control mediated by p53 (29). Our data show, that mono-allelic TP53 deletion cannot be considered a major driver for tumor progression in esophageal cancer while there is no mutation in the remaining TP53 allele.
Since the poor outcome of patients with strong p53 immunostaining was independent of the TP53 deletion status, it is tempting to speculate that these cancers may at least carry dominant negative mutations with complete inactivation of wild-type p53 protein through complex formation with mutant p53 protein. This mechanism is known to lead to massive nuclear accumulation of inactive p53 complexes composed of mutated and non-mutated p53 protein (29). While IHC reveals both mutated and non-mutated p53, cells with accumulation of inactive p53 complexes are the ones that will predominantly show strong staining. Thus, strong p53 immunostaining in our samples indicates an accumulation of unfunctional p53.
Alterations of p53 have been found in virtually every region of the protein but only a handful of the most frequently occurring mutations haven been studied in depth for their contribution to cancer progression (32). In EC, the majority of patients present with TP53 exon single nucleotide mutations (approximately 70%) while frameshift mutations are much less frequent. Only 15% of cases have been reported to have insertions or deletions as found by genome wide sequencing and PCR exon analysis (6,32). Numerous in vitro and in vivo studies confirmed the ability of mutant p53 to drive enhanced cancer invasion and motility, with evidence that mutant p53 can enhance signaling through receptors such as transforming growth factor β (TGFβ) or epidermal growth factor receptor (EGFR). Additionally, although mutated p53 has generally lost the ability to bind p53 DNA binding regions in target gene promoters, various p53 mutants can bind directly to DNA with some degree of selectivity and may thereby control the transcription of certain genes (33,34). Furthermore, there is increasing evidence that mutated p53 has an inhibitory effect on transcription factors, such as TAp63, which is of regulative importance for numerous miRNA with important roles in cancer invasion and progression (35,36).
In contrast to other cancers, such as prostate cancer, the combination of gene deletion and mutation status does not increase the malignant potential in esophageal cancer (22). In esophageal adenocarcinoma only 53 (21%) patients showed TP53 deletion combined with strong p53 immunostaining, the rate being even lower in SCC with only 11%. These low frequencies suggest that biallelic inactivation, which is associated with a total loss of functional p53, is a catastrophic cellular event related with a high level of apoptosis. Studies using transgenic mouse models carrying heterozygous TP53 deletions show larger and more mammalian tumors than animals with two wild type TP53 alleles (37). Moreover, the results from animal studies confirm the findings in our study, as these results indicate that loss of both TP53 alleles is not a prerequisite for tumor formation and that mere reduction in p53 levels may be sufficient to promote tumorigenesis.
Other studies have examined serum levels of p53 antibodies and their correlation to outcome. Shimada et al (38) reported on 28 patients with SCC that were positive for serum p53 antibodies out of a cohort of 105 cancers. These patients' survival was significantly worse than that of seronegative patients. To our knowledge, no correlation between serum p53 antibody levels and p53 expression in cancer tissue has yet been described for esophageal cancer. Our study does not provide any data on this issue since serum p53 antibodies were not examined in our cohort.
Seeing that cancers with strong p53 expression are correlated with poor survival independent of the established clinic-pathological prognostic markers supports the notion that these patients might profit from neoadjuvant and adjuvant therapeutic regimes in a multimodal setting. Tumor size, nodal status and resection margin appear to not be the only relevant markers for prediction of survival in EC and therefore the search for prognostic biomarkers is warranted even in this highly malignant disease with poor overall survival even in less advanced stages.
In summary, the results of our large-scale TMA analysis in esophageal adeno- and squamous cell carcinoma show that different types of p53 alterations characterize subgroups of patients with different outcomes. Strong p53 expression is correlated with unfavorable prognosis in esophageal cancer and represents an independent prognosticator in SCC. Furthermore, homozygous TP53 deletions are catastrophic cellular events related with a high level of apoptosis.