Clinical significance of p53 protein expression and TP53 variation status in colorectal cancer

In the present study, we investigated the immunohistochemical expression of p53 and the variational status of TP53 by NGS in CRC patients. In the 204 CRC patients, TP53 variations were detected in 73% of patients (149/204), with 108 (72.5%) patients harboring missense variation and 41 (27.5%) patients with nonsense or frameshift variation. (2) The cutoff value for p53 IHC expression reflecting missense variations was 80%, and the cutoff value for nonsense/frameshift variations was 0%. Subdividing p53 expression into missense (p53 proportion, ≥80%) and nonsense/frameshift (p53 proportion, 0%) patterns showed significant correlation with missense and nonsense/frameshift TP53 variations, respectively. (3) TP53 variation and p53 IHC expression showed correlation with poor prognostic factors such as higher N stage and TNM stage. (4) Univariate and multivariate survival analyses indicated positive p53 IHC expression (p53 proportion, > 55%) as an independent factor for poor OS in patients with CRC. (5) Nonsense/frameshift (p53 proportion, 0%) expression pattern of p53 showed a significantly better prognosis than wild type or missense p53 IHC expression pattern.

Currently, immunohistochemical staining for p53 is the tool used most often for evaluating TP53 variation status. However, after introduction of NGS, sequencing of the TP53 gene in cancer has been increasing rapidly. Previous reports have demonstrated the correlation between p53 expression and TP53 variation detection by NGS. In a study on ovarian carcinoma, the authors classified p53 expression into wild type, overexpression, and complete absence [15]. The p53 IHC expression showed good concordance with the variation status of TP53. The sensitivity of IHC for detecting gain-of-function variations, loss-of-function variation, and the wild type expression of p53 was 100, 76, and 100%, respectively [15]. The specificity of IHC for detecting gain-of-function variations, loss-of-function variations, and wild type expression of p53 was 95, 100, and 96%, respectively [15]. In gastric cancer, the IHC of p53 expression showed a significant correlation with TP53 variation detected by NGS. In brain glioma, the sensitivity of p53 IHC for detecting TP53 variation was 87% [18]. The cut-off point for p53 IHC differs according to organ studied. The cut-off point was 50% in ovarian cancer, 10% in brain glioma, and 50% in gastric cancer. In the present study, we performed ROC curve analysis to set a cut-off point for p53 IHC. The cut-off point was 80 and 1% for missense variation and nonsense/frameshift variation, respectively. On the other hand, there was also a report that the IHC of p53 expression cannot be used to predict TP53 variations [19]. However, precise validation of the cut-offs related to percent positivity of p53 IHC has been limited in CRC. The reports regarding the correlation between immunohistochemical expression of p53 and TP53 variation status is summarized in Table 10. To the best of our knowledge, this is the first study to report a correlation between immunohistological expression of p53 and variational status of TP53 gene in CRC patients. In line with previous reports, our data showed a significant correlation between IHC expression of p53 and variational status of the TP53 gene. Moreover, we set the cut-off point for IHC of p53 expression by analyzing the ROC curve for variational status of TP53. Subclassifying p53 expression into three types (missense, nonsense/frameshift, and wild type) showed better accuracy for detecting TP53 variations than did subdividing p53 expression into two types, such as positive/negative or wild/aberrant type. Based on these results, if the cut-off point for p53 IHC is appropriately set, the IHC of p53 expression can predict the variational status of TP53 with high probability.

Table 10 Previous reports regarding the association between TP53 variation status and IHC expression of p53

Additionally, in predicting TP53 variation, the sensitivity, specificity, and accuracy of p53 IHC expression show different results depending on the different clones of the p53 antibody. As shown in Table 10, in the study conducted in gastric carcinoma, p53 IHC using SP5 clone predicted the TP53 variation most accurately. Also, in the present study, it was found that the SP5 clone of the p53 antibody was the best predictor of the TP53 mutation state. These results suggest that not all p53 antibodies are acceptable in predicting TP53. Therefore, when conducting future studies, it is recommended to set the conditions that can most effectively predict the TP53 variation through the combination of staining conditions and different p53 antibody clones.

The p53 protein, it has been established as a tumor suppressor by extensive studies [20]. Generally, tumor suppressor genes such as BRCA1, RB, and APC lose function through deletions or truncating variations in cancer cells. However, unlike other tumor suppressor genes, the majority of TP53 variations in cancers is missense variation [21, 22], and most of these occur in the DBD [23]. Our data supported this, showing that 98.1% of the missense variations were located in the DBD. Many studies have confirmed that missense variations can induce tumor progression by a gain-of-function mechanism through regulating proliferation, metastasis, genomic instability, differentiation, metabolism, and immune reactions [23]. In addition, if there is a product missense variation of the TP53 gene, the mutant protein product is relatively resistant to MDM2-mediated ubiquitination and accumulates in the nucleus of cancer cells, leading to overexpression of p53 [21]. There have been previous reports that p53 overexpression is related to poor survival or progression of CRC in patients [24, 25]. In our study, we investigated the prognosis of CRC patients according to the status of p53 IHC and TP53 variations.

As with previous reports, our data showed that the CRC patients with negative p53 expression have better OS than CRC patients with positive p53 expression. In addition, multivariate analysis confirmed that positive p53 IHC is an independent poor prognostic factor for CRC patients. However, no other criteria for p53 IHC (wild type pattern/aberrant type pattern) or variational status of TP53 affected the prognosis of CRC patients. The IHC of p53 expression reveals not only the variational status of TP53, but also the post-transcriptional status of the p53 protein. Some reports emphasize the importance of post-translational modification of p53 in tumorigenesis or tumor progression [26, 27]. Our findings and previous reports suggest that the expression status of the p53 protein has a greater impact on the prognosis of CRC patients than does the TP53 variation itself.

Another interesting finding in our study was that CRC patients with a nonsense/frameshift pattern of DO-7 clone of p53 expression showed significantly better OS than patients with a missense pattern or a wild type pattern of p53 expression. Many studies have been reported on the effect of immunohistochemical expression of p53 on the prognosis of CRC patients. Most of those studies report that CRC patients with p53 overexpression, that is, missense pattern expression, have a poor prognosis. However, there are very limited reports that patients with no or reduced p53 expression have a better prognosis than CRC patients with wild type or missense pattern expression, as in the present study. The p53 protein is actively involved in various DNA damage-response mechanisms [28]. When cells are under stress and experience DNA damage, p53 induces cell-cycle arrest, activates DNA-repair mechanisms, and restores genomic stability [28]. In addition, various DNA-repair systems can be directly activated by the p53 protein [28]. The main adjuvant chemotherapeutic agent for advanced CRC in our institute is oxaliplatin. This agent induces DNA damage by preventing DNA replication. There are numerous reports that mutant p53 (mainly with gain-of-function missense variations) is associated with chemoresistance via various pathways [29,30,31]. However, we could not find any reports about increased sensitivity to chemotherapy in cells with nonsense/frameshift TP53 variation or absence of p53 expression. In this study, CRC patients without p53 expression had better OS than patients with p53 expression. Based on these results and the results of previous studies indicating that p53 overexpression is related to chemoresistance, we considered the possibility that the group with no p53 expression had better OS through chemosensitivity (or low chemoresistance). However, further studies are needed to determine the chemotherapy susceptibility in cancer cells lacking p53 expression.

Although variations of p53 protein are investigated in the present study, isoforms of p53 protein have been proven to be dysregulated in several human tumors including CRC [32]. Various isoforms of p53 are reported to be involved in development and progression of CRCs [32]. Cell functions affected by the p53 isoforms include apoptosis, autophagy, DNA repair, invasion, angiogenesis, metabolism, and senescence [32]. Moreover, although not much research has been conducted yet, it has been reported that a specific p53 isoform affects the prognosis of CRC. The antibodies used in the present study can capture some isoforms as well (DO7 and Bp53–11 recognize p53β and p53γ; for SP5 the epitope is not determined). However, in this study, the effect of p53 isoforms in CRC patients was not investigated. Therefore, in the future, not only studies on TP53 variations but also studies on the effect of the various p53 isoforms on the prognosis and treatment of CRC patients might be considered.

In conclusion, our study showed that IHC of p53 expression can predict TP53 variation status. To predict the prognosis of CRC patients, p53 protein expression is thought to provide more information than the variation itself. In our study, CRC patients without p53 expression had a better prognosis. Further studies are needed to establish the mechanism for differences in OS in CRC patients with or without p53 expression.

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