TP53 mutation analysis
We used targeted NGS (by OCAv1) to characterize CRC for TP53 mutations. For this method, TP53 mutation analysis was performed on the entire exome. The TP53 mutations were observed in 73% (149/204) and are summarized in Table 1. Of the TP53 mutations, 108 (72.5%) were missense mutations (MS), 23 (15.4%) were nonsense mutations (NS), and 18 (12.1%) were frameshift mutations (FS). Among the functional domains of TP53, mutations were observed most often in the DNA binding domain (DBD), in 86.6% of patients (129/149 cases). By mutation type, 98.2% of MS mutations (106/108), 43.5% of NS mutations (10/23), and 72.2% of FS mutations (13/18) were observed in DBD. Compared to MS or FS mutations, NS mutations were more commonly observed in domains other than the DBD, 30.4% (7/23) in the tetramerization domain and 26.1% (6/23) in the nuclear localization signaling (NLS) region. MS mutations were evenly distributed in the subregions within the DBD (L2, L3, LSH, and other). However, NS and FS mutations were observed mostly in non-zinc binding regions (excluding L2, L3, and LSH), at 91.3% (21/23) and 83.3% (15/18), respectively.
Table 1
Summary of TP53 mutations for 204 colorectal carcinoma patients
| Total n = 149 | Missense n = 108 | Nonsense n = 23 | Frameshift n = 18 |
Functional domains | | | | |
Transactivation | 0 | 0 | 0 | 0 |
Proline rich region | 3 | 0 (0%) | 0 (0%) | 3 (16.7%) |
DNA binding region | 129 | 106 (98.1%) | 10 (43.5%) | 13 (72.2%) |
Nuclear localization signalling | 6 | 0 (0%) | 6 (26.1%) | 0 (0%) |
Tetramerization | 9 | 1 (0.9%) | 7 (30.4%) | 1 (5.6%) |
Regulatory | 2 | 1 (0.9%) | 0 (0%) | 1 (5.6%) |
Sub-regions of DB domain | | | | |
L2 | 31 | 28 (25.9%) | 1 (4.3%) | 2 (11.1%) |
L3 | 22 | 20 (18.5%) | 1 (4.3%) | 1 (5.6%) |
LSH | 31 | 31 (28.7%) | 0 (0%) | 0 (0%) |
Other | 65 | 29 (26.9%) | 21 (91.3%) | 15 (83.3%) |
Exons | | | | |
Exon4 | 9 | 1 (0.9%) | 1 (4.3%) | 7 (38.9%) |
Exon5 | 36 | 33 (30.6%) | 1 (4.3%) | 2 (11.1%) |
Exon6 | 22 | 12 (11.1%) | 7 (30.4%) | 3 (16.7%) |
Exon7 | 26 | 24 (22.2%) | 0 (0%) | 2 (11.1%) |
Exon8 | 44 | 35 (32.4%) | 7 (30.4%) | 2 (11.1%) |
Exon9 | 1 | 0 (0%) | 1 (4.3%) | 0 (0%) |
Exon10 | 10 | 2 (1.9%) | 6 (26.1%) | 1 (5.6%) |
Exon11 | 2 | 1 (0.9%) | 0 (0%) | 1 (5.6%) |
Correlation between immunohistochemical p53 expression and TP53 mutation
Despite the increased incidence of NGS testing in CRCs, IHC is used most commonly to evaluate TP53 status. Therefore, we investigated the correlation of immunohistochemical expression of p53 with TP53 mutations. In this study, we classified p53 expression based on two criteria. First, p53 expression was categorized as wild type pattern (p53 proportion, 1 ~ 79%) or aberrant type pattern (0% or ≥ 80%), and this classification showed significant correlation with TP53 mutation (P < 0.001) (Table 2). Sensitivity and specificity for detecting TP53 mutation using this criterion were 87.2% and 61.8%, respectively (Table 3). The other criterion of classifying p53 expression into positive (p53 proportion, > 55%) and negative (p53 proportion, ≤ 55%) groups was also significantly correlated with TP53 mutation (P < 0.001) (Table 2). Sensitivity and specificity for detecting TP53 mutation using this criterion were 71.8% and 69.1%, respectively (Table 3).
Table 3
Sensitivity, specificity, and accuracy of p53 immunohistochemistry for detecting TP53 mutation
Mutation type | Sensitivity | Specificity | Accuracy |
Binary (IHC: wild/aberrant) | 87.2% | 61.8% | 80.4% |
Binary (IHC: positive/negative) | 71.8% | 69.1% | 71.1% |
Missense mutation | 88% | 84.4% | 86.3% |
Nonsense/frameshift mutation | 70.7% | 92.6% | 88.2% |
Wild type | 61.8% | 87.2% | 80.4% |
As mentioned above, TP53 mutation can be further classified into missense and nonsense/frameshift types. Accordingly, we subdivided the p53 aberrant type pattern into missense (p53 proportion, ≥ 80%) and nonsense/frameshift (p53 proportion, 0%) type. This subgrouping of p53 expression showed a significant correlation with TP53 mutation types (Table 4). The sensitivity, specificity, and accuracy for detecting TP53 mutations are shown in Table 3.
Table 4
Correlation between p53 immunohistochemical expression pattern and TP53 mutation type
| | p53 IHC | p |
Wild type pattern (1 ~ 79%) | Aberrant type pattern |
Nonsense/frameshift pattern (0%) | Missense pattern (≥ 80%) |
All cases | | 53 | 41 | 110 | |
TP53 mutation status | | | | | |
Wild type | 55 | 34 (61.8%) | 11 (20%) | 10 (18.2%) | |
Missense mutation | 108 | 12 (11.1%) | 1 (0.9%) | 95 (88%) | |
Nonsense/frameshift mutation | 41 | 7 (17.1%) | 29 (70.7%) | 5 (12.2%) | < 0.001 |
Immunohistochemical expression of p53 and TP53 mutation in CRC patients and their correlation with clinicopathologic characteristics
Correlation between the clinicopathologic factors of TP53 mutation and p53 expression is summarized in Table 5. In the immunohistochemical staining of p53, an aberrant expression pattern was identified in 151 (74%) patients, and 124 (60.8%) patients were classified in the positive (> 55%) expression group. The aberrant p53 expression pattern was significantly correlated with lower histologic grade, higher N stage, and TNM stage. The positive p53 expression group showed a significant correlation with left-side CRC, higher N stage, and TNM stage. The TP53 mutation showed a significant correlation with smaller tumor size, higher N stage, and TNM stage.
Table 5
Correlation between clinicopathologic factors to TP53 mutation and p53 IHC expressions
Characteristics | Total | p53 IHC | p | p53 IHC | p | TP53 mutation | p |
Wild type pattern (1 ~ 79%) | Aberrant type pattern (0% or ≥ 80%) | Negative ≤ 55% | Positive > 55% | Wild | Mutant |
All cases | 204 | 53 (26%) | 151 (74%) | | 80 (39.2%) | 124 (60.8%) | | 55 (27%) | 149 (73%) | |
Sex | | | | | | | | | | |
Male | 117 | 34 (29.1%) | 83 (70.9%) | | 50 (42.7%) | 67 (57.3%) | | 35 (29.9%) | 82 (70.1%) | |
Female | 87 | 19 (21.8%) | 68 (78.2%) | 0.245 | 30 (34.5%) | 57 (65.5%) | 0.232 | 20 (23%) | 67 (77%) | 0.27 |
Age (years) | | | | | | | | | | |
< 50 | 12 | 2 (16.7%) | 10 (83.3%) | | 5 (41.7%) | 7 (58.3%) | | 4 (33.3%) | 8 (66.7%) | |
≥ 50 | 192 | 51 (26.6%) | 141 (73.4%) | 0.448 | 75 (39.1%) | 117 (60.9%) | 0.858 | 51 (26.6%) | 141 (73.4%) | 0.608 |
Histologic grade | | | | | | | | | | |
Well or Moderate | 167 | 38 (22.8%) | 129 (77.2%) | | 64 (38.3%) | 103 (61.7%) | | 42 (25.1%) | 125 (74.9%) | |
Poor | 37 | 15 (40.5%) | 22 (59.5%) | 0.026 | 16 (43.2%) | 21 (56.8%) | 0.579 | 13 (35.1%) | 24 (64.9%) | 0.216 |
Site | | | | | | | | | | |
Right side | 69 | 23 (33.3%) | 46 (66.7%) | | 34 (49.3%) | 35 (50.7%) | | 19 (27.5%) | 50 (72.5%) | |
Left side | 135 | 30 (22.2%) | 105 (77.8%) | 0.087 | 46 (34.1%) | 89 (65.9%) | 0.035 | 36 (26.7%) | 99 (73.3%) | 0.895 |
Tumor size | | | | | | | | | | |
< 4.5cm | 114 | 25 (21.9%) | 89 (78.1%) | | 43 (37.7%) | 71 (62.3%) | | 24 (21.1%) | 90 (78.9%) | |
≥ 4.5cm | 90 | 28 (31.1%) | 62 (68.9%) | | 37 (41.1%) | 53 (58.9%) | 0.622 | 31 (34.4%) | 59 (65.6%) | 0.032 |
CEA | | | | | | | | | | |
< 5 ng/ml | 158 | 40 (25.3%) | 118 (74.7%) | | 64 (40.5%) | 94 (59.5%) | | 38 (24.1%) | 120 (75.9%) | |
≥ 5 ng/ml | 46 | 13 (28.3%) | 33 (71.7%) | 0.689 | 16 (34.8%) | 30 (65.2%) | 0.484 | 17 (37%) | 29 (63%) | 0.083 |
T stage | | | | | | | | | | |
T1-3 | 171 | 43 (25.1%) | 128 (74.9%) | | 68 (39.8%) | 103 (60.2%) | | 45 (26.3%) | 126 (73.7%) | |
T4 | 33 | 10 (30.3%) | 23 (69.7%) | 0.536 | 12 (36.4%) | 21 (63.6%) | 0.714 | 10 (30.3%) | 23 (69.7%) | 0.637 |
N stage | | | | | | | | | | |
N0 | 107 | 37 (34.6%) | 70 (65.4%) | | 49 (45.8%) | 58 (54.2%) | | 36 (33.6%) | 71 (66.4%) | |
N1-3 | 97 | 16 (16.5%) | 81 (83.5%) | 0.003 | 31 (32%) | 66 (68%) | 0.043 | 19 (19.6%) | 78 (80.4%) | 0.024 |
TNM stage | | | | | | | | | | |
Stage I, II | 110 | 37 (33.6%) | 73 (66.4%) | | 51 (46.4%) | 59 (53.6%) | | 37 (33.6%) | 73 (66.4%) | |
Stage III, IV | 94 | 16 (17%) | 78 (83%) | 0.007 | 29 (30.9%) | 65 (69.1%) | 0.024 | 18 (19.1%) | 76 (80.9%) | 0.02 |
Since p53 expression and TP53 mutation showed significant correlations with N stage and TNM stage, we subdivided the p53 aberrant type pattern into missense (p53 proportion, ≥ 80%) and nonsense/frameshift (p53 proportion, 0%) types and TP53 mutation into missense and nonsense/frameshift mutations and analyzed the correlation between N stage and TNM stage (Tables 6, 7). For p53 IHC, missense pattern and nonsense/frameshift pattern were significantly associated with higher N stage compared to wild type pattern, and missense pattern p53 expression was significantly higher in high TNM stage (Table 6). For TP53 mutation, nonsense/frameshift mutation showed significant correlations to higher N stage and TNM stage (Table 7). Missense mutations of TP53 were significantly related with higher TNM stage but not with N stage (Table 7).
Table 6
Correlation between p53 immunohistochemical expression pattern and lymph node stage and TNM stage.
Characteristics | Total | p53 IHC | p | Total | p53 IHC | p |
Wild type pattern (1 ~ 79%) | Missense pattern (≥ 80%) | Wild type pattern (1 ~ 79%) | Nonsense/frameshift pattern (0%) |
N stage | | | | | | | | |
N0 | 88 | 37 (42%) | 51 (58%) | | 56 | 37 (66.1%) | 19 (33.9%) | |
N1-3 | 76 | 16 (21.1%) | 60 (78.9%) | 0.004 | 37 | 16 (43.2%) | 21 (56.8%) | 0.03 |
TNM stage | | | | | | | | |
Stage I, II | 90 | 37 (41.1%) | 53 (58.9%) | | 57 | 37 (64.9%) | 20 (35.1%) | |
Stage III, IV | 74 | 16 (21.6%) | 58 (78.4%) | 0.008 | 36 | 16 (44.4%) | 20 (55.6%) | 0.052 |
Table 7
Correlation of TP53 mutation type with lymph node stage and TNM stage.
Characteristics | Total | TP53 mutation status | p | Total | TP53 mutation status | p |
Wild type | Missense | Wild type | Nonsense/frameshift |
N stage | | | | | | | | |
N0 | 90 | 36 (40%) | 54 (60%) | | 53 | 36 (67.9%) | 17 (32.1%) | |
N1-3 | 73 | 19 (26%) | 54 (74%) | 0.061 | 43 | 19 (44.2%) | 24 (55.8%) | 0.019 |
TNM stage | | | | | | | | |
Stage I, II | 92 | 37 (40.2%) | 55 (59.8%) | | 55 | 37 (67.3%) | 18 (32.7%) | |
Stage III, IV | 71 | 18 (25.4%) | 53 (74.6%) | 0.047 | 41 | 18 (43.9%) | 23 (56.1%) | 0.022 |
Prognostic impact of immunohistochemical expression of p53 and TP53 mutation in CRC patients
Table 8 shows univariate analysis for OS and RFS of CRC patients. Histologic grade, T stage, N stage, TNM stage, and positive p53 expression (P = 0.018) were significantly associated with OS of CRC patients. The CRC patients with positive p53 expression had a 4.35-fold [95% confidence interval (95% CI); 1.29–14.71, P = 0.018] increased risk of death. Tumor size, T stage, N stage, and TNM stage were significantly correlated with the RFS of CRC patients by univariate analysis. CRC patients with an aberrant p53 expression pattern showed a tendency (P = 0.081) for shorter RFS. The mutational status of TP53 was not associated with OS or RFS.
Table 8
Univariate Cox proportional hazards regression analysis for overall survival and relapsefree survival in colorectal cancer patients.
Characteristics | OS | RFS |
| HR (95% CI) | p | HR (95% CI) | p |
Sex, female (vs. male) | 0.646 (0.263–1.588) | 0.341 | 0.985 (0.473–2.052) | 0.968 |
Age, y ≥ 50 (vs. < 50) | 1.394 (0.187–10.369) | 0.745 | 1.874 (0.255–13.759) | 0.537 |
Histologic grade, Poor (vs. Well or Moderate) | 3.324 (1.419–7.784) | 0.006 | 1.211 (0.494–2.964) | 0.676 |
Site, Left side (vs. Right side) | 0.725 (0.31–1.696) | 0.725 | 1.003 (0.469–2.144) | 0.993 |
Tumor size, ≥ 4.5cm (vs. < 4.5cm) | 1.95 (0.833–4.565) | 0.124 | 2.109 (1.014–4.387) | 0.046 |
T stage, T4 (vs. T1-3) | 3.262 (1.367–7.779) | 0.008 | 2.695 (1.231–5.899) | 0.013 |
N stage, N1-3 (vs. N0) | 3.129 (1.224- 8) | 0.017 | 4.262 (1.826–9.943) | 0.001 |
TNM Stage, III or IV (vs. TNM Stage, I or II) | 3.326 (1.301–8.503) | 0.012 | 3.775 (1.679–8.489) | 0.001 |
CEA, < 5 ng/ml (vs. ≥ 5 ng/ml) | 1.389 (0.542–3.557) | 0.493 | 1.048 (0.425–2.584) | 0.919 |
p53 IHC, Aberrant pattern (vs. Wild pattern) | 0.96 (0.376–2.454) | 0.932 | 3.591 (0.854–15.097) | 0.081 |
p53 IHC, positive, > 55% (vs. negative, ≤ 55%) | 4.352 (1.288–14.712) | 0.018 | 0.919 (0.446–1.894) | 0.818 |
TP53, Wild type (vs. Mutant type) | 1.739 (0.588–5.142) | 0.317 | 1.36 (0.583–3.174) | 0.477 |
Kaplan-Meier survival analysis curves for OS and RFS of CRC patients according to TP53 status or p53 IHC are presented in Fig. 1. For OS, the group with positive expression for p53 had significantly shorter OS than the negative expression group (P = 0.01). The OS of CRC patients with TP53 mutation or aberrant p53 expression pattern did not show a significant difference from the TP53 wild type or p53 wild type expression pattern. The mutation status of TP53 and p53 expression showed no difference on the RFS of CRC patients.
In addition, we further divided the p53 aberrant type pattern into missense and nonsense/frameshift type and TP53 mutation into missense and nonsense/frameshift mutation and performed Kaplan-Meier analysis for OS of CRC patients (Fig. 2). The p53 expression patterns were significantly associated with OS survival of CRC patients (P = 0.04). The CRC patients with nonsense/frameshift pattern of p53 expression showed significantly better prognosis compared to patients with missense or wild type pattern (P = 0.012, P = 0.025, respectively). However, there was no significant difference in OS of CRC patients according to type of TP53 mutation (Fig. 2).
We performed multivariate analysis for OS and RFS of CRC patients (Table 9). Multivariate analysis included sex, age, histologic grade, site, tumor size, T stage, N stage, and TNM stage. Along with the above-listed variables, multivariate analysis was performed and included p53 expression pattern (wild/aberrant), positive/negative p53 expression group, and TP53 mutational status in models 1–3. For the OS of CRC patients, histologic grade, TNM stage, and positive/negative p53 expression were independent prognostic factors. The group with positive expression for p53 had a 4.1-fold (95% CI; 1.2-14.03, P = 0.025) increased risk of death compared to the group with negative expression. For the RFS of CRC patients, only N stage was an independent prognostic factor.
Table 9
Multivariate Cox regression analysis for overall survival and relapsefree survival in colorectal cancer patients.
Characteristics | OS | RFS |
| HR (95% CI) | p | HR (95% CI) | p |
Model 1 | | | | |
Histologic grade, Poor (vs. Well or Moderate) | 3.077 (1.312–7.221) | 0.01 | 0.846 (0.336–2.131) | 0.723 |
N stage, 1–3 (vs. N stage, 0) | 0.766 (0.1-5.873) | 0.798 | 4.262 (1.826–9.943) | 0.001 |
TNM Stage, III or IV (vs. TNM Stage, I or II) | 3.122 (1.219-8) | 0.018 | 1.088 (0.205–5.768) | 0.921 |
Model 2 | | | | |
Histologic grade, Poor (vs. Well or Moderate) | 3.375 (1.431–7.964) | 0.005 | 0.828 (0.328–2.092) | 0.69 |
N stage, 1–3 (vs. N stage, 0) | 0.61 (0.094–3.982) | 0.606 | 4.262 (1.826–9.943) | 0.001 |
TNM Stage, III or IV (vs. TNM Stage, I or II) | 2.543 (0.981–6.592) | 0.055 | 1.123 (0.212–5.946) | 0.892 |
p53 IHC, positive, > 55% (vs. negative, ≤ 55%) | 4.098 (1.197–14.031) | 0.025 | 0.77 (0.371–1.599) | 0.483 |
Model 3 | | | | |
Histologic grade, Poor (vs. Well or Moderate) | 3.077 (1.312–7.221) | 0.01 | 0.836 (0.33–2.114) | 0.705 |
N stage, 1–3 (vs. N stage, 0) | 0.722 (0.083–6.314) | 0.769 | 4.262 (1.826–9.943) | 0.001 |
TNM Stage, III or IV (vs. TNM Stage, I or II) | 3.122 (1.219-8) | 0.018 | 1.115 (0.211–5.878) | 0.898 |