# The High Expression of p53 Is Predictive of Poor Survival RatherTP53 Mutation in Esophageal Squamous Cell Carcinoma

Authors: Yan Jin; Xueke Zhao; Xin Song; Ran Wang; Zongmin Fan; Panpan Wang; Miaomiao Yang; Fuyou Zhou; Qide Bao; Lidong Wang
Journal: Journal of Oncology
 (2023)

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## Abstract

TP53 is a well-known tumor suppressor gene and one of the most common genetic alterations in human cancers. However, the role of p53 as a prognostic marker of esophageal squamous cell carcinoma (ESCC) is controversial in the association between TP53 alterations and clinical outcomes. To address this issue, we evaluated TP53 mutations, p53 protein expression, clinicopathological parameters, and survivals rates in a large scale of patients with ESCC. Two cohorts were included in this study: TP53 mutations were detected by next-generation sequencing in 316 ESCC patients, and p53 protein expression was tested by immunohistochemistry in 6,028 ESCC patients. Survival analysis was performed using the Kaplan–Meier curve and the Cox proportional hazards model. TP53 mutations were found in ESCC patients from 241 of 316 (76.3%), and the rate of positive expression of p53 protein was 59.1% in 6,028 ESCC patients (including 1819 with high expression of p53 protein), respectively. Most mutations were missense, which has a high expression of p53 protein. Compared with wild-typeTP53, TP53 gene mutations were not significantly associated with survival time (p=0.083). In multivariate analysis, the p53 protein expression was an independent prognostic factor for ESCC. The high-expression group of p53 protein has poor survival (p<0.001) compared to low-expression group in patients with ESCC. The high expression of the p53 protein, not the TP53 mutation, is predictive of poor survival in patients with ESCC, and p53 protein expression might have the potential to be a prognosis biomarker and therapy target in ESCC.


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## Body

## 1. Introduction

Esophageal carcinoma is one of the most aggressive cancers and the sixth leading cause of cancer death [1]. Esophageal cancer (EC) is the fourth most common malignancy associated with cancer-related death, and esophageal squamous cell carcinoma (ESCC) is the most common pathological subtypes (>90%) in China [2–5]. With limited early clinical diagnosis approaches and few targeted therapies, although the five-year survival rate has improved during past decades, it still remains dismal, only at 10%–30% in most countries [6]. The most effective way to improve the survival rate of ESCC patients is through early detection and treatment. Therefore, the discovery of molecular markers for early screening, prognosis, and efficacy of evaluation to provide personalized treatment for ESCC patients is one of the entry points to reduce the incidence and improve the survival rate of patients with ESCC.TP53 is one of the most frequently mutated genes in human cancers and has occurred in more than 50% of all human cancers [7, 8]. A recent study analyzed the TP53 mutational spectra of 7,525 pan-cancer tissues and found TP53 mutations in 35% of all 30 tumor-type samples, of which the most mutated cohorts are more than 80% and the lowest percentage of TP53 mutation was less than 1% [9]. Even in the same tumor, the heterogeneity including differences in histology, molecular subtype, pathogenic factors, tumor stage, and degrees of differentiation can also affect the evaluation of the frequency of TP53 mutations [10]. Majority mutations of TP53 cause the loss of function (LOF) of wild-typeTP53 and abrogate their ability to bind on specific DNA motif and perform its tumor-suppressive function. Gain of function (GOF), dominant negative effect on the wild-typeTP53 allele, the loss of heterozygosity of TP53, and interactions with viral proteins also result in the activity loss of wild-typeTP53 [11, 12].Recent studies using whole-genome and exome sequencing in patients with EC revealed that the most mutated gene isTP53, which means that at least TP53 has a significant influence on EC pathogenicity [13, 14]. In general, TP53 mutations often cause changes on the amino acid sequence of the p53 protein, thus disrupting the function of p53 for tumor inhibition. Under normal conditions, it is difficult to detect the expression of wild-type p53 protein due to its short half-life. Therefore, we usually detect the expression level of p53 protein (mutant-type) using immunohistochemistry (IHC) in clinical diagnosis, but in ESCC, the positive rate of p53 protein expression varies greatly in different reports, ranging from 27 to 75% [15]. At present, the role of p53 as a prognostic marker of ESCC is controversial. To date, the mechanism and correlation between the expression of the p53 protein and mutation of the TP53 gene or prognostic effect in ESCC are limited and contradictory. The clinical value of TP53 mutation and p53 protein expression is worth further exploration.Herein, we conducted an analysis ofTP53 mutation and p53 protein expression in two-large ESCC cohorts, using a series of methods including whole-genome sequencing (WGS), whole exon sequencing (WES), regionally targeted sequencing (TRS) and IHC, to further clarify the association between TP53 mutation status/ the expression of p53 protein and clinicopathologic phenotype and prognosis. This study provided effective and reliable evidence showing TP53 alterations as a biomarker for clinical diagnosis and treatment in ESCC.


## 2. Materials and Methods

### 2.1. Patients and Samples

All of the patients and samples were selected from the tissue bank and database of about 500,000 esophageal and gastric cardia carcinomas (1973–2020), established by the State Key Laboratory of Esophageal Cancer Prevention & Treatment and the Henan Key Laboratory for Esophageal Cancer Research of The First Affiliated Hospital, Zhengzhou University [16–18]. The database was reviewed to select the present study cohort. All medical records were collected, including detailed clinical, pathological, and survival information. Carcinoma and adjacent noncancerous tissues of 335 patients, which included frozen tissue samples and paraffin tissue samples (for using WGS, WES, and TRS) were collected after surgery. Postoperative tumor paraffin-embedded tissues from 6,252 patients were used for producing tissue microarray (TMA). During the cases of screening, patients were excluded from the database according to the following standard: non-ESCC patients, lacking T, N, M stage, preoperative treatment, incomplete following-up, without tissue for IHC staining in TMA, or failed staining. The detailed flow chart for patient enrollment is shown in Figure 1.Figure 1
Flow chart of patient selection. Patients who hadTP53 mutations by sequencing (a) and p53 protein expression by IHC (b) were included on the basis of clinical and histopathological characteristics and survival status. ∗ the TP53 mutation analysis was included in 316 patients. # the p53 protein expression analysis was included in 276 patients.
(a)(b)For the first cohort, a total of 316 surgically resected ESCC tissue specimens (paired primary malignant and adjacent normal) were collected. The tissues were analyzed on a different platform: 316 paired primary malignant and adjacent normal tissues, including 19 cases with frozen tissues that had WGS data available; 92 samples (51 cases of frozen tissue and 41 cases of paraffin-embedded tissues) that had WES data available; and 205 samples (203 cases of frozen tissue and 2 cases of paraffin-embedded tissues) had TRS data available. Among the 316 patients, 276 were used to make TMA. The remaining 40 had no tumor specimen available (owing to a lack of specimen or insufficient tumor cells). The second cohort that contains 6,028 from the 6,252 patients with ESCC TMA was included in the final statistical analysis.All the patients enrolled for this study were staged using the Union for International Cancer Control (UICC) staging standards, 6th (2002), for esophageal cancer. All patients were followed up after diagnosis until the date of death or December 2019.


### 2.2. Genomic DNA Extraction

Before DNA extraction, tissue was stained with hematoxylin and eosin (H&E) in order to assess the accurate histopathology for each case. The samples confirmed by a pathologist and in which tumor cells accounted for ≥50%, were chosen for DNA extraction. DNA was isolated from frozen tissue and formalin-fixed, paraffin-embedded (FFPE) tissue. Frozen tissue specimens were collected during surgery, snap-frozen in liquid nitrogen, and stored at −80°C. DNA was extracted from frozen tissue using the phenol-chloroform protocol [19]. For FFPE tissue, DNA was extracted from 8 sections with a 10 μm thickness of each paraffin block using the QIAamp DNA FFPE tissue kit (Qiagen, Hilden, Germany).


### 2.3. Detection ofTP53 Mutation by Next Generation Sequencing

Extracted genomic DNA was examined on agarose gel and by Nanodrop 2000 and Qubit 2.0. Mutations ofTP53 were determined using NGS, including WGS, WES, and TRS. DNA library was constructed and captured using TruSeq Nano DNA HT Sample Prep Kit for WGS sequencing, and Agilent SureSelect Human All Exon V6 for WES sequencing, and the SureSelect XT2 Target Enrichment System for the Illumina Multiplexed Sequencing Platform for TRS sequencing following the manufacturer’s recommendations, and then sequenced by an Illumina HiSeqPE150 or Illumina HiSeq 2500 sequencing platform (collaborating with Novogene Bioinformatics Technology Co., Tianjin, China).


### 2.4. Immunohistochemical Staining for p53 Protein Expression

IHC staining of 4μm TMA sections was performed by a 2-step protocol using a p53 antibody (1 : 100 dilution) and DAB detection kit (both from Wuhan Servicebio Technology Co., Wuhan, China). In each experiment, both positive and negative controls were included. All images were captured by CaseViewer 2.2 for Windows (3DHISTECH, Budapest, Hungary, Figure 2).Figure 2
Immunohistochemical staining scoring criteria for p53 protein in ESCC (A, D, G, J, M, P, S, V, magnification ×80, B, E, H, K, N, Q, T, W, magnification ×200, C, F, I, L, O, R, U, X, magnification ×400). (a–l) Scoring criteria of pattern of immunostaining: (a–c) entirely negative staining; (d–f) scattered; (g–i) focal; (j–l) diffuse. (m–x) Scoring criteria of intensity of immunostaining: (m–o) negative staining; (p–q) weak staining; (s–u) moderate staining; (v–x) strong staining.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)(k)(l)(m)(n)(o)(p)(q)(r)(s)(t)(u)(v)(w)(x)


### 2.5. Establishment of Scoring Criterion for p53 Immunohistochemical Staining

The scoring of IHC staining was completed independently by two experienced pathologists. Staining location, intensity, and patterns were reviewed. Tumor cells having a dark brown precipitate in their nuclei were the criterion for a positive reaction. The intensity of staining was grouped into four grades: 0 = entirely negative; 1 = weak; 2 = moderate; 3 = strong. The immunostaining patterns were divided into four terms: 0 = entirely negative; 1 = scattered, meaning only some isolates were positive cells; 2 = focal, where clusters of positive cells were seen in some areas; 3 = diffuse, in which the sheets of positive cells were found throughout most of the areas (Figure2) [20]. The final results were multiplied by the scores of the immunostaining patterns and staining intensity. Patients were categorized as “high expression (>4)” or “low expression (0∼4)” by the use of IHC scoring criteria.


### 2.6. Statistical Analyses

Statistical analysis was processed by SPSS for Windows, version 25.0. TheT-test and chi-square test or Fisher exact test were used to compare the association of categorical and continuous variables between different groups, respectively. The Kaplan–Meier method analyzed survival tendency and used the log-rank test to compare the survival curves. Cox proportional-hazard models were used for the univariate and multivariate analyses to estimate the hazard ratio of each clinicopathological feature for overall survival (OS). All predictors with p value <0.1 in univariate Cox were selected in multivariate Cox analysis. p values were 2-tailed and considered statistically significant with less than 0.05.


## 2.1. Patients and Samples

All of the patients and samples were selected from the tissue bank and database of about 500,000 esophageal and gastric cardia carcinomas (1973–2020), established by the State Key Laboratory of Esophageal Cancer Prevention & Treatment and the Henan Key Laboratory for Esophageal Cancer Research of The First Affiliated Hospital, Zhengzhou University [16–18]. The database was reviewed to select the present study cohort. All medical records were collected, including detailed clinical, pathological, and survival information. Carcinoma and adjacent noncancerous tissues of 335 patients, which included frozen tissue samples and paraffin tissue samples (for using WGS, WES, and TRS) were collected after surgery. Postoperative tumor paraffin-embedded tissues from 6,252 patients were used for producing tissue microarray (TMA). During the cases of screening, patients were excluded from the database according to the following standard: non-ESCC patients, lacking T, N, M stage, preoperative treatment, incomplete following-up, without tissue for IHC staining in TMA, or failed staining. The detailed flow chart for patient enrollment is shown in Figure 1.Figure 1
Flow chart of patient selection. Patients who hadTP53 mutations by sequencing (a) and p53 protein expression by IHC (b) were included on the basis of clinical and histopathological characteristics and survival status. ∗ the TP53 mutation analysis was included in 316 patients. # the p53 protein expression analysis was included in 276 patients.
(a)(b)For the first cohort, a total of 316 surgically resected ESCC tissue specimens (paired primary malignant and adjacent normal) were collected. The tissues were analyzed on a different platform: 316 paired primary malignant and adjacent normal tissues, including 19 cases with frozen tissues that had WGS data available; 92 samples (51 cases of frozen tissue and 41 cases of paraffin-embedded tissues) that had WES data available; and 205 samples (203 cases of frozen tissue and 2 cases of paraffin-embedded tissues) had TRS data available. Among the 316 patients, 276 were used to make TMA. The remaining 40 had no tumor specimen available (owing to a lack of specimen or insufficient tumor cells). The second cohort that contains 6,028 from the 6,252 patients with ESCC TMA was included in the final statistical analysis.All the patients enrolled for this study were staged using the Union for International Cancer Control (UICC) staging standards, 6th (2002), for esophageal cancer. All patients were followed up after diagnosis until the date of death or December 2019.


## 2.2. Genomic DNA Extraction

Before DNA extraction, tissue was stained with hematoxylin and eosin (H&E) in order to assess the accurate histopathology for each case. The samples confirmed by a pathologist and in which tumor cells accounted for ≥50%, were chosen for DNA extraction. DNA was isolated from frozen tissue and formalin-fixed, paraffin-embedded (FFPE) tissue. Frozen tissue specimens were collected during surgery, snap-frozen in liquid nitrogen, and stored at −80°C. DNA was extracted from frozen tissue using the phenol-chloroform protocol [19]. For FFPE tissue, DNA was extracted from 8 sections with a 10 μm thickness of each paraffin block using the QIAamp DNA FFPE tissue kit (Qiagen, Hilden, Germany).


## 2.3. Detection ofTP53 Mutation by Next Generation Sequencing

Extracted genomic DNA was examined on agarose gel and by Nanodrop 2000 and Qubit 2.0. Mutations ofTP53 were determined using NGS, including WGS, WES, and TRS. DNA library was constructed and captured using TruSeq Nano DNA HT Sample Prep Kit for WGS sequencing, and Agilent SureSelect Human All Exon V6 for WES sequencing, and the SureSelect XT2 Target Enrichment System for the Illumina Multiplexed Sequencing Platform for TRS sequencing following the manufacturer’s recommendations, and then sequenced by an Illumina HiSeqPE150 or Illumina HiSeq 2500 sequencing platform (collaborating with Novogene Bioinformatics Technology Co., Tianjin, China).


## 2.4. Immunohistochemical Staining for p53 Protein Expression

IHC staining of 4μm TMA sections was performed by a 2-step protocol using a p53 antibody (1 : 100 dilution) and DAB detection kit (both from Wuhan Servicebio Technology Co., Wuhan, China). In each experiment, both positive and negative controls were included. All images were captured by CaseViewer 2.2 for Windows (3DHISTECH, Budapest, Hungary, Figure 2).Figure 2
Immunohistochemical staining scoring criteria for p53 protein in ESCC (A, D, G, J, M, P, S, V, magnification ×80, B, E, H, K, N, Q, T, W, magnification ×200, C, F, I, L, O, R, U, X, magnification ×400). (a–l) Scoring criteria of pattern of immunostaining: (a–c) entirely negative staining; (d–f) scattered; (g–i) focal; (j–l) diffuse. (m–x) Scoring criteria of intensity of immunostaining: (m–o) negative staining; (p–q) weak staining; (s–u) moderate staining; (v–x) strong staining.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)(k)(l)(m)(n)(o)(p)(q)(r)(s)(t)(u)(v)(w)(x)


## 2.5. Establishment of Scoring Criterion for p53 Immunohistochemical Staining

The scoring of IHC staining was completed independently by two experienced pathologists. Staining location, intensity, and patterns were reviewed. Tumor cells having a dark brown precipitate in their nuclei were the criterion for a positive reaction. The intensity of staining was grouped into four grades: 0 = entirely negative; 1 = weak; 2 = moderate; 3 = strong. The immunostaining patterns were divided into four terms: 0 = entirely negative; 1 = scattered, meaning only some isolates were positive cells; 2 = focal, where clusters of positive cells were seen in some areas; 3 = diffuse, in which the sheets of positive cells were found throughout most of the areas (Figure2) [20]. The final results were multiplied by the scores of the immunostaining patterns and staining intensity. Patients were categorized as “high expression (>4)” or “low expression (0∼4)” by the use of IHC scoring criteria.


## 2.6. Statistical Analyses

Statistical analysis was processed by SPSS for Windows, version 25.0. TheT-test and chi-square test or Fisher exact test were used to compare the association of categorical and continuous variables between different groups, respectively. The Kaplan–Meier method analyzed survival tendency and used the log-rank test to compare the survival curves. Cox proportional-hazard models were used for the univariate and multivariate analyses to estimate the hazard ratio of each clinicopathological feature for overall survival (OS). All predictors with p value <0.1 in univariate Cox were selected in multivariate Cox analysis. p values were 2-tailed and considered statistically significant with less than 0.05.


## 3. Results

### 3.1. The Clinicopathological Distributions of ESCC Patients Results

To discover the genetic alteration ofTP53 in ESCC, we collected tumor samples to set up two cohorts, including 316 patients for sequencing analysis (276 patients out of 316 patients performed IHC to detect the expression of p53 protein), and 6,028 patients for the expression of p53 protein in TMA, for investigation in this study (Figure 1). Detailed clinicopathological data are listed in Figure 3 and Table 1. The follow-up period of all those patients ranged from 0.08 years to 30.87 years, and the median survival time was 2.98 years.Figure 3
Baseline characteristics of patients with ESCC were included in both study cohorts. The proportions of the two study cohorts are shown according to sex (a), age at diagnosis (b), high/low incidence area (c), cigarette smoking (d), alcohol consumption (e), family history (f), location (g), differentiation (h), T stage (i), N stage (j), M stage (k), UICC stage (l), cancer embolus (m), and treatment (n), respectively.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)(k)(l)(m)(n)Table 1
Baseline characteristics of patients with ESCC.
CharacteristicsThe patients undergoingTP53 mutation analysis,The patients with p53 IHC staining in TMA,n = 316(%)n = 6028(%)SexFemale11737.0210735.0Male19963.0392165.0Age at diagnosis (mean ± SD)61.39 ± 8.1860.92 ± 8.28≤6013643.0291748.4>6018057.0311151.6High/low incidence area★Low6420.3211935.2High25279.7390964.8Cigarette smokingNegative17856.3316052.4Positive13643.1273145.3Missing20.61372.3Alcohol consumptionNegative18458.2410368.1Positive13041.1179129.7Missing20.61342.2Family historyPositive13141.5203733.8Negative17455.0387564.3Missing113.51161.9LocationCervical + upper4514.295415.8Middle18257.6409267.9Lower6821.587914.6Multiple154.7510.8No record61.9520.9DifferentiationWell differentiated257.95018.3Moderate differentiated19561.7330354.8Poor differentiated9128.8193532.1No record51.62894.8Pathological T stageTis + T1185.72053.4T28326.3146624.3T320364.2424270.4T4123.81151.9Pathological N stageN017455.1335455.6N114244.9267444.4Pathological M stageM030897.5581496.4M182.52143.6UICC stage (6th)0 + I113.81442.4II18859.5369161.2III10934.2197932.8IV82.52143.6Cancer embolusPositive216.62564.2Negative29593.4577295.8Type of treatmentSurgical29392.7500683.0Surgical + chemo113.54938.2Surgical + radio82.53956.6Surgical + chemo + radio41.31342.2★areas with esophageal cancer incidence ≥50/100,000 are high-incidence area for esophageal cancer; on the contrary, areas with esophageal cancer incidence <50/100,000 is low-incidence area for esophageal cancer.


### 3.2. Distribution ofTP53 Somatic Mutations and p53 Protein

We evaluatedTP53 somatic alterations in the first cohort, and the results showed that 241 of the 316 ESCC patients (76.3%) exhibited a total of 276 TP53 somatic mutations, which included 208 cases having only one mutation and 33 having multiple (two and three) mutations (Tables 2 and 3). TP53 mutations were mainly located in the exon 5–8 (79.4%), meaning that most mutations occurred in the DBD, and very few mutations occurred in the AD1, AD2, and TET domains. Mutations were mostly clustered in exon 5 (23.6%) and exon 8 (23.6%), followed by exon 6 (19.2%) (Figure 4(a)).Table 2
Comparison of clinicopathologic features between mutation and wild-typeTP53 in patients with ESCC.
CharacteristicsAll patients,Wild-typeTP53,MutationTP53,p valuen = 316n = 75(%)n = 241(%)Sex0.736Female1172924.88875.2Male1994623.115376.9Age at diagnosis (mean ± SD)0.543≤601363022.110677.9>601804525.013575.0High/low incidence area0.551Low641726.64773.4High2525823.019477.0Cigarette smoking0.277Negative1784625.813274.2Positive1362820.610879.4Alcohol consumption0.211Negative1844826.113673.9Positive1302620.010480.0Family history0.984Negative1744123.613376.4Positive1313123.710076.3Location0.904∗Cervical + upper451226.73373.3Middle1824223.114076.9Lower681522.15377.9Multiple15426.71173.3Differentiation0.260Well differentiated25312.02288.0Moderate differentiated1955025.614574.4Poor differentiated911920.97279.1Pathological T stage0.239∗Tis + T118738.91161.1T2831720.56679.5T3 + T42155123.716476.3Pathological N stage0.130N01744727.012773.0N11422819.711480.3Pathological M stage1.000∗M03087323.723576.3M18225.0675.0UICC stage (6th)0.053∗0 + I11545.5654.5II1884926.113973.9III + IV1172117.99682.1Cancer embolus0.590∗Negative2956923.422676.6Positive21628.61571.4Type of treatment0.211Surgical2937224.622175.4Surgical + chemo/radio23313.02087.0∗the differences among categoric variables were analyzed using the Fisher exact test.Table 3
Characteristics of the ESCC patients undergoingTP53 mutation detection.
NoPatient IDSexAgeUICC stageSequence sourceMutation statusNumber of mutationsp53 IHC (positive/negative)p53 IHC (low/high)Survival statusSurvival time (years)1CE12TM72IVBWESTP53 wild-type0PositiveLowDead1.702CE18TM50IIIWESTP53 mutation1NegativeLowDead4.733CE16TM69IIAWESTP53 wild-type0PositiveLowDead1.814CE4TM64IIBWESTP53 mutation1NegativeLowDead3.955CE7TM70IIIWESTP53 mutation1PositiveLowDead5.006CE15TM57IIIWESTP53 mutation1PositiveLowDead5.817CE30TM56IIAWESTP53 wild-type0NegativeLowAlive10.518CE20TM72IIAWESTP53 mutation1PositiveLowDead4.079CE26TM71IIIWESTP53 mutation2PositiveHighDead6.1610CE5TF75IIIWESTP53 mutation1PositiveLowDead5.1111CE25TM64IIAWESTP53 wild-type0PositiveHighDead2.0512CE24TM58IIAWESTP53 mutation1PositiveHighAlive11.0413CE21TM54IIAWESTP53 mutation2NegativeLowDead6.5014CE27TM64IIIWESTP53 mutation1PositiveHighDead2.4115EC888TM59IIITRSTP53 wild-type0PositiveHighAlive5.0516EC889TM62IIITRSTP53 mutation1PositiveHighDead0.7917EC891TM55IIBTRSTP53 wild-type0NegativeLowDead1.7518EC892TM60IIITRSTP53 mutation1PositiveHighDead3.1519EC893TF64IIATRSTP53 mutation1PositiveHighAlive4.7120EC1038TM74IVTRSTP53 mutation1PositiveLowDead1.4921EC1035TM65IIITRSTP53 wild-type0PositiveHighDead2.2722EC1032TF51IIATRSTP53 mutation1PositiveHighDead2.2723EC632TM59IIATRSTP53 mutation1PositiveHighAlive4.7824EC636TM64IIBTRSTP53 mutation1NegativeLowAlive4.7725EC090TM47IIIWESTP53 mutation1PositiveHighAlive1.4126EC633TF63ITRSTP53 mutation1PositiveLowAlive4.8027EC640TM58IIATRSTP53 mutation1NegativeLowAlive4.8128EC643TF67IIATRSTP53 mutation1PositiveLowDead2.5429EC644TM61IIITRSTP53 mutation1NegativeLowDead2.3330EC641TF60IIATRSTP53 mutation1NegativeLowAlive4.8431EC967TM69IIATRSTP53 mutation2NegativeLowAlive4.8432EC968TF63IIATRSTP53 mutation1PositiveLowAlive4.8533EC966TM69IIATRSTP53 mutation2PositiveHighDead2.3734EC965TF71IIATRSTP53 mutation1PositiveHighDead0.9535EC1024TF65IVATRSTP53 mutation1PositiveLowDead4.0336EC190TM50IIAWESTP53 mutation2PositiveLowAlive10.8037EC969TM63IIITRSTP53 mutation1PositiveHighAlive4.8838EC970TM66IIITRSTP53 wild-type0NegativeLowDead0.1339EC971TM66IIITRSTP53 mutation1NegativeLowAlive5.2140EC973TF63IIBTRSTP53 mutation1NegativeLowDead0.8841EC975TM62IIITRSTP53 mutation1PositiveLowAlive4.8842EC976TF79IIATRSTP53 mutation1PositiveLowDead1.8943EC978TM57IIATRSTP53 mutation1NegativeLowAlive5.2644EC981TM69IIITRSTP53 mutation1NegativeLowAlive4.9045EC982TM64IIBTRSTP53 mutation1PositiveLowAlive4.9046EC983TM44IIATRSTP53 mutation1NegativeLowAlive3.9547EC191TM59IIIWESTP53 mutation1PositiveLowDead4.1848EC984TM63IIITRSTP53 mutation1PositiveLowDead1.3649EC985TM51IIITRSTP53 mutation1NegativeLowAlive5.3050EC986TM72IIATRSTP53 mutation1NegativeLowDead3.2451EC987TM67IIITRSTP53 mutation1PositiveLowAlive4.9452EC990TM73IIITRSTP53 mutation1NegativeLowDead1.6453EC991TM67IVATRSTP53 mutation1PositiveLowDead2.4254EC993TM56IIITRSTP53 wild-type0NegativeLowAlive3.1255EC992TM59IIATRSTP53 mutation1PositiveLowAlive4.9656EC994TM47IIITRSTP53 mutation1PositiveLowAlive2.8157EC995TM68IIATRSTP53 wild-type0NegativeLowAlive4.9858EC088TM58IIIWESTP53 mutation1NegativeLowDead6.0759EC996TF74IIATRSTP53 mutation1PositiveHighAlive4.9860EC630TF57IIATRSTP53 mutation1PositiveHighDead2.7961EC997TF61IIATRSTP53 mutation1PositiveLowAlive4.9862EC629TM67IIBTRSTP53 mutation1PositiveLowDead2.4363EC1006TM58IIITRSTP53 mutation2PositiveHighAlive5.0464EC885TM65IIBTRSTP53 mutation2PositiveLowDead4.0165EC1005TF73IIITRSTP53 mutation1PositiveLowDead2.9266EC886TM62IIBTRSTP53 wild-type0PositiveHighDead1.5667G008_TM71IIAWESTP53 mutation1PositiveHighDead2.5368NR131225_2TF70IIIWGSTP53 mutation1NegativeLowAlive5.2969EC080TM70IWESTP53 mutation1PositiveLowDead2.7070NR131225_3TM55IIAWGSTP53 mutation1PositiveHighAlive5.3271NR131225_5TF67IIAWGSTP53 mutation1NegativeLowDead0.9372NR131225_9TM67IIAWGSTP53 mutation1NegativeLowAlive5.6773NR131225_4TM74IIAWGSTP53 mutation1PositiveLowDead3.6174NR131225_10TM77IIIWGSTP53 wild-type0NegativeLowDead2.5075NR131225_1TF57IIIWGSTP53 mutation1PositiveHighDead3.7076NR131225_8TM58IIIWGSTP53 wild-type0PositiveLowAlive5.3677NR131225_6TM60IIAWGSTP53 mutation1PositiveHighDead2.8378NR131121_10TM53IIAWGSTP53 mutation1PositiveHighDead0.6679NR131121_9TM66IIBWGSTP53 mutation1PositiveHighDead1.0080EC076TM60IIIWESTP53 wild-type0PositiveLowAlive11.1981NR131121_8TM60IIAWGSTP53 mutation1PositiveLowDead4.0082NR131121_7TM59IIBWGSTP53 mutation1PositiveLowDead5.2783NR131121_6TM69IIAWGSTP53 mutation1PositiveHighDead1.0184NR131121_5TF64IIIWGSTP53 mutation1PositiveLowAlive4.8085NR131121_4TM65IIIWGSTP53 mutation1PositiveHighDead0.5886NR131121_3TF72IIAWGSTP53 mutation1PositiveLowAlive5.5787NR131121_2TM61IIAWGSTP53 mutation2PositiveHighDead4.4988NR131121_1TM63IIAWGSTP53 mutation1PositiveHighDead2.0389TW2352AFM55IIAWESTP53 mutation1PositiveLowAlive7.8790TW2351BFM46IIAWESTP53 mutation1PositiveHighAlive8.2891EC062TF59IIIWESTP53 mutation1PositiveLowDead6.4992EC129TM54IIAWESTP53 mutation1NegativeLowAlive10.3093EC127TF72IIBWESTP53 mutation1NegativeLowDead8.4594MC16TM62IIAWESTP53 mutation1NegativeLowDead7.8095MC17TM50IIAWESTP53 mutation2PositiveLowDead6.8796MC13TM54IIIWESTP53 mutation1PositiveHighDead4.2597MC7TM53IIIWESTP53 mutation2PositiveHighDead0.7598TW258AFM40IIIWESTP53 mutation1PositiveHighDead0.6099FE0008TM64IIITRSTP53 mutation1PositiveHighDead0.83100EC123TM56IIAWESTP53 mutation1PositiveLowDead4.06101G556TM64IIITRSTP53 mutation1PositiveLowAlive2.65102G406TM69IIITRSTP53 mutation1PositiveHighDead0.46103FG0067TM68IIITRSTP53 mutation2PositiveHighAlive1.10104EC745TM64IIATRSTP53 wild-type0NegativeLowAlive2.95105G414TM57IIITRSTP53 mutation1NegativeLowAlive3.11106EC727TM70IIATRSTP53 mutation1PositiveHighAlive3.14107EC720TF74IIATRSTP53 mutation1NegativeLowDead1.16108EC718TM64IIITRSTP53 wild-type0NegativeLowDead1.66109EC721TM56IIITRSTP53 mutation1PositiveHighDead0.48110EC943TM65IIATRSTP53 mutation1PositiveHighAlive3.79111EC121TM59IIIWESTP53 mutation1PositiveHighDead1.00112EC854TF64IIATRSTP53 mutation2PositiveHighAlive3.44113EC868TF52IIITRSTP53 mutation1PositiveLowAlive3.44114EC686TF59IIATRSTP53 mutation2PositiveLowAlive3.43115EC687TF58IIATRSTP53 wild-type0PositiveHighDead0.92116EC685TF78IIATRSTP53 wild-type0PositiveLowAlive3.47117EC884TM63IIATRSTP53 wild-type0NegativeLowAlive3.45118EC883TM62IIITRSTP53 mutation2PositiveHighAlive3.49119EC881TF62IIITRSTP53 mutation1PositiveHighDead1.24120EC877TM68IIITRSTP53 mutation1PositiveHighAlive3.51121EC880TM68IIATRSTP53 mutation1PositiveHighAlive3.47122EC122TM52IIIWESTP53 mutation1NegativeLowAlive11.47123EC876TF47IIATRSTP53 wild-type0NegativeLowAlive3.51124EC871TM78IIATRSTP53 mutation1PositiveHighAlive3.51125EC874TF64IIATRSTP53 mutation1NegativeLowAlive3.53126EC855TF67ITRSTP53 wild-type0PositiveHighDead1.00127EC870TF65IIITRSTP53 wild-type0PositiveHighAlive3.91128EC872TM64IIITRSTP53 mutation1PositiveHighDead0.89129EC875TM68IIATRSTP53 mutation1PositiveLowDead1.99130EC869TM64ITRSTP53 mutation1PositiveHighDead1.53131EC811TF65IIATRSTP53 wild-type0PositiveHighDead2.22132G479TM77IVTRSTP53 mutation1PositiveLowDead0.57133EC124TM62IIIWESTP53 mutation1PositiveHighDead2.07134EC814TF61IIATRSTP53 mutation1NegativeLowAlive3.58135EC813TF60IIATRSTP53 mutation1PositiveHighAlive3.58136EC816TF66IIATRSTP53 wild-type0NegativeLowDead3.21137EC812TF72IIATRSTP53 wild-type0PositiveHighDead0.64138EC822TM63IIATRSTP53 mutation1PositiveLowAlive3.95139EC818TF53IIATRSTP53 mutation2PositiveLowAlive3.58140EC821TF64IIATRSTP53 mutation1PositiveHighAlive3.59141EC853TF65IIATRSTP53 wild-type0PositiveHighAlive2.64142EC832TF72IIATRSTP53 mutation1NegativeLowDead0.25143EC851TF67IIATRSTP53 mutation1NegativeLowAlive3.64144EC120TM65IIBWESTP53 mutation1PositiveHighDead3.49145EC835TM63IIATRSTP53 mutation1NegativeLowDead1.26146EC834TF59IIATRSTP53 mutation1NegativeLowAlive3.66147EC859TM61IIITRSTP53 mutation1NegativeLowDead1.02148EC850TM72IIATRSTP53 wild-type0PositiveLowAlive3.69149EC838TM58IIBTRSTP53 mutation1PositiveHighAlive2.67150EC849TM64IIATRSTP53 mutation1PositiveHighDead1.26151EC848TM53IIITRSTP53 mutation1PositiveHighAlive3.73152EC847TF65IIATRSTP53 mutation1NegativeLowAlive3.72153EC845TM57ITRSTP53 mutation1NegativeLowAlive3.75154EC932TF60IIATRSTP53 mutation1NegativeLowDead2.10155EC116TF58IIAWESTP53 mutation1PositiveHighAlive11.53156EC934TM70IIATRSTP53 wild-type0NegativeLowDead1.44157EC935TF64ITRSTP53 wild-type0NegativeLowAlive3.76158EC933TF66IIATRSTP53 mutation1NegativeLowDead3.20159EC941TM62IIITRSTP53 mutation1NegativeLowDead2.57160EC942TF73IIATRSTP53 mutation1NegativeLowAlive3.78161EC945TM66IIATRSTP53 mutation1PositiveHighAlive3.80162EC944TF65IIBTRSTP53 mutation1PositiveHighDead1.31163EC936TF58IIATRSTP53 wild-type0PositiveHighAlive3.79164EC937TF60IIBTRSTP53 wild-type0PositiveHighAlive3.79165EC948TF60IIBTRSTP53 mutation1NegativeLowAlive3.81166EC117TM50IIIWESTP53 mutation3PositiveHighDead1.03167EC146TF51IIIWESTP53 mutation1PositiveLowDead3.25168EC940TF69IIATRSTP53 mutation1NegativeLowDead3.09169EC938TF63IIATRSTP53 wild-type0PositiveLowDead1.08170EC939TF52IIATRSTP53 wild-type0PositiveHighDead0.92171EC951TF63IIATRSTP53 mutation2PositiveHighAlive3.85172EC952TF61IIATRSTP53 mutation1NegativeLowAlive3.85173EC963TF56IIATRSTP53 mutation2NegativeLowAlive3.86174EC958TF71IIITRSTP53 mutation1NegativeLowAlive3.86175EC959TF57IIITRSTP53 wild-type0NegativeLowDead1.32176EC961TF48IIATRSTP53 mutation2PositiveHighAlive3.88177EC962TM52IIATRSTP53 mutation1PositiveLowAlive4.24178EC118TF65IIBWESTP53 mutation1NegativeLowDead3.05179EC956TM55IIATRSTP53 wild-type0PositiveLowAlive3.91180EC960TM51IIITRSTP53 mutation2PositiveHighDead1.98181EC954TF69IIATRSTP53 wild-type0PositiveLowAlive3.91182EC955TF52IIBTRSTP53 mutation1NegativeLowDead1.20183EC953TM61IIATRSTP53 wild-type0PositiveHighDead1.27184EC802TF63IIITRSTP53 mutation1NegativeLowDead1.48185EC793TM69IIATRSTP53 mutation1NegativeLowAlive3.95186EC789TM63IIATRSTP53 mutation1NegativeLowAlive3.95187EC797TM58IIITRSTP53 mutation1NegativeLowAlive4.33188EC798TF59IIATRSTP53 mutation1NegativeLowAlive3.96189EC115TF69IIAWESTP53 mutation2PositiveHighDead1.48190EC788TF58IIATRSTP53 wild-type0PositiveLowDead2.98191EC792TM70IIATRSTP53 mutation1PositiveLowDead3.21192EC791TM61IIITRSTP53 mutation3PositiveHighDead1.25193EC790TM79IIITRSTP53 mutation1NegativeLowDead1.78194EC794TM65IIITRSTP53 mutation2PositiveHighDead1.74195EC783TM75IIITRSTP53 mutation1PositiveHighAlive3.97196EC782TM67IIATRSTP53 wild-type0PositiveLowDead2.42197EC795TF69IIATRSTP53 mutation1PositiveHighDead1.51198EC787TM59IIITRSTP53 mutation1PositiveHighDead0.84199G521TF65IIATRSTP53 mutation1NegativeLowAlive3.63200EC113TM64IIIWESTP53 mutation2PositiveLowAlive11.54201EC780TM49IIATRSTP53 mutation1NegativeLowDead1.96202EC786TM66IIATRSTP53 mutation1PositiveHighAlive4.05203EC781TM72IIATRSTP53 mutation1PositiveHighDead1.33204EC785TM47IIATRSTP53 wild-type0NegativeLowAlive4.06205EC779TF52IIITRSTP53 mutation1PositiveHighAlive4.42206EC766TF62IIITRSTP53 mutation1PositiveLowDead1.61207EC769TF68IIATRSTP53 mutation1PositiveHighAlive4.46208EC807TF82IIATRSTP53 mutation1PositiveHighAlive4.11209EC900TF64IIATRSTP53 mutation1NegativeLowAlive4.11210EC898TM59IIITRSTP53 mutation1NegativeLowAlive4.13211EC015TM63IIIWESTP53 wild-type0PositiveLowDead2.25212EC904TF48IIATRSTP53 mutation1PositiveHighAlive4.12213EC899TM60IIBTRSTP53 wild-type0PositiveHighAlive4.12214EC908TF56IIITRSTP53 mutation1PositiveHighDead1.88215EC907TM60IIATRSTP53 mutation1PositiveHighDead15.80216EC901TF76IIITRSTP53 wild-type0PositiveLowDead3.13217EC902TM48IIITRSTP53 mutation1PositiveLowAlive4.15218EC906TM49IIATRSTP53 mutation1Not detectedNot detectedAlive4.51219EC972TM63IIATRSTP53 wild-type0PositiveHighDead0.98220EC910TM65IIATRSTP53 wild-type0NegativeLowAlive4.14221G048_TF64IIIWESTP53 wild-type0PositiveHighDead0.70222TW094BFM40IIIWESTP53 mutation2PositiveLowDead2.74223G042_TM65IIIWESTP53 mutation1NegativeLowAlive4.16224EC912TF63IIATRSTP53 wild-type0PositiveHighDead2.14225EC911TF52IIATRSTP53 wild-type0NegativeLowDead0.46226EC909TF56IIBTRSTP53 mutation1NegativeLowAlive3.12227EC916TF62IIATRSTP53 mutation1PositiveLowAlive4.18228EC915TM67IIATRSTP53 wild-type0NegativeLowAlive4.19229EC913TM70IIATRSTP53 mutation1NegativeLowDead3.05230EC917TF62IIATRSTP53 wild-type0PositiveHighDead3.65231EC920TF56IIITRSTP53 mutation1PositiveHighDead2.15232EC918TM68IIITRSTP53 mutation1PositiveHighDead2.21233TW890BFM65IIIWESTP53 mutation1PositiveLowDead2.35234G039_TM64IIAWESTP53 wild-type0NegativeLowAlive4.24235EC922TM74IIBTRSTP53 wild-type0PositiveLowDead3.49236EC921TF60IIBTRSTP53 mutation1NegativeLowDead2.00237EC923TM68IIITRSTP53 wild-type0NegativeLowDead2.90238EC924TM57IIATRSTP53 mutation1PositiveHighDead3.30239EC683TM46IIATRSTP53 wild-type0NegativeLowAlive4.42240EC684TM56IIITRSTP53 mutation1NegativeLowDead3.84241EC682TM63IIATRSTP53 mutation1NegativeLowAlive4.42242EC679TM59IIITRSTP53 wild-type0NegativeLowDead2.31243EC680TF62IIATRSTP53 mutation1NegativeLowAlive4.42244EC055TM45IIIWESTP53 mutation1PositiveHighDead0.88245EC681TM70IIATRSTP53 mutation1NegativeLowAlive4.44246EC678TF48IIBTRSTP53 wild-type0PositiveLowDead1.37247EC676TM57IIITRSTP53 mutation1NegativeLowDead0.98248EC677TM46IIITRSTP53 mutation1NegativeLowAlive4.44249EC672TM57IIITRSTP53 mutation1NegativeLowDead1.83250EC666TF67IVATRSTP53 mutation1NegativeLowDead1.54251EC660TF66IIATRSTP53 mutation1PositiveHighAlive4.50252EC658TM59ITRSTP53 wild-type0PositiveHighAlive4.52253EC659TF63IIITRSTP53 mutation1PositiveHighAlive4.87254EC1002TF62IIITRSTP53 mutation1PositiveHighAlive4.65255EC039TM74IIAWESTP53 wild-type0NegativeLowDead0.35256EC656TF72IIATRSTP53 wild-type0NegativeLowAlive4.51257EC654TF61IIITRSTP53 wild-type0PositiveHighDead0.96258EC651TM51IIBTRSTP53 mutation1PositiveHighAlive4.53259EC645TF57IIATRSTP53 mutation1NegativeLowAlive4.55260EC928TF61IIBTRSTP53 mutation2PositiveLowAlive4.58261EC926TM73IIATRSTP53 mutation1NegativeLowAlive4.96262EC929TF64ITRSTP53 mutation2PositiveHighDead1.54263EC927TM60IIBTRSTP53 mutation1NegativeLowDead1.99264EC1003TM64IIITRSTP53 wild-type0PositiveHighDead1.78265EC038TM68IIAWESTP53 mutation1NegativeLowDead1.47266EC930TF58IIATRSTP53 mutation1PositiveHighAlive4.91267EC931TF57IIATRSTP53 wild-type0NegativeLowAlive4.63268EC1000TM67IIATRSTP53 mutation2PositiveLowAlive4.64269EC999TF75IIBTRSTP53 mutation1PositiveLowAlive4.64270EC1001TF68IIITRSTP53 mutation2PositiveHighDead1.92271G017_TF67IIIWESTP53 mutation1NegativeLowAlive4.70272EC894TM66IIATRSTP53 mutation1PositiveHighAlive4.68273EC887TM77IIATRSTP53 wild-type0NegativeLowAlive4.68274EC895TM73IIITRSTP53 mutation1PositiveHighDead2.11275EC890TF64IIATRSTP53 mutation1NegativeLowAlive4.69276EC036TM49IWESTP53 wild-type0PositiveHighAlive7.88277EC027TM47IIAWESTP53 mutation1PositiveLowAlive11.89278TW258BFM40IIAWESTP53 mutation1Not detectedNot detectedDead1.39279EC114TM64IIIWESTP53 mutation1Not detectedNot detectedAlive10.78280EC099TM58IIAWESTP53 mutation1Not detectedNot detectedDead1.76281TW1375BFM55IWESTP53 mutation1Not detectedNot detectedAlive9.72282EC067TM50IVBWESTP53 wild-type0Not detectedNot detectedDead1.98283EC107TM56IIIWESTP53 mutation1Not detectedNot detectedDead3.06284MC23TF59IIIWESTP53 wild-type0Not detectedNot detectedDead0.06285EC188TM64IIAWESTP53 mutation1Not detectedNot detectedDead1.93286MC27TM56IIBWESTP53 wild-type0Not detectedNot detectedDead1.99287EC101TM73IIAWESTP53 wild-type0Not detectedNot detectedDead2.51288EC081TF68IIBWESTP53 mutation1Not detectedNot detectedDead2.62289MC30TF41IIBWESTP53 wild-type0Not detectedNot detectedAlive8.52290EC145TM51IIIWESTP53 mutation1Not detectedNot detectedAlive2.88291EC131TM49IVAWESTP53 mutation2Not detectedNot detectedAlive12.35292TW470BFF49IIIWESTP53 mutation1Not detectedNot detectedAlive15.36293EC086TM36IIAWESTP53 mutation1Not detectedNot detectedAlive11.00294EC013TF69IIAWESTP53 wild-type0Not detectedNot detectedDead2.92295EC126TF63IIIWESTP53 mutation1Not detectedNot detectedAlive11.37296EC040TM59IIIWESTP53 mutation2Not detectedNot detectedAlive8.24297TW1371BFM44IIAWESTP53 mutation1Not detectedNot detectedDead2.22298TW1371AFM43IIBWESTP53 mutation1Not detectedNot detectedDead0.92299MC34TM51IIAWESTP53 mutation1Not detectedNot detectedDead7.54300EC925TM59IIBTRSTP53 wild-type0Not detectedNot detectedDead1.77301CE2TM54IIIWESTP53 mutation1Not detectedNot detectedAlive13.37302MC29TM60IIBWESTP53 mutation1Not detectedNot detectedDead4.26303EC034TM62IIBWESTP53 mutation1Not detectedNot detectedDead6.17304MC4TM69IIAWESTP53 wild-type0Not detectedNot detectedDead2.81305TW2309BFF45IIAWESTP53 mutation1Not detectedNot detectedAlive11.74306EC128TF51IIIWESTP53 mutation1Not detectedNot detectedDead4.00307EC192TM57IIIWESTP53 mutation1Not detectedNot detectedDead2.78308EC061TM76IIAWESTP53 wild-type0Not detectedNot detectedDead5.83309TW002BFM59IWESTP53 wild-type0Not detectedNot detectedAlive10.24310MC26TM44IIIWESTP53 mutation1Not detectedNot detectedDead1.66311EC060TM50IIAWESTP53 mutation1Not detectedNot detectedAlive6.87312TW829AFF60IIAWESTP53 mutation1Not detectedNot detectedAlive14.09313TW1099AFM48IIAWESTP53 mutation2Not detectedNot detectedAlive11.02314EC094TF58IIAWESTP53 mutation2Not detectedNot detectedDead5.70315EC165TM55IIIWESTP53 wild-type0Not detectedNot detectedDead9.95316EC012TF51IIAWESTP53 mutation1Not detectedNot detectedDead1.36Figure 4
Type of somaticTP53 mutation in ESCC patients. (a) Distribution of TP53 mutations according to the affected exons. (b) Pie chart showing the proportion of the different types of somatic TP53 mutations. (c) Different types of mutations in the spectrum of TP53 mutations. (d) Graphical representation of the site of mutations in the coding sequence of TP53: red lollypop symbols in the lower panel representing missense mutations, blue, yellow, green, pink, and purple lollypop symbols in the upper panel representing others mutations (including nonsense, frameshift del, frameshift ins, silent, mixed type, respectively). Mutations occurring six or more are labeled: TA1, TA2, transactivation domain; DBD, DNA binding domain; TET, tetramerization domain. The figure was generated by the Illustrator for biological sequences version 1.0. (e) Summary of patients’ characteristics and alterations for TP53 and p53 protein in ESCC. Each column represents one patient sample. The phenotypic information in 316 ESCC patients is shown in the upper panel. Alterations for the TP53 gene and p53 protein are shown in the bottom panel.
(a)(b)(c)(d)(e)Next, we investigated the overall pattern of the 276 somatic mutations identified in theTP53, in which 145 were missense (52.5%), 54 were nonsense (19.6%), 33 were splices (12.0%), 24 were frameshift deletion (8.7%), eleven were frame-shit insertion (4.0%), five were silent (1.8%), two were nonframe deletion (0.7%), one was nonframe insertion (0.4%), and one was splice site insertion and deletion (0.4%) (Figure 4(b) and Table 4). For the protein domain distribution of missense mutations, the majority of which occurred in the DBD domain. However, the main type of mutation varied with different exons. In exons 5, 7, and 8, missense mutations accounted for more than 70% (73.8%, 75.0%, and 70.7%, respectively).We observed that the proportions of theTP53 mutation spectrum were 49/237 (20.7%) for C > A/G > T, 15/237 (6.3%) for C > G/G > C, 117/237 (49.4%) for C > T/G > A, 17/237 (7.2%) for T > A/A > T, 27/237 (11.4%) for T > C/A > G, 12/237 (5.1%) for T > G/A > C. The transition was predominant (144/237, 60.8%), followed by transversion (93/237, 39.2%) (Figure 4(c) and Table 4).Table 4
Descriptive features and functional effects ofTP53 mutated in ESCC patients.
NoPatient IDNumber of mutationsMutationsMutation typeexonMutation annotation (cDNA)TP53 mutation (protein)TA classAlign GVGDp53 IHC (positive/negative)p53 IHC (low/high)Survival statusSurvival time (years)2CE18T17577509C > ANonsenseexon7c.G772Tp.E258X——NegativeLowDead4.734CE4T17578212G > ANonsenseexon6c.C637Tp.R213X——NegativeLowDead3.955CE7T17579528C > TNonsenseexon4c.G159Ap.W53X——PositiveLowDead5.006CE15T17578461C > AMissenseexon5c.G469Tp.V157FNot-functionalC0PositiveLowDead5.818CE20T17578413C > TMissenseexon5c.G517Ap.V173MNot-functionalC15PositiveLowDead4.079CE26T27577556C > AMissenseexon7c.G725Tp.C242FNot-functionalC65PositiveHighDead6.167578406C > TMissenseexon5c.G524Ap.R175HNot-functionalC2510CE5T17577105G > AMissenseexon8c.C833Tp.P278LNot-functionalC65PositiveLowDead5.1112CE24T17577539G > AMissenseexon7c.C742Tp.R248WNot-functionalC65PositiveHighAlive11.0413CE21T27577603G > CSilentexon7c.C678Gp.G226G——NegativeLowDead6.507577610T > ASplice_siteexon8c.673-2A > T———14CE27T17578406C > TMissenseexon5c.G524Ap.R175HNot-functionalC25PositiveHighDead2.4116EC889T17577156C > ASplice_siteexon9c.783-1G > T———PositiveHighDead0.7918EC892T17579529C > TNonsenseexon4c.G158Ap.W53X——PositiveHighDead3.1519EC893T17578406C > TMissenseexon5c.G524Ap.R175HNot-functionalC25PositiveHighAlive4.7120EC1038T17579414C > TNonsenseexon4c.G273Ap.W91X——PositiveLowDead1.4922EC1032T17578403C > AMissenseexon5c.G527Tp.C176FPartially functionalC65PositiveHighDead2.2723EC632T17577085C > TMissenseexon8c.G853Ap.E285KNot-functionalC55PositiveHighAlive4.7824EC636T17579691TGT​CCT​TAC​CA > TFrame_Shift_Delexon3c.95_96delp.L32fs——NegativeLowAlive4.7725EC090T17578268A > CMissenseexon6c.T581Gp.L194RNot-functionalC65PositiveHighAlive1.4126EC633T17577022G > ANonsenseexon8c.C916Tp.R306X——PositiveLowAlive4.8027EC640T17579539T > TGFrame_Shift_Insexon4c.147_148insCp.I50fs——NegativeLowAlive4.8128EC643T17578235T > CMissenseexon6c.A614Gp.Y205CNot-functionalC65PositiveLowDead2.5429EC644T17579310A > GSplice_siteexon5c.375+2T >C———NegativeLowDead2.3330EC641T17578188C > ANonsenseexon6c.G661Tp.E221X——NegativeLowAlive4.8431EC967T27576897G > ANonsenseexon9c.C949Tp.Q317X——NegativeLowAlive4.847579494T > ANonsenseexon4c.A193Tp.R65X——32EC968T17577511A > CMissenseexon7c.T770Gp.L257RNot-functionalC65PositiveLowAlive4.8533EC966T27574029C > TMissenseexon10c.G998Ap.R333HFunctionalC0PositiveHighDead2.377577580T > CMissenseexon7c.A701Gp.Y234CNot-functionalC4534EC965T17578478G > CMissenseexon5c.C452Gp.P151RNot-functionalC65PositiveHighDead0.9535EC1024T17578395G > AMissenseexon5c.C535Tp.H179YPartially functionalC65PositiveLowDead4.0336EC190T27578520A > TMissenseexon5c.T410Ap.L137QFunctionalC65PositiveLowAlive10.807579313G > AMissenseexon4c.C374Tp.T125MNot-functionalC6537EC969T17577539G > AMissenseexon7c.C742Tp.R248WNot-functionalC65PositiveHighAlive4.8839EC971T17579311C > TSplice_Siteexon5c.375+1G >A———NegativeLowAlive5.2140EC973T17579415C > TNonsenseexon4c.G272Ap.W91X——NegativeLowDead0.8841EC975T17577058C > ANonsenseexon8c.G880Tp.E294X——PositiveLowAlive4.8842EC976T17577575A > GMissenseexon7c.T706Cp.Y236HNot-functionalC35PositiveLowDead1.8943EC978T17579722C > TSplice_Siteexon4c.75-1G >A———NegativeLowAlive5.2644EC981T17578370C > TSplice_Siteexon6c.559+1G >A———NegativeLowAlive4.9045EC982T17577105G > CMissenseexon8c.C833Gp.P278RNot-functionalC65PositiveLowAlive4.9046EC983T17574003G > ANonsenseexon10c.C1024Tp.R342X——NegativeLowAlive3.9547EC191T17578412A > GMissenseexon5c.T518Cp.V173APartially functionalC55PositiveLowDead4.1848EC984T17578466G > TMissenseexon5c.C464Ap.T155NNot-functionalC0PositiveLowDead1.3649EC985T17577511A > GMissenseexon7c.T770Cp.L257PNot-functionalC65NegativeLowAlive5.3050EC986T17578275G > ANonsenseexon6c.C574Tp.Q192X——NegativeLowDead3.2451EC987T17577018C > TSplice_Siteexon9c.919+1G >A———PositiveLowAlive4.9452EC990T17577539G > AMissenseexon7c.C742Tp.R248WNot-functionalC65NegativeLowDead1.6453EC991T17577503AGT​CTT​CCA​GT > AFrame_Shift_Delexon7c.768_777delp.T256fs——PositiveLowDead2.4255EC992T17577094G > AMissenseexon8c.C844Tp.R282WNot-functionalC65PositiveLowAlive4.9656EC994T17578394T > CMissenseexon5c.A536Gp.H179RNot-functionalC25PositiveLowAlive2.8158EC088T17578469CCGGG > CFrame_Shift_Delexon5c.457_460delp.P153fs——NegativeLowDead6.0759EC996T17578403C > AMissenseexon5c.G527Tp.C176FPartially functionalC65PositiveHighAlive4.9860EC630T17577153C > AMissenseexon8c.G785Tp.G262VNot-functionalC65PositiveHighDead2.7961EC997T17578406C > TMissenseexon5c.G524Ap.R175HNot-functionalC25PositiveLowAlive4.9862EC629T17577156C > TSplice_Siteexon9c.783-1G >A———PositiveLowDead2.4363EC1006T27577106G > TMissenseexon8c.C832Ap.P278TNot-functionalC35PositiveHighAlive5.047578475G > AMissenseexon5c.C455Tp.P152LNot-functionalC6564EC885T27577548C > TMissenseexon7c.G733Ap.G245SNot-functionalC55PositiveLowDead4.017576888T > ANonsenseexon9c.A958Tp.K320X——65EC1005T17578536T > CMissenseexon5c.A394Gp.K132ENot-functionalC55PositiveLowDead2.9267G008_T17578406C > TMissenseexon5c.G524Ap.R175HNot-functionalC25PositiveHighDead2.5368NR131225_2T17578290C > TSplice_Siteexon7c.560-1G >A———NegativeLowAlive5.2969EC080T17577143CAGT > Cnon_Frame_Delexon8c.792_794delp.264_265del——PositiveLowDead2.7070NR131225_3T17577082C > TMissenseexon8c.G856Ap.E286KNot-functionalC55PositiveHighAlive5.3271NR131225_5T17576870C > ANonsenseexon9c.G976Tp.E326X——NegativeLowDead0.9372NR131225_9T17578239C > ANonsenseexon6c.G610Tp.E204X——NegativeLowAlive5.6773NR131225_4T17577120C > TMissenseexon8c.G818Ap.R273HNot-functionalC25PositiveLowDead3.6175NR131225_1T17578403C > AMissenseexon5c.G527Tp.C176FPartially functionalC65PositiveHighDead3.7077NR131225_6T17577120C > AMissenseexon8c.G818Tp.R273LNot-functionalC65PositiveHighDead2.8378NR131121_10T17577565T > CMissenseexon7c.A716Gp.N239SNot-functionalC45PositiveHighDead0.6679NR131121_9T17577538C > TMissenseexon7c.G743Ap.R248QNot-functionalC35PositiveHighDead1.0081NR131121_8T17577120C > TMissenseexon8c.G818Ap.R273HNot-functionalC25PositiveLowDead4.0082NR131121_7T17579355A > TMissenseexon4c.T332Ap.L111QNot-functionalC65PositiveLowDead5.2783NR131121_6T17577142C > GMissenseexon8c.G796Cp.G266RNot-functionalC65PositiveHighDead1.0184NR131121_5T17577575A > CMissenseexon7c.T706Gp.Y236DNot-functionalC55PositiveLowAlive4.8085NR131121_4T17577117A > CMissenseexon8c.T821Gp.V274GNot-functionalC65PositiveHighDead0.5886NR131121_3T17577046C > ANonsenseexon8c.G892Tp.E298X——PositiveLowAlive5.5787NR131121_2T27578446T > AMissenseexon5c.A484Tp.I162FNot-functionalC0PositiveHighDead4.497578484G > TMissenseexon5c.C446Ap.S149YFunctionalC088NR131121_1T17577586A > TMissenseexon7c.T695Ap.I232NNot-functionalC35PositiveHighDead2.0389TW2352AF17578208T > CMissenseexon6c.A641Gp.H214RNot-functionalC0PositiveLowAlive7.8790TW2351BF17577085C > TMissenseexon8c.G853Ap.E285KNot-functionalC55PositiveHighAlive8.2891EC062T17578392C > ANonsenseexon5c.G538Tp.E180X——PositiveLowDead6.4992EC129T17578212G > ANonsenseexon6c.C637Tp.R213X——NegativeLowAlive10.3093EC127T17578239C > ANonsenseexon6c.G610Tp.E204X——NegativeLowDead8.4594MC16T17577609C > TSplice_Siteexon8c.673-1G >A———NegativeLowDead7.8095MC17T27578469C > AMissenseexon5c.G461Tp.G154VNot-functionalC65PositiveLowDead6.877579312C > TSilentexon4c.G375Ap.T125T——96MC13T17577090C > GMissenseexon8c.G848Cp.R283PNot-functionalC35PositiveHighDead4.2597MC7T27577106G > AMissenseexon8c.C832Tp.P278SNot-functionalC65PositiveHighDead0.757577538C > GMissenseexon7c.G743Cp.R248PNot-functionalC6598TW258AF17578406C > TMissenseexon5c.G524Ap.R175HNot-functionalC25PositiveHighDead0.6099FE0008T17577094G > AMissenseexon8c.C844Tp.R282WNot-functionalC65PositiveHighDead0.83100EC123T17573991C > ANonsenseexon10c.G1036Tp.E346X——PositiveLowDead4.06101G556T17579419AG > AFrame_Shift_Delexon4c.267delCp.P89fs——PositiveLowAlive2.65102G406T17577118C > AMissenseexon8c.G820Tp.V274FNot-functionalC45PositiveHighDead0.46103FG0067T27578449C > TMissenseexon5c.G481Ap.A161TPartially functionalC55PositiveHighAlive1.107579362A > CMissenseexon4c.T325Gp.F109VNot-functionalC0105G414T17578524G > ANonsenseexon5c.C406Tp.Q136X——NegativeLowAlive3.11106EC727T17577121G > AMissenseexon8c.C817Tp.R273CNot-functionalC65PositiveHighAlive3.14107EC720T17578275G > ANonsenseexon6c.C574Tp.Q192X——NegativeLowDead1.16109EC721T17578203C > TMissenseexon6c.G646Ap.V216MNot-functionalC15PositiveHighDead0.48110EC943T17578206T > CMissenseexon6c.A643Gp.S215GNot-functionalC0PositiveHighAlive3.79111EC121T17578271T > GMissenseexon6c.A578Cp.H193PNot-functionalC65PositiveHighDead1.00112EC854T27577124C > AMissenseexon8c.G814Tp.V272LNot-functionalC25PositiveHighAlive3.447578290C > GSplice_Siteexon7c.560-1G >C———113EC868T17577120C > TMissenseexon8c.G818Ap.R273HNot-functionalC25PositiveLowAlive3.44114EC686T27577124C > AMissenseexon8c.G814Tp.V272LNot-functionalC25PositiveLowAlive3.437578275G > ANonsenseexon6c.C574Tp.Q192X——118EC883T27577539G > AMissenseexon7c.C742Tp.R248WNot-functionalC65PositiveHighAlive3.497578192G > GGG​CAC​CAC​CAC​ACT​ATG​TCFrame_Shift_Insexon6c.656_657insGACATAGTGTGGTGGTGCCp.P219_Y220delinsPTX——119EC881T17577153C > AMissenseexon8c.G785Tp.G262VNot-functionalC65PositiveHighDead1.24120EC877T17578413C > AMissenseexon5c.G517Tp.V173LNot-functionalC25PositiveHighAlive3.51121EC880T17578443A > GMissenseexon5c.T487Cp.Y163HNot-functionalC65PositiveHighAlive3.47122EC122T17579485C > ANonsenseexon4c.G202Tp.E68X——NegativeLowAlive11.47124EC871T17577094G > AMissenseexon8c.C844Tp.R282WNot-functionalC65PositiveHighAlive3.51125EC874T17578211CGA > CFrame_Shift_Delexon6c.636_637delp.F212fs——NegativeLowAlive3.53128EC872T17577094G > AMissenseexon8c.C844Tp.R282WNot-functionalC65PositiveHighDead0.89129EC875T17574003G > ANonsenseexon10c.C1024Tp.R342X——PositiveLowDead1.99130EC869T17577106G > AMissenseexon8c.C832Tp.P278SNot-functionalC65PositiveHighDead1.53132G479T17578263G > ANonsenseexon6c.C586Tp.R196X——PositiveLowDead0.57133EC124T17577569A > TMissenseexon7c.T712Ap.C238SNot-functionalC65PositiveHighDead2.07134EC814T17579591C > ASplice_Siteexon5c.97-1G >T———NegativeLowAlive3.58135EC813T17578212G > ANonsenseexon6c.C637Tp.R213X——PositiveHighAlive3.58138EC822T17578190T > CMissenseexon6c.A659Gp.Y220CNot-functionalC65PositiveLowAlive3.95139EC818T27577094G > AMissenseexon8c.C844Tp.R282WNot-functionalC65PositiveLowAlive3.587578246CAA > CFrame_Shift_Delexon6c.601_602delp.L201fs——140EC821T17577538C > TMissenseexon7c.G743Ap.R248QNot-functionalC35PositiveHighAlive3.59142EC832T17577610T > CSplice_Siteexon8c.673-2A >G———NegativeLowDead0.25143EC851T17578212G > ANonsenseexon6c.C637Tp.R213X——NegativeLowAlive3.64144EC120T17577120C > TMissenseexon8c.G818Ap.R273HNot-functionalC25PositiveHighDead3.49145EC835T17578221TTC > TFrame_Shift_Delexon6c.626_627delp.R209fs——NegativeLowDead1.26146EC834T17579360G > GAFrame_Shift_Insexon4c.326dupTp.F109fs——NegativeLowAlive3.66147EC859T17577058C > ANonsenseexon8c.G880Tp.E294X——NegativeLowDead1.02149EC838T17577570C > TMissenseexon7c.G711Ap.M237INot-functionalC0PositiveHighAlive2.67150EC849T17578394T > CMissenseexon5c.A536Gp.H179RNot-functionalC25PositiveHighDead1.26151EC848T17578266TAAGATGCTG > Tnon_Frame_Delexon6c.574_582delp.192_194del——PositiveHighAlive3.73152EC847T17578263G > ANonsenseexon6c.C586Tp.R196X——NegativeLowAlive3.72153EC845T17578371C > TMissenseexon5c.G559Ap.G187SFunctionalC0NegativeLowAlive3.75154EC932T17579400G > GAAFrame_Shift_Insexon4c.286_287insTTp.S96fs——NegativeLowDead2.10155EC116T17577094G > AMissenseexon8c.C844Tp.R282WNot-functionalC65PositiveHighAlive11.53158EC933T17578440T > ANonsenseexon5c.A490Tp.K164X——NegativeLowDead3.20159EC941T17577609C > GSplice_Siteexon8c.673-1G >C———NegativeLowDead2.57160EC942T17577149AT > AFrame_Shift_Delexon8c.788delAp.N263fs——NegativeLowAlive3.78161EC945T17577082C > TMissenseexon8c.G856Ap.E286KNot-functionalC55PositiveHighAlive3.80162EC944T17577539G > AMissenseexon7c.C742Tp.R248WNot-functionalC65PositiveHighDead1.31165EC948T17576927C > TSplice_Siteexon10c.920-1G >A———NegativeLowAlive3.81166EC117T37577556C > AMissenseexon7c.G725Tp.C242FNot-functionalC65PositiveHighDead1.037577558G > ASilentexon7c.C723Tp.S241S——7578406C > TMissenseexon5c.G524Ap.R175HNot-functionalC25167EC146T17578395G > AMissenseexon5c.C535Tp.H179YPartially functionalC65PositiveLowDead3.25168EC940T17579591C > GSplice_Siteexon5c.97-1G >C———NegativeLowDead3.09171EC951T27577144A > CMissenseexon8c.T794Gp.L265RNot-functionalC65PositiveHighAlive3.857578446T > TGGCnon_Frame_Insexon5c.483_484insGCCp.I162delinsAI——172EC952T17579699C > TSplice_Siteexon4c.96+1G >A———NegativeLowAlive3.85173EC963T27576927C > TSplice_Siteexon10c.920-1G >A———NegativeLowAlive3.867578212G > ANonsenseexon6c.C637Tp.R213X——174EC958T17579470C > CGFrame_Shift_Insexon4c.216dupCp.V73fs——NegativeLowAlive3.86176EC961T27577121G > AMissenseexon8c.C817Tp.R273CNot-functionalC65PositiveHighAlive3.887578263G > ANonsenseexon6c.C586Tp.R196X——177EC962T17574029CG > CFrame_Shift_Delexon10c.997delCp.R333fs——PositiveLowAlive4.24178EC118T17578212G > ANonsenseexon6c.C637Tp.R213X——NegativeLowDead3.05180EC960T27578242C > AMissenseexon6c.G607Tp.V203LPartially functionalC0PositiveHighDead1.987576865A > CNonsenseexon9c.T981Gp.Y327X——182EC955T17578249AT > AFrame_Shift_Delexon6c.599delAp.N200fs——NegativeLowDead1.20184EC802T17578368CA > CSplice_Site InDelexon6c.559+2T > -———NegativeLowDead1.48185EC793T17577610T > GSplice_Siteexon8c.673-2A >C———NegativeLowAlive3.95186EC789T17576927C > ASplice_Siteexon10c.920-1G >T———NegativeLowAlive3.95187EC797T17578176C > TSplice_Siteexon7c.672+1G >A———NegativeLowAlive4.33188EC798T17576928T > CSplice_Siteexon10c.920-2A >G———NegativeLowAlive3.96189EC115T27577105G > AMissenseexon8c.C833Tp.P278LNot-functionalC65PositiveHighDead1.487578538T > AMissenseexon5c.A392Tp.N131INot-functionalC65191EC792T17574029CG > CFrame_Shift_Delexon10c.997delCp.R333fs——PositiveLowDead3.21192EC791T37578215A > CMissenseexon6c.T634Gp.F212VPartially functionalC0PositiveHighDead1.257577022G > ANonsenseexon8c.C916Tp.R306X——7579699C > ASplice_Siteexon4c.96+1G >T———193EC790T17578419C > ANonsenseexon5c.G511Tp.E171X——NegativeLowDead1.78194EC794T27578406C > TMissenseexon5c.G524Ap.R175HNot-functionalC25PositiveHighDead1.747578370CCA​TCG​CTA​TCT​GAG​CAG > CFrame_Shift_Delexon5c.543_559delp.R181fs——195EC783T17577121G > CMissenseexon8c.C817Gp.R273GNot-functionalC65PositiveHighAlive3.97197EC795T17578388C > GMissenseexon5c.G542Cp.R181PNot-functionalC65PositiveHighDead1.51198EC787T17577547C > TMissenseexon7c.G734Ap.G245DNot-functionalC65PositiveHighDead0.84199G521T17579311C > ASplice_Siteexon5c.375+1G >T———NegativeLowAlive3.63200EC113T27577518T > AMissenseexon7c.A763Tp.I255FNot-functionalC15PositiveLowAlive11.547578212G > ANonsenseexon6c.C637Tp.R213X——201EC780T17577093CG > CFrame_Shift_Delexon8c.844delCp.R282fs——NegativeLowDead1.96202EC786T17578271T > CMissenseexon6c.A578Gp.H193RNot-functionalC65PositiveHighAlive4.05203EC781T17578524G > CMissenseexon5c.C406Gp.Q136ENot-functionalC25PositiveHighDead1.33205EC779T17577120C > TMissenseexon8c.G818Ap.R273HNot-functionalC25PositiveHighAlive4.42206EC766T17577035TG > TFrame_Shift_Delexon8c.902delCp.P301fs——PositiveLowDead1.61207EC769T17577106G > TMissenseexon8c.C832Ap.P278TNot-functionalC35PositiveHighAlive4.46208EC807T17578454G > AMissenseexon5c.C476Tp.A159VNot-functionalC0PositiveHighAlive4.11209EC900T17579414C > TNonsenseexon4c.G273Ap.W91X——NegativeLowAlive4.11210EC898T17578212G > ANonsenseexon6c.C637Tp.R213X——NegativeLowAlive4.13212EC904T17577142C > TMissenseexon8c.G796Ap.G266RNot-functionalC65PositiveHighAlive4.12214EC908T17574018G > AMissenseexon10c.C1009Tp.R337CNot-functionalC45PositiveHighDead1.88215EC907T17577124C > AMissenseexon8c.G814Tp.V272LNot-functionalC25PositiveHighDead15.80217EC902T17577094G > CMissenseexon8c.C844Gp.R282GNot-functionalC65PositiveLowAlive4.15218EC906T17578437G > ANonsenseexon5c.C493Tp.Q165X——Not detectedNot detectedAlive4.51222TW094BF27576857A > CMissenseexon9c.T989Gp.L330RNot-functionalC65PositiveLowDead2.747578176C > ASplice_Siteexon7c.672 + 1G >T———223G042_T17578427T > CMissenseexon5c.A503Gp.H168RNot-functionalC25NegativeLowAlive4.16226EC909T17578177C > TSilentexon6c.G672Ap.E224E——NegativeLowAlive3.12227EC916T17578395G > AMissenseexon5c.C535Tp.H179YPartially functionalC65PositiveLowAlive4.18229EC913T17578249AT > AFrame_Shift_Delexon6c.599delAp.N200fs——NegativeLowDead3.05231EC920T17578442T > CMissenseexon5c.A488Gp.Y163CNot-functionalC65PositiveHighDead2.15232EC918T17578406C > TMissenseexon5c.G524Ap.R175HNot-functionalC25PositiveHighDead2.21233TW890BF17577121G > AMissenseexon8c.C817Tp.R273CNot-functionalC65PositiveLowDead2.35236EC921T17579592T > ASplice_Siteexon5c.97-2A >T———NegativeLowDead2.00238EC924T17577121G > AMissenseexon8c.C817Tp.R273CNot-functionalC65PositiveHighDead3.30240EC684T17578223CT > CFrame_Shift_Delexon6c.625delAp.R209fs——NegativeLowDead3.84241EC682T17577152AC > AFrame_Shift_Delexon8c.785delGp.G262fs——NegativeLowAlive4.42243EC680T17579419AG > AFrame_Shift_Delexon4c.267delCp.P89fs——NegativeLowAlive4.42244EC055T17577124C > TMissenseexon8c.G814Ap.V272MNot-functionalC15PositiveHighDead0.88245EC681T17577610T > ASplice_Siteexon8c.673-2A >T———NegativeLowAlive4.44247EC676T17579311C > TSplice_Siteexon5c.375+1G >A———NegativeLowDead0.98248EC677T17576851A > GSplice_Siteexon10c.993+2T >C———NegativeLowAlive4.44249EC672T17576851A > GSplice_Siteexon10c.993+2T >C———NegativeLowDead1.83250EC666T17578212G > ANonsenseexon6c.C637Tp.R213X——NegativeLowDead1.54251EC660T17579358C > AMissenseexon4c.G329Tp.R110LNot-functionalC25PositiveHighAlive4.50253EC659T17578479G > AMissenseexon5c.C451Tp.P151SNot-functionalC65PositiveHighAlive4.87254EC1002T17577094G > AMissenseexon8c.C844Tp.R282WNot-functionalC65PositiveHighAlive4.65258EC651T17578413C > AMissenseexon5c.G517Tp.V173LNot-functionalC25PositiveHighAlive4.53259EC645T17579528C > TNonsenseexon4c.G159Ap.W53X——NegativeLowAlive4.55260EC928T27577141C > TMissenseexon8c.G797Ap.G266ENot-functionalC65PositiveLowAlive4.587578263G > ANonsenseexon6c.C586Tp.R196X——261EC926T17578265A > ATFrame_Shift_Insexon6c.583dupAp.I195fs——NegativeLowAlive4.96262EC929T27579355A > GMissenseexon4c.T332Cp.L111PNot-functionalC65PositiveHighDead1.547579354C > ASilentexon4c.G333Tp.L111L——263EC927T17578175A > GSplice_Siteexon7c.672+2T >C———NegativeLowDead1.99265EC038T17579335TC > TFrame_Shift_Delexon4c.351delGp.G117fs——NegativeLowDead1.47266EC930T17578265A > TMissenseexon6c.T584Ap.I195NNot-functionalC65PositiveHighAlive4.91268EC1000T27577105G > CMissenseexon8c.C833Gp.P278RNot-functionalC65PositiveLowAlive4.647578406C > TMissenseexon5c.G524Ap.R175HNot-functionalC25269EC999T17578469C > AMissenseexon5c.G461Tp.G154VNot-functionalC65PositiveLowAlive4.64270EC1001T27578413C > TMissenseexon5c.G517Ap.V173MNot-functionalC15PositiveHighDead1.927578263G > ANonsenseexon6c.C586Tp.R196X——271G017_T17578212G > ANonsenseexon6c.C637Tp.R213X——NegativeLowAlive4.70272EC894T17577094G > AMissenseexon8c.C844Tp.R282WNot-functionalC65PositiveHighAlive4.68274EC895T17578265A > GMissenseexon6c.T584Cp.I195TNot-functionalC55PositiveHighDead2.11275EC890T17578245G > GCFrame_Shift_Insexon6c.603dupGp.R202fs——NegativeLowAlive4.69277EC027T17578526C > AMissenseexon5c.G404Tp.C135FNot-functionalC65PositiveLowAlive11.89278TW258BF17577609C > TSplice_Siteexon8c.673-1G >A———Not detectedNot detectedDead1.39279EC114T17577144A > CMissenseexon8c.T794Gp.L265RNot-functionalC65Not detectedNot detectedAlive10.78280EC099T17578384G > TNonsenseexon5c.C546Ap.C182X——Not detectedNot detectedDead1.76281TW1375BF17578263G > ANonsenseexon6c.C586Tp.R196X——Not detectedNot detectedAlive9.72283EC107T17579388T > TGFrame_Shift_Insexon4c.298dupCp.Q100fs——Not detectedNot detectedDead3.06285EC188T17579315G > GCFrame_Shift_Insexon4c.371dupGp.C124fs——Not detectedNot detectedDead1.93288EC081T17578265A > GMissenseexon6c.T584Cp.I195TNot-functionalC55Not detectedNot detectedDead2.62290EC145T17578413C > TMissenseexon5c.G517Ap.V173MNot-functionalC15Not detectedNot detectedAlive2.88291EC131T27578413C > AMissenseexon5c.G517Tp.V173LNot-functionalC25Not detectedNot detectedAlive12.357574000C > ANonsenseexon10c.G1027Tp.E343X——292TW470BF17577544A > TMissenseexon7c.T737Ap.M246KNot-functionalC65Not detectedNot detectedAlive15.36293EC086T17579372GC > GFrame_Shift_Delexon4c.314delGp.G105fs——Not detectedNot detectedAlive11.00295EC126T17579470CG > CFrame_Shift_Delexon4c.216delCp.P72fs——Not detectedNot detectedAlive11.37296EC040T27577568C > GMissenseexon7c.G713Cp.C238SNot-functionalC65Not detectedNot detectedAlive8.247577022G > ANonsenseexon8c.C916Tp.R306X——297TW1371BF17578483GGAAT > GFrame_Shift_Delexon5c.443_446delp.D148fs——Not detectedNot detectedDead2.22298TW1371AF17578271T > AMissenseexon6c.A578Tp.H193LNot-functionalC65Not detectedNot detectedDead0.92299MC34T17577538C > AMissenseexon7c.G743Tp.R248LNot-functionalC65Not detectedNot detectedDead7.54301CE2T17578205CTA > CFrame_Shift_Delexon6c.642_643delp.H214fs——Not detectedNot detectedAlive13.37302MC29T17579345CA > CFrame_Shift_Delexon4c.341delTp.L114fs——Not detectedNot detectedDead4.26303EC034T17577539G > AMissenseexon7c.C742Tp.R248WNot-functionalC65Not detectedNot detectedDead6.17305TW2309BF17577022G > ANonsenseexon8c.C916Tp.R306X——Not detectedNot detectedAlive11.74306EC128T17578406C > TMissenseexon5c.G524Ap.R175HNot-functionalC25Not detectedNot detectedDead4.00307EC192T17578395G > AMissenseexon5c.C535Tp.H179YPartially functionalC65Not detectedNot detectedDead2.78310MC26T17578265A > GMissenseexon6c.T584Cp.I195TNot-functionalC55Not detectedNot detectedDead1.66311EC060T17578179C > ANonsenseexon6c.G670Tp.E224X——Not detectedNot detectedAlive6.87312TW829AF17579315G > GCFrame_Shift_Insexon4c.371dupGp.C124fs——Not detectedNot detectedAlive14.09313TW1099AF27578208T > CMissenseexon6c.A641Gp.H214RNot-functionalC0Not detectedNot detectedAlive11.027579485C > ANonsenseexon4c.G202Tp.E68X——314EC094T27577539G > AMissenseexon7c.C742Tp.R248WNot-functionalC65Not detectedNot detectedDead5.707576928T > ASplice_Siteexon10c.920-2A >T———316EC012T17579315G > GCFrame_Shift_Insexon4c.371dupGp.C124fs——Not detectedNot detectedDead1.36Notably, we found the frequency of nine protein mutations of p53 protein, including four hotspot mutations (p.R175, p.R248, p.R273, and p.R282) and five nonhotspot mutations (p.V173, p.H179, p.R196, p.R213, and p.P278) [21, 22], were more than five, respectively. Hotspot mutations comprised 29.0% (42/145) of all p53 missense mutations (Figure 4(d)).


### 3.3. Correlation of p53 Protein Expression with TA Class and Align GVGD Classifications

To speculate on the effect of the protein function of theTP53 missense mutation sites, we submitted queries to the IARC TP53 Database (https://p53.iarc.fr) [23–25]. According to TA classification, 145 TP53 missense mutation sites were divided into three categories: functional (4/145, 2.7%), partially functional (11/145, 7.6%), and not-functional (130/145, 89.7%) based on the TA class of the protein function (Table 4). We further observed that four of the 145 sites (in 135 patients) with missense mutations didn’t affect the function of the wild-p53 protein. However, only one of the four sites was a single mutation, and its p53 protein expression was negative. The other three sites were multiple mutations, and their p53 protein expression was positively detected (two were high, and one was low). Among the 130 sites (126 patients) with not-functional (108 patients with p53 protein expression tests), 105 patients were positive p53 protein expression (105/108, 97.2%), including 74 patients with p53 protein high expression (74/108, 68.5%). The p53 protein expression level is consistent with the predicted function based on the mutation sites. In the Align GVGD classification, we did not observe a significant difference in the positive rate of p53 protein in each group.


### 3.4. Association betweenTP53 Mutation and Protein Expression with Clinicopathological Parameters and Survival in ESCC

In the first cohort, we did not find any remarkable association between theTP53 mutation and these clinicopathological characteristics in ESCC (Table 2). Although there was no statistically significant difference in survival time when comparing the patients with and without TP53 mutations (Figure 5(a)), surprisingly, the results showed that the patients who had high protein expression of p53 exhibited significantly worse survival than those with low expression when the population narrow down to 276 patients (p=0.002, Figure 5(b)). The median survival time for 108 patients with high protein expression of p53 and for 168 patients who had low expression was 2.79 ± 0.63 and 5.27 ± 0.52 years, respectively. In addition, no significant difference was observed in the survival time of different mutation types of TP53 (p>0.05, Figure 5(c)), and no significant difference was observed in survival time between the hotspot and nonhotspot in TP53 missense mutations patients (p>0.05, Figure 5(d)).Figure 5
Kaplan–Meier curves survival analysis of ESCC patients with differentTP53 mutations and p53 protein expression. (a) OS of TP53 mutation and wild-type in 316 patients with sequencing data. (b) OS of p53 protein high and low expression in 276 patients who have TP53 mutation and p53 protein expression. (c) OS of different types of mutation in 316 patients with sequencing data. (d) OS of nonhotspot and hotspot mutation in 135 patients who have missense mutation. (e) OS of different groups of TP53 mutation status/p53 protein expression in 276 patients who have TP53 mutation and p53 protein expression. (f) OS of p53 protein high and low expression in 6,028 patients who have p53 expression in TMAs.
(a)(b)(c)(d)(e)(f)To better understand the factors which contribute to the association betweenTP53 mutations/high p53 expression in patients and survival, we compared the mutation types of p53 patients with ESCC. The high expression of the p53 protein was 66.9% among the 121 patients with TP53 gene missense mutations and only 5.6% in the 36 cases with nonsense mutations. Other types of mutations in the 55 patients (including frame shift, silent, and splice) were 3.6%. Apparently, missense mutation was most likely the cause of p53 protein mutation, leading to the high expression of p53 protein. Other mutation types were possible causes of low expression of the p53 protein. Surprisingly, in the 64 cases of TP53wild-type, high expression of p53 protein accounted for 35.9%, while low expression was 64.1% (41 patients, of which 26 were not expressed at all) (Figure 4(e) and Table 5).Table 5
Association betweenTP53 mutation and p53 protein expression in 276 ESCC patients.
p53 protein expressionTP53 MutationTP53wild-type, n = 64Missense,n = 121Nonsense,n = 36Others,n = 55Totalp53 high/low expressionp53 high expression81228523p53 low expression40345312741p53 positive/negative expressionp53 expression (+)117111013838p53 expression (−)425457426Note. When a missense mutation occurs, patients with multiple mutations are preferentially included in the missense mutation group. When a nonsense mutation occurs, the remaining patients are preferentially included in the nonsense mutation group.


### 3.5. A New Classification for Evaluating the Association betweenTP53 Mutation/p53 Protein and Survival

To address the controversial role ofTP53 mutation in ESCC, in this study, we established a new classification for evaluating TP53-related survival in ESCC by combining TP53 mutations and p53 protein expression analysis. The results clearly showed that 276 ESCC patients were divided into four groups as follows: TP53 mutation/p53 high expression (85/276, 30.8%), TP53 mutation/p53 low expression (127/276, 46.0%), TP53 wild-type/p53 high expression (23/276, 8.3%), TP53 wild-type/p53 low expression (41/276, 14.9%) (Table 5). Furthermore, the total survival time of the four groups was significantly different (p<0.01), among which the TP53 mutation/p53 high expression group had the worst survival time, followed by the TP53 wild-type/p53 high expression group (Figure 5(e)).


### 3.6. Association between p53 Protein Expression and Clinicopathological Changes in Cohort with Large-Scale ESCC Patients

To further validate our discovery of the association between the p53 expression-related prognosis and clinicopathological changes, we assessed additional cohorts to make an analysis (Table6). The positive expression of p53 protein was observed in 3,562 (59.1%) ESCC patients, of which 1,819 patients (51.1%) showed high expression of p53 protein. Moreover, the high expression of p53 protein in high-incidence areas was more common than in low-incidence areas with a rate and p value of 31.5% vs. 27.7%, p=0.003. High expression of p53 protein was closely correlated with poor tumor differentiation (p<0.001). The frequency of high expression of p53 protein in the early ESCC (0 + I stage) was 1.27-fold higher than that in the advanced ESCC (38.2% vs. 30.0%, p=0.034). Furthermore, the rate of positive cancer embolus in high-expression groups was 1.31-fold higher than in low-expression groups (5.1% vs. 3.9%, p=0.028).Table 6
Association between p53 protein expression and clinicopathologic features in patients with ESCC in tissue microarray.
CharacteristicsAll patients,p53 protein expressionp valueLow expression,High expression,n = 6028n = 4209(%)n = 1819(%)Sex0.525Female2107148270.362529.7Male3921272769.5119430.5Age at diagnosis0.492≤602917204970.286829.8>603111216069.495130.6High/low incidence area0.003Low2119153172.358827.7High3909267868.5123131.5Cigarette smoking0.393Negative3160221770.294329.8Positive2731188869.184330.9Alcohol consumption0.901Negative4103285769.6124630.4Positive1791125069.854130.2Family history0.615Negative3875270369.8117230.2Positive2037140869.162930.9Location0.667Cervical + upper95467871.127628.9Middle4092284269.5125030.5Lower87961369.726630.3Mix513364.71835.3Differentiation<0.001Well differentiated50138576.811623.2Moderate differentiated3303235071.195328.9Poor differentiated1935128066.165533.9Pathological T stage0.335Tis + T120513465.47134.6T21466102269.744430.3T34242297870.2126429.8T41157565.24034.8Pathological N stage0.494N03354235470.2100029.8N12674185569.481930.6Pathological M stage0.930M05814405969.8175530.2M121415070.16429.9UICC stage (6th)0.1440 + I1448961.85538.2II3691260070.4109129.6III1979137069.260930.8IV21415070.16429.9Cancer embolus0.028Negative5772404670.1172629.9Positive25616363.79336.3Type of treatment0.596Surgical5006349969.9150730.1Surgical + chemo49335271.414128.6Surgical + radio39526968.112631.9Surgical + chemo + radio1348966.44533.6


### 3.7. Independent Prognosis Marker Role of p53 Protein on Survival Analysis in 6,028 ESCC Patients

To determine whether a high expression level of the p53 protein could be used as a prognosis marker in patients with ESCC, we conducted the analysis using the p53 expression in 6,028 ESCC patients. In this cohort, the analysis showed median OS time in patients with p53 high expression and with low was 2.71 ± 0.09 and 3.08 ± 0.06 years, respectively. The presence of high expression of p53 protein was significantly associated with decreased OS (p<0.001, Figure 5(f)).


### 3.8. Cox Univariate and Multivariate Regression Analyses

Cox proportional-hazard models were chosen for the univariate and multivariate analyses. The univariate Cox regression analysis demonstrated that the sex, age, high/low incidence area, cigarette smoking, alcohol consumption, location, differentiation, T stage, N stage, M stage, UICC stage, cancer embolus, and p53 protein high/low expression were dependent prognostic factors (p<0.05, Figures 6(a), 6(b), and 7). In multivariate Cox regression analysis, as compared with the low expression of p53 protein, the patients with high expression (HR = 1.134, 95% CI 1.065 to 1.207) were associated with decreased survival after adjustment for the age, high/low incidence area, cigarette smoking, differentiation, and T and N stages as independent prognostic factors (p<0.05, Figure 6(c)).Figure 6
Forest plot demonstrating univariate and multivariate analysis of factors that influence OS in 6,028 ESCC patients. The results of the univariate analysis of factors that influence OS in 6,028 ESCC patients are shown in (a) and (b). The results of the multivariate analysis of factors that influence OS in 6,028 ESCC patients are shown in (c).
(a)(b)(c)Figure 7
Kaplan–Meier curves analysis estimating OS following stratifying 6,028 patients with ESCC according to sex (a), age at diagnosis (b), high/low incidence area (c), cigarette smoking (d), alcohol consumption (e), family history (f), location (g), differentiation (h), T stage (i), N stage (j), M stage (k), UICC stage (l), cancer embolus (m), and treatment (n), respectively.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)(k)(l)(m)(n)


### 3.9. Stratified Survival Analysis by within Independent Prognostic Factors

We performed a stratified analysis to eliminate the influence of the independent prognostic factors on evaluating the expression of the p53 protein on prognosis. When patients were stratified according to high/low incidence area, cigarette smoking, and N stages, the consistent trend of OS was observed in different stratification (Figures8(a)–8(f)). When stratified according to patients’ age at diagnosis, degree of differentiation, and T stage, we only found that patients the high expression of p53 protein in >60 (p<0.001, Figure 8(g)), medium and low differentiation (p<0.001, Figures 8(h) and 8(i)), and T3 (p<0.001, Figure 8(j)) had shorter OS than those with low expression.Figure 8
Kaplan–Meier curves survival analysis of ESCC patients with high/low p53 protein expression after stratification based on independent prognostic factors. OS of patients with p53 protein high vs. low expression in patients with high-incidence area (a) and low-incidence area (b). OS of patients with p53 protein high vs. low expression in patients with (c) and without cigarette smoking (d). OS of patients with p53 protein high vs. low expression in patients with N0 (e) and N1 stage (f). OS of patients with p53 protein high vs. low expression in patients >60 years (g). OS of patients with p53 protein high vs. low expression in patients with moderate (h) and poor differentiation (i). OS of patients with p53 protein high vs. low expression in patients with T3 stage (j).
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)


## 3.1. The Clinicopathological Distributions of ESCC Patients Results

To discover the genetic alteration ofTP53 in ESCC, we collected tumor samples to set up two cohorts, including 316 patients for sequencing analysis (276 patients out of 316 patients performed IHC to detect the expression of p53 protein), and 6,028 patients for the expression of p53 protein in TMA, for investigation in this study (Figure 1). Detailed clinicopathological data are listed in Figure 3 and Table 1. The follow-up period of all those patients ranged from 0.08 years to 30.87 years, and the median survival time was 2.98 years.Figure 3
Baseline characteristics of patients with ESCC were included in both study cohorts. The proportions of the two study cohorts are shown according to sex (a), age at diagnosis (b), high/low incidence area (c), cigarette smoking (d), alcohol consumption (e), family history (f), location (g), differentiation (h), T stage (i), N stage (j), M stage (k), UICC stage (l), cancer embolus (m), and treatment (n), respectively.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)(k)(l)(m)(n)Table 1
Baseline characteristics of patients with ESCC.
CharacteristicsThe patients undergoingTP53 mutation analysis,The patients with p53 IHC staining in TMA,n = 316(%)n = 6028(%)SexFemale11737.0210735.0Male19963.0392165.0Age at diagnosis (mean ± SD)61.39 ± 8.1860.92 ± 8.28≤6013643.0291748.4>6018057.0311151.6High/low incidence area★Low6420.3211935.2High25279.7390964.8Cigarette smokingNegative17856.3316052.4Positive13643.1273145.3Missing20.61372.3Alcohol consumptionNegative18458.2410368.1Positive13041.1179129.7Missing20.61342.2Family historyPositive13141.5203733.8Negative17455.0387564.3Missing113.51161.9LocationCervical + upper4514.295415.8Middle18257.6409267.9Lower6821.587914.6Multiple154.7510.8No record61.9520.9DifferentiationWell differentiated257.95018.3Moderate differentiated19561.7330354.8Poor differentiated9128.8193532.1No record51.62894.8Pathological T stageTis + T1185.72053.4T28326.3146624.3T320364.2424270.4T4123.81151.9Pathological N stageN017455.1335455.6N114244.9267444.4Pathological M stageM030897.5581496.4M182.52143.6UICC stage (6th)0 + I113.81442.4II18859.5369161.2III10934.2197932.8IV82.52143.6Cancer embolusPositive216.62564.2Negative29593.4577295.8Type of treatmentSurgical29392.7500683.0Surgical + chemo113.54938.2Surgical + radio82.53956.6Surgical + chemo + radio41.31342.2★areas with esophageal cancer incidence ≥50/100,000 are high-incidence area for esophageal cancer; on the contrary, areas with esophageal cancer incidence <50/100,000 is low-incidence area for esophageal cancer.


## 3.2. Distribution ofTP53 Somatic Mutations and p53 Protein

We evaluatedTP53 somatic alterations in the first cohort, and the results showed that 241 of the 316 ESCC patients (76.3%) exhibited a total of 276 TP53 somatic mutations, which included 208 cases having only one mutation and 33 having multiple (two and three) mutations (Tables 2 and 3). TP53 mutations were mainly located in the exon 5–8 (79.4%), meaning that most mutations occurred in the DBD, and very few mutations occurred in the AD1, AD2, and TET domains. Mutations were mostly clustered in exon 5 (23.6%) and exon 8 (23.6%), followed by exon 6 (19.2%) (Figure 4(a)).Table 2
Comparison of clinicopathologic features between mutation and wild-typeTP53 in patients with ESCC.
CharacteristicsAll patients,Wild-typeTP53,MutationTP53,p valuen = 316n = 75(%)n = 241(%)Sex0.736Female1172924.88875.2Male1994623.115376.9Age at diagnosis (mean ± SD)0.543≤601363022.110677.9>601804525.013575.0High/low incidence area0.551Low641726.64773.4High2525823.019477.0Cigarette smoking0.277Negative1784625.813274.2Positive1362820.610879.4Alcohol consumption0.211Negative1844826.113673.9Positive1302620.010480.0Family history0.984Negative1744123.613376.4Positive1313123.710076.3Location0.904∗Cervical + upper451226.73373.3Middle1824223.114076.9Lower681522.15377.9Multiple15426.71173.3Differentiation0.260Well differentiated25312.02288.0Moderate differentiated1955025.614574.4Poor differentiated911920.97279.1Pathological T stage0.239∗Tis + T118738.91161.1T2831720.56679.5T3 + T42155123.716476.3Pathological N stage0.130N01744727.012773.0N11422819.711480.3Pathological M stage1.000∗M03087323.723576.3M18225.0675.0UICC stage (6th)0.053∗0 + I11545.5654.5II1884926.113973.9III + IV1172117.99682.1Cancer embolus0.590∗Negative2956923.422676.6Positive21628.61571.4Type of treatment0.211Surgical2937224.622175.4Surgical + chemo/radio23313.02087.0∗the differences among categoric variables were analyzed using the Fisher exact test.Table 3
Characteristics of the ESCC patients undergoingTP53 mutation detection.
NoPatient IDSexAgeUICC stageSequence sourceMutation statusNumber of mutationsp53 IHC (positive/negative)p53 IHC (low/high)Survival statusSurvival time (years)1CE12TM72IVBWESTP53 wild-type0PositiveLowDead1.702CE18TM50IIIWESTP53 mutation1NegativeLowDead4.733CE16TM69IIAWESTP53 wild-type0PositiveLowDead1.814CE4TM64IIBWESTP53 mutation1NegativeLowDead3.955CE7TM70IIIWESTP53 mutation1PositiveLowDead5.006CE15TM57IIIWESTP53 mutation1PositiveLowDead5.817CE30TM56IIAWESTP53 wild-type0NegativeLowAlive10.518CE20TM72IIAWESTP53 mutation1PositiveLowDead4.079CE26TM71IIIWESTP53 mutation2PositiveHighDead6.1610CE5TF75IIIWESTP53 mutation1PositiveLowDead5.1111CE25TM64IIAWESTP53 wild-type0PositiveHighDead2.0512CE24TM58IIAWESTP53 mutation1PositiveHighAlive11.0413CE21TM54IIAWESTP53 mutation2NegativeLowDead6.5014CE27TM64IIIWESTP53 mutation1PositiveHighDead2.4115EC888TM59IIITRSTP53 wild-type0PositiveHighAlive5.0516EC889TM62IIITRSTP53 mutation1PositiveHighDead0.7917EC891TM55IIBTRSTP53 wild-type0NegativeLowDead1.7518EC892TM60IIITRSTP53 mutation1PositiveHighDead3.1519EC893TF64IIATRSTP53 mutation1PositiveHighAlive4.7120EC1038TM74IVTRSTP53 mutation1PositiveLowDead1.4921EC1035TM65IIITRSTP53 wild-type0PositiveHighDead2.2722EC1032TF51IIATRSTP53 mutation1PositiveHighDead2.2723EC632TM59IIATRSTP53 mutation1PositiveHighAlive4.7824EC636TM64IIBTRSTP53 mutation1NegativeLowAlive4.7725EC090TM47IIIWESTP53 mutation1PositiveHighAlive1.4126EC633TF63ITRSTP53 mutation1PositiveLowAlive4.8027EC640TM58IIATRSTP53 mutation1NegativeLowAlive4.8128EC643TF67IIATRSTP53 mutation1PositiveLowDead2.5429EC644TM61IIITRSTP53 mutation1NegativeLowDead2.3330EC641TF60IIATRSTP53 mutation1NegativeLowAlive4.8431EC967TM69IIATRSTP53 mutation2NegativeLowAlive4.8432EC968TF63IIATRSTP53 mutation1PositiveLowAlive4.8533EC966TM69IIATRSTP53 mutation2PositiveHighDead2.3734EC965TF71IIATRSTP53 mutation1PositiveHighDead0.9535EC1024TF65IVATRSTP53 mutation1PositiveLowDead4.0336EC190TM50IIAWESTP53 mutation2PositiveLowAlive10.8037EC969TM63IIITRSTP53 mutation1PositiveHighAlive4.8838EC970TM66IIITRSTP53 wild-type0NegativeLowDead0.1339EC971TM66IIITRSTP53 mutation1NegativeLowAlive5.2140EC973TF63IIBTRSTP53 mutation1NegativeLowDead0.8841EC975TM62IIITRSTP53 mutation1PositiveLowAlive4.8842EC976TF79IIATRSTP53 mutation1PositiveLowDead1.8943EC978TM57IIATRSTP53 mutation1NegativeLowAlive5.2644EC981TM69IIITRSTP53 mutation1NegativeLowAlive4.9045EC982TM64IIBTRSTP53 mutation1PositiveLowAlive4.9046EC983TM44IIATRSTP53 mutation1NegativeLowAlive3.9547EC191TM59IIIWESTP53 mutation1PositiveLowDead4.1848EC984TM63IIITRSTP53 mutation1PositiveLowDead1.3649EC985TM51IIITRSTP53 mutation1NegativeLowAlive5.3050EC986TM72IIATRSTP53 mutation1NegativeLowDead3.2451EC987TM67IIITRSTP53 mutation1PositiveLowAlive4.9452EC990TM73IIITRSTP53 mutation1NegativeLowDead1.6453EC991TM67IVATRSTP53 mutation1PositiveLowDead2.4254EC993TM56IIITRSTP53 wild-type0NegativeLowAlive3.1255EC992TM59IIATRSTP53 mutation1PositiveLowAlive4.9656EC994TM47IIITRSTP53 mutation1PositiveLowAlive2.8157EC995TM68IIATRSTP53 wild-type0NegativeLowAlive4.9858EC088TM58IIIWESTP53 mutation1NegativeLowDead6.0759EC996TF74IIATRSTP53 mutation1PositiveHighAlive4.9860EC630TF57IIATRSTP53 mutation1PositiveHighDead2.7961EC997TF61IIATRSTP53 mutation1PositiveLowAlive4.9862EC629TM67IIBTRSTP53 mutation1PositiveLowDead2.4363EC1006TM58IIITRSTP53 mutation2PositiveHighAlive5.0464EC885TM65IIBTRSTP53 mutation2PositiveLowDead4.0165EC1005TF73IIITRSTP53 mutation1PositiveLowDead2.9266EC886TM62IIBTRSTP53 wild-type0PositiveHighDead1.5667G008_TM71IIAWESTP53 mutation1PositiveHighDead2.5368NR131225_2TF70IIIWGSTP53 mutation1NegativeLowAlive5.2969EC080TM70IWESTP53 mutation1PositiveLowDead2.7070NR131225_3TM55IIAWGSTP53 mutation1PositiveHighAlive5.3271NR131225_5TF67IIAWGSTP53 mutation1NegativeLowDead0.9372NR131225_9TM67IIAWGSTP53 mutation1NegativeLowAlive5.6773NR131225_4TM74IIAWGSTP53 mutation1PositiveLowDead3.6174NR131225_10TM77IIIWGSTP53 wild-type0NegativeLowDead2.5075NR131225_1TF57IIIWGSTP53 mutation1PositiveHighDead3.7076NR131225_8TM58IIIWGSTP53 wild-type0PositiveLowAlive5.3677NR131225_6TM60IIAWGSTP53 mutation1PositiveHighDead2.8378NR131121_10TM53IIAWGSTP53 mutation1PositiveHighDead0.6679NR131121_9TM66IIBWGSTP53 mutation1PositiveHighDead1.0080EC076TM60IIIWESTP53 wild-type0PositiveLowAlive11.1981NR131121_8TM60IIAWGSTP53 mutation1PositiveLowDead4.0082NR131121_7TM59IIBWGSTP53 mutation1PositiveLowDead5.2783NR131121_6TM69IIAWGSTP53 mutation1PositiveHighDead1.0184NR131121_5TF64IIIWGSTP53 mutation1PositiveLowAlive4.8085NR131121_4TM65IIIWGSTP53 mutation1PositiveHighDead0.5886NR131121_3TF72IIAWGSTP53 mutation1PositiveLowAlive5.5787NR131121_2TM61IIAWGSTP53 mutation2PositiveHighDead4.4988NR131121_1TM63IIAWGSTP53 mutation1PositiveHighDead2.0389TW2352AFM55IIAWESTP53 mutation1PositiveLowAlive7.8790TW2351BFM46IIAWESTP53 mutation1PositiveHighAlive8.2891EC062TF59IIIWESTP53 mutation1PositiveLowDead6.4992EC129TM54IIAWESTP53 mutation1NegativeLowAlive10.3093EC127TF72IIBWESTP53 mutation1NegativeLowDead8.4594MC16TM62IIAWESTP53 mutation1NegativeLowDead7.8095MC17TM50IIAWESTP53 mutation2PositiveLowDead6.8796MC13TM54IIIWESTP53 mutation1PositiveHighDead4.2597MC7TM53IIIWESTP53 mutation2PositiveHighDead0.7598TW258AFM40IIIWESTP53 mutation1PositiveHighDead0.6099FE0008TM64IIITRSTP53 mutation1PositiveHighDead0.83100EC123TM56IIAWESTP53 mutation1PositiveLowDead4.06101G556TM64IIITRSTP53 mutation1PositiveLowAlive2.65102G406TM69IIITRSTP53 mutation1PositiveHighDead0.46103FG0067TM68IIITRSTP53 mutation2PositiveHighAlive1.10104EC745TM64IIATRSTP53 wild-type0NegativeLowAlive2.95105G414TM57IIITRSTP53 mutation1NegativeLowAlive3.11106EC727TM70IIATRSTP53 mutation1PositiveHighAlive3.14107EC720TF74IIATRSTP53 mutation1NegativeLowDead1.16108EC718TM64IIITRSTP53 wild-type0NegativeLowDead1.66109EC721TM56IIITRSTP53 mutation1PositiveHighDead0.48110EC943TM65IIATRSTP53 mutation1PositiveHighAlive3.79111EC121TM59IIIWESTP53 mutation1PositiveHighDead1.00112EC854TF64IIATRSTP53 mutation2PositiveHighAlive3.44113EC868TF52IIITRSTP53 mutation1PositiveLowAlive3.44114EC686TF59IIATRSTP53 mutation2PositiveLowAlive3.43115EC687TF58IIATRSTP53 wild-type0PositiveHighDead0.92116EC685TF78IIATRSTP53 wild-type0PositiveLowAlive3.47117EC884TM63IIATRSTP53 wild-type0NegativeLowAlive3.45118EC883TM62IIITRSTP53 mutation2PositiveHighAlive3.49119EC881TF62IIITRSTP53 mutation1PositiveHighDead1.24120EC877TM68IIITRSTP53 mutation1PositiveHighAlive3.51121EC880TM68IIATRSTP53 mutation1PositiveHighAlive3.47122EC122TM52IIIWESTP53 mutation1NegativeLowAlive11.47123EC876TF47IIATRSTP53 wild-type0NegativeLowAlive3.51124EC871TM78IIATRSTP53 mutation1PositiveHighAlive3.51125EC874TF64IIATRSTP53 mutation1NegativeLowAlive3.53126EC855TF67ITRSTP53 wild-type0PositiveHighDead1.00127EC870TF65IIITRSTP53 wild-type0PositiveHighAlive3.91128EC872TM64IIITRSTP53 mutation1PositiveHighDead0.89129EC875TM68IIATRSTP53 mutation1PositiveLowDead1.99130EC869TM64ITRSTP53 mutation1PositiveHighDead1.53131EC811TF65IIATRSTP53 wild-type0PositiveHighDead2.22132G479TM77IVTRSTP53 mutation1PositiveLowDead0.57133EC124TM62IIIWESTP53 mutation1PositiveHighDead2.07134EC814TF61IIATRSTP53 mutation1NegativeLowAlive3.58135EC813TF60IIATRSTP53 mutation1PositiveHighAlive3.58136EC816TF66IIATRSTP53 wild-type0NegativeLowDead3.21137EC812TF72IIATRSTP53 wild-type0PositiveHighDead0.64138EC822TM63IIATRSTP53 mutation1PositiveLowAlive3.95139EC818TF53IIATRSTP53 mutation2PositiveLowAlive3.58140EC821TF64IIATRSTP53 mutation1PositiveHighAlive3.59141EC853TF65IIATRSTP53 wild-type0PositiveHighAlive2.64142EC832TF72IIATRSTP53 mutation1NegativeLowDead0.25143EC851TF67IIATRSTP53 mutation1NegativeLowAlive3.64144EC120TM65IIBWESTP53 mutation1PositiveHighDead3.49145EC835TM63IIATRSTP53 mutation1NegativeLowDead1.26146EC834TF59IIATRSTP53 mutation1NegativeLowAlive3.66147EC859TM61IIITRSTP53 mutation1NegativeLowDead1.02148EC850TM72IIATRSTP53 wild-type0PositiveLowAlive3.69149EC838TM58IIBTRSTP53 mutation1PositiveHighAlive2.67150EC849TM64IIATRSTP53 mutation1PositiveHighDead1.26151EC848TM53IIITRSTP53 mutation1PositiveHighAlive3.73152EC847TF65IIATRSTP53 mutation1NegativeLowAlive3.72153EC845TM57ITRSTP53 mutation1NegativeLowAlive3.75154EC932TF60IIATRSTP53 mutation1NegativeLowDead2.10155EC116TF58IIAWESTP53 mutation1PositiveHighAlive11.53156EC934TM70IIATRSTP53 wild-type0NegativeLowDead1.44157EC935TF64ITRSTP53 wild-type0NegativeLowAlive3.76158EC933TF66IIATRSTP53 mutation1NegativeLowDead3.20159EC941TM62IIITRSTP53 mutation1NegativeLowDead2.57160EC942TF73IIATRSTP53 mutation1NegativeLowAlive3.78161EC945TM66IIATRSTP53 mutation1PositiveHighAlive3.80162EC944TF65IIBTRSTP53 mutation1PositiveHighDead1.31163EC936TF58IIATRSTP53 wild-type0PositiveHighAlive3.79164EC937TF60IIBTRSTP53 wild-type0PositiveHighAlive3.79165EC948TF60IIBTRSTP53 mutation1NegativeLowAlive3.81166EC117TM50IIIWESTP53 mutation3PositiveHighDead1.03167EC146TF51IIIWESTP53 mutation1PositiveLowDead3.25168EC940TF69IIATRSTP53 mutation1NegativeLowDead3.09169EC938TF63IIATRSTP53 wild-type0PositiveLowDead1.08170EC939TF52IIATRSTP53 wild-type0PositiveHighDead0.92171EC951TF63IIATRSTP53 mutation2PositiveHighAlive3.85172EC952TF61IIATRSTP53 mutation1NegativeLowAlive3.85173EC963TF56IIATRSTP53 mutation2NegativeLowAlive3.86174EC958TF71IIITRSTP53 mutation1NegativeLowAlive3.86175EC959TF57IIITRSTP53 wild-type0NegativeLowDead1.32176EC961TF48IIATRSTP53 mutation2PositiveHighAlive3.88177EC962TM52IIATRSTP53 mutation1PositiveLowAlive4.24178EC118TF65IIBWESTP53 mutation1NegativeLowDead3.05179EC956TM55IIATRSTP53 wild-type0PositiveLowAlive3.91180EC960TM51IIITRSTP53 mutation2PositiveHighDead1.98181EC954TF69IIATRSTP53 wild-type0PositiveLowAlive3.91182EC955TF52IIBTRSTP53 mutation1NegativeLowDead1.20183EC953TM61IIATRSTP53 wild-type0PositiveHighDead1.27184EC802TF63IIITRSTP53 mutation1NegativeLowDead1.48185EC793TM69IIATRSTP53 mutation1NegativeLowAlive3.95186EC789TM63IIATRSTP53 mutation1NegativeLowAlive3.95187EC797TM58IIITRSTP53 mutation1NegativeLowAlive4.33188EC798TF59IIATRSTP53 mutation1NegativeLowAlive3.96189EC115TF69IIAWESTP53 mutation2PositiveHighDead1.48190EC788TF58IIATRSTP53 wild-type0PositiveLowDead2.98191EC792TM70IIATRSTP53 mutation1PositiveLowDead3.21192EC791TM61IIITRSTP53 mutation3PositiveHighDead1.25193EC790TM79IIITRSTP53 mutation1NegativeLowDead1.78194EC794TM65IIITRSTP53 mutation2PositiveHighDead1.74195EC783TM75IIITRSTP53 mutation1PositiveHighAlive3.97196EC782TM67IIATRSTP53 wild-type0PositiveLowDead2.42197EC795TF69IIATRSTP53 mutation1PositiveHighDead1.51198EC787TM59IIITRSTP53 mutation1PositiveHighDead0.84199G521TF65IIATRSTP53 mutation1NegativeLowAlive3.63200EC113TM64IIIWESTP53 mutation2PositiveLowAlive11.54201EC780TM49IIATRSTP53 mutation1NegativeLowDead1.96202EC786TM66IIATRSTP53 mutation1PositiveHighAlive4.05203EC781TM72IIATRSTP53 mutation1PositiveHighDead1.33204EC785TM47IIATRSTP53 wild-type0NegativeLowAlive4.06205EC779TF52IIITRSTP53 mutation1PositiveHighAlive4.42206EC766TF62IIITRSTP53 mutation1PositiveLowDead1.61207EC769TF68IIATRSTP53 mutation1PositiveHighAlive4.46208EC807TF82IIATRSTP53 mutation1PositiveHighAlive4.11209EC900TF64IIATRSTP53 mutation1NegativeLowAlive4.11210EC898TM59IIITRSTP53 mutation1NegativeLowAlive4.13211EC015TM63IIIWESTP53 wild-type0PositiveLowDead2.25212EC904TF48IIATRSTP53 mutation1PositiveHighAlive4.12213EC899TM60IIBTRSTP53 wild-type0PositiveHighAlive4.12214EC908TF56IIITRSTP53 mutation1PositiveHighDead1.88215EC907TM60IIATRSTP53 mutation1PositiveHighDead15.80216EC901TF76IIITRSTP53 wild-type0PositiveLowDead3.13217EC902TM48IIITRSTP53 mutation1PositiveLowAlive4.15218EC906TM49IIATRSTP53 mutation1Not detectedNot detectedAlive4.51219EC972TM63IIATRSTP53 wild-type0PositiveHighDead0.98220EC910TM65IIATRSTP53 wild-type0NegativeLowAlive4.14221G048_TF64IIIWESTP53 wild-type0PositiveHighDead0.70222TW094BFM40IIIWESTP53 mutation2PositiveLowDead2.74223G042_TM65IIIWESTP53 mutation1NegativeLowAlive4.16224EC912TF63IIATRSTP53 wild-type0PositiveHighDead2.14225EC911TF52IIATRSTP53 wild-type0NegativeLowDead0.46226EC909TF56IIBTRSTP53 mutation1NegativeLowAlive3.12227EC916TF62IIATRSTP53 mutation1PositiveLowAlive4.18228EC915TM67IIATRSTP53 wild-type0NegativeLowAlive4.19229EC913TM70IIATRSTP53 mutation1NegativeLowDead3.05230EC917TF62IIATRSTP53 wild-type0PositiveHighDead3.65231EC920TF56IIITRSTP53 mutation1PositiveHighDead2.15232EC918TM68IIITRSTP53 mutation1PositiveHighDead2.21233TW890BFM65IIIWESTP53 mutation1PositiveLowDead2.35234G039_TM64IIAWESTP53 wild-type0NegativeLowAlive4.24235EC922TM74IIBTRSTP53 wild-type0PositiveLowDead3.49236EC921TF60IIBTRSTP53 mutation1NegativeLowDead2.00237EC923TM68IIITRSTP53 wild-type0NegativeLowDead2.90238EC924TM57IIATRSTP53 mutation1PositiveHighDead3.30239EC683TM46IIATRSTP53 wild-type0NegativeLowAlive4.42240EC684TM56IIITRSTP53 mutation1NegativeLowDead3.84241EC682TM63IIATRSTP53 mutation1NegativeLowAlive4.42242EC679TM59IIITRSTP53 wild-type0NegativeLowDead2.31243EC680TF62IIATRSTP53 mutation1NegativeLowAlive4.42244EC055TM45IIIWESTP53 mutation1PositiveHighDead0.88245EC681TM70IIATRSTP53 mutation1NegativeLowAlive4.44246EC678TF48IIBTRSTP53 wild-type0PositiveLowDead1.37247EC676TM57IIITRSTP53 mutation1NegativeLowDead0.98248EC677TM46IIITRSTP53 mutation1NegativeLowAlive4.44249EC672TM57IIITRSTP53 mutation1NegativeLowDead1.83250EC666TF67IVATRSTP53 mutation1NegativeLowDead1.54251EC660TF66IIATRSTP53 mutation1PositiveHighAlive4.50252EC658TM59ITRSTP53 wild-type0PositiveHighAlive4.52253EC659TF63IIITRSTP53 mutation1PositiveHighAlive4.87254EC1002TF62IIITRSTP53 mutation1PositiveHighAlive4.65255EC039TM74IIAWESTP53 wild-type0NegativeLowDead0.35256EC656TF72IIATRSTP53 wild-type0NegativeLowAlive4.51257EC654TF61IIITRSTP53 wild-type0PositiveHighDead0.96258EC651TM51IIBTRSTP53 mutation1PositiveHighAlive4.53259EC645TF57IIATRSTP53 mutation1NegativeLowAlive4.55260EC928TF61IIBTRSTP53 mutation2PositiveLowAlive4.58261EC926TM73IIATRSTP53 mutation1NegativeLowAlive4.96262EC929TF64ITRSTP53 mutation2PositiveHighDead1.54263EC927TM60IIBTRSTP53 mutation1NegativeLowDead1.99264EC1003TM64IIITRSTP53 wild-type0PositiveHighDead1.78265EC038TM68IIAWESTP53 mutation1NegativeLowDead1.47266EC930TF58IIATRSTP53 mutation1PositiveHighAlive4.91267EC931TF57IIATRSTP53 wild-type0NegativeLowAlive4.63268EC1000TM67IIATRSTP53 mutation2PositiveLowAlive4.64269EC999TF75IIBTRSTP53 mutation1PositiveLowAlive4.64270EC1001TF68IIITRSTP53 mutation2PositiveHighDead1.92271G017_TF67IIIWESTP53 mutation1NegativeLowAlive4.70272EC894TM66IIATRSTP53 mutation1PositiveHighAlive4.68273EC887TM77IIATRSTP53 wild-type0NegativeLowAlive4.68274EC895TM73IIITRSTP53 mutation1PositiveHighDead2.11275EC890TF64IIATRSTP53 mutation1NegativeLowAlive4.69276EC036TM49IWESTP53 wild-type0PositiveHighAlive7.88277EC027TM47IIAWESTP53 mutation1PositiveLowAlive11.89278TW258BFM40IIAWESTP53 mutation1Not detectedNot detectedDead1.39279EC114TM64IIIWESTP53 mutation1Not detectedNot detectedAlive10.78280EC099TM58IIAWESTP53 mutation1Not detectedNot detectedDead1.76281TW1375BFM55IWESTP53 mutation1Not detectedNot detectedAlive9.72282EC067TM50IVBWESTP53 wild-type0Not detectedNot detectedDead1.98283EC107TM56IIIWESTP53 mutation1Not detectedNot detectedDead3.06284MC23TF59IIIWESTP53 wild-type0Not detectedNot detectedDead0.06285EC188TM64IIAWESTP53 mutation1Not detectedNot detectedDead1.93286MC27TM56IIBWESTP53 wild-type0Not detectedNot detectedDead1.99287EC101TM73IIAWESTP53 wild-type0Not detectedNot detectedDead2.51288EC081TF68IIBWESTP53 mutation1Not detectedNot detectedDead2.62289MC30TF41IIBWESTP53 wild-type0Not detectedNot detectedAlive8.52290EC145TM51IIIWESTP53 mutation1Not detectedNot detectedAlive2.88291EC131TM49IVAWESTP53 mutation2Not detectedNot detectedAlive12.35292TW470BFF49IIIWESTP53 mutation1Not detectedNot detectedAlive15.36293EC086TM36IIAWESTP53 mutation1Not detectedNot detectedAlive11.00294EC013TF69IIAWESTP53 wild-type0Not detectedNot detectedDead2.92295EC126TF63IIIWESTP53 mutation1Not detectedNot detectedAlive11.37296EC040TM59IIIWESTP53 mutation2Not detectedNot detectedAlive8.24297TW1371BFM44IIAWESTP53 mutation1Not detectedNot detectedDead2.22298TW1371AFM43IIBWESTP53 mutation1Not detectedNot detectedDead0.92299MC34TM51IIAWESTP53 mutation1Not detectedNot detectedDead7.54300EC925TM59IIBTRSTP53 wild-type0Not detectedNot detectedDead1.77301CE2TM54IIIWESTP53 mutation1Not detectedNot detectedAlive13.37302MC29TM60IIBWESTP53 mutation1Not detectedNot detectedDead4.26303EC034TM62IIBWESTP53 mutation1Not detectedNot detectedDead6.17304MC4TM69IIAWESTP53 wild-type0Not detectedNot detectedDead2.81305TW2309BFF45IIAWESTP53 mutation1Not detectedNot detectedAlive11.74306EC128TF51IIIWESTP53 mutation1Not detectedNot detectedDead4.00307EC192TM57IIIWESTP53 mutation1Not detectedNot detectedDead2.78308EC061TM76IIAWESTP53 wild-type0Not detectedNot detectedDead5.83309TW002BFM59IWESTP53 wild-type0Not detectedNot detectedAlive10.24310MC26TM44IIIWESTP53 mutation1Not detectedNot detectedDead1.66311EC060TM50IIAWESTP53 mutation1Not detectedNot detectedAlive6.87312TW829AFF60IIAWESTP53 mutation1Not detectedNot detectedAlive14.09313TW1099AFM48IIAWESTP53 mutation2Not detectedNot detectedAlive11.02314EC094TF58IIAWESTP53 mutation2Not detectedNot detectedDead5.70315EC165TM55IIIWESTP53 wild-type0Not detectedNot detectedDead9.95316EC012TF51IIAWESTP53 mutation1Not detectedNot detectedDead1.36Figure 4
Type of somaticTP53 mutation in ESCC patients. (a) Distribution of TP53 mutations according to the affected exons. (b) Pie chart showing the proportion of the different types of somatic TP53 mutations. (c) Different types of mutations in the spectrum of TP53 mutations. (d) Graphical representation of the site of mutations in the coding sequence of TP53: red lollypop symbols in the lower panel representing missense mutations, blue, yellow, green, pink, and purple lollypop symbols in the upper panel representing others mutations (including nonsense, frameshift del, frameshift ins, silent, mixed type, respectively). Mutations occurring six or more are labeled: TA1, TA2, transactivation domain; DBD, DNA binding domain; TET, tetramerization domain. The figure was generated by the Illustrator for biological sequences version 1.0. (e) Summary of patients’ characteristics and alterations for TP53 and p53 protein in ESCC. Each column represents one patient sample. The phenotypic information in 316 ESCC patients is shown in the upper panel. Alterations for the TP53 gene and p53 protein are shown in the bottom panel.
(a)(b)(c)(d)(e)Next, we investigated the overall pattern of the 276 somatic mutations identified in theTP53, in which 145 were missense (52.5%), 54 were nonsense (19.6%), 33 were splices (12.0%), 24 were frameshift deletion (8.7%), eleven were frame-shit insertion (4.0%), five were silent (1.8%), two were nonframe deletion (0.7%), one was nonframe insertion (0.4%), and one was splice site insertion and deletion (0.4%) (Figure 4(b) and Table 4). For the protein domain distribution of missense mutations, the majority of which occurred in the DBD domain. However, the main type of mutation varied with different exons. In exons 5, 7, and 8, missense mutations accounted for more than 70% (73.8%, 75.0%, and 70.7%, respectively).We observed that the proportions of theTP53 mutation spectrum were 49/237 (20.7%) for C > A/G > T, 15/237 (6.3%) for C > G/G > C, 117/237 (49.4%) for C > T/G > A, 17/237 (7.2%) for T > A/A > T, 27/237 (11.4%) for T > C/A > G, 12/237 (5.1%) for T > G/A > C. The transition was predominant (144/237, 60.8%), followed by transversion (93/237, 39.2%) (Figure 4(c) and Table 4).Table 4
Descriptive features and functional effects ofTP53 mutated in ESCC patients.
NoPatient IDNumber of mutationsMutationsMutation typeexonMutation annotation (cDNA)TP53 mutation (protein)TA classAlign GVGDp53 IHC (positive/negative)p53 IHC (low/high)Survival statusSurvival time (years)2CE18T17577509C > ANonsenseexon7c.G772Tp.E258X——NegativeLowDead4.734CE4T17578212G > ANonsenseexon6c.C637Tp.R213X——NegativeLowDead3.955CE7T17579528C > TNonsenseexon4c.G159Ap.W53X——PositiveLowDead5.006CE15T17578461C > AMissenseexon5c.G469Tp.V157FNot-functionalC0PositiveLowDead5.818CE20T17578413C > TMissenseexon5c.G517Ap.V173MNot-functionalC15PositiveLowDead4.079CE26T27577556C > AMissenseexon7c.G725Tp.C242FNot-functionalC65PositiveHighDead6.167578406C > TMissenseexon5c.G524Ap.R175HNot-functionalC2510CE5T17577105G > AMissenseexon8c.C833Tp.P278LNot-functionalC65PositiveLowDead5.1112CE24T17577539G > AMissenseexon7c.C742Tp.R248WNot-functionalC65PositiveHighAlive11.0413CE21T27577603G > CSilentexon7c.C678Gp.G226G——NegativeLowDead6.507577610T > ASplice_siteexon8c.673-2A > T———14CE27T17578406C > TMissenseexon5c.G524Ap.R175HNot-functionalC25PositiveHighDead2.4116EC889T17577156C > ASplice_siteexon9c.783-1G > T———PositiveHighDead0.7918EC892T17579529C > TNonsenseexon4c.G158Ap.W53X——PositiveHighDead3.1519EC893T17578406C > TMissenseexon5c.G524Ap.R175HNot-functionalC25PositiveHighAlive4.7120EC1038T17579414C > TNonsenseexon4c.G273Ap.W91X——PositiveLowDead1.4922EC1032T17578403C > AMissenseexon5c.G527Tp.C176FPartially functionalC65PositiveHighDead2.2723EC632T17577085C > TMissenseexon8c.G853Ap.E285KNot-functionalC55PositiveHighAlive4.7824EC636T17579691TGT​CCT​TAC​CA > TFrame_Shift_Delexon3c.95_96delp.L32fs——NegativeLowAlive4.7725EC090T17578268A > CMissenseexon6c.T581Gp.L194RNot-functionalC65PositiveHighAlive1.4126EC633T17577022G > ANonsenseexon8c.C916Tp.R306X——PositiveLowAlive4.8027EC640T17579539T > TGFrame_Shift_Insexon4c.147_148insCp.I50fs——NegativeLowAlive4.8128EC643T17578235T > CMissenseexon6c.A614Gp.Y205CNot-functionalC65PositiveLowDead2.5429EC644T17579310A > GSplice_siteexon5c.375+2T >C———NegativeLowDead2.3330EC641T17578188C > ANonsenseexon6c.G661Tp.E221X——NegativeLowAlive4.8431EC967T27576897G > ANonsenseexon9c.C949Tp.Q317X——NegativeLowAlive4.847579494T > ANonsenseexon4c.A193Tp.R65X——32EC968T17577511A > CMissenseexon7c.T770Gp.L257RNot-functionalC65PositiveLowAlive4.8533EC966T27574029C > TMissenseexon10c.G998Ap.R333HFunctionalC0PositiveHighDead2.377577580T > CMissenseexon7c.A701Gp.Y234CNot-functionalC4534EC965T17578478G > CMissenseexon5c.C452Gp.P151RNot-functionalC65PositiveHighDead0.9535EC1024T17578395G > AMissenseexon5c.C535Tp.H179YPartially functionalC65PositiveLowDead4.0336EC190T27578520A > TMissenseexon5c.T410Ap.L137QFunctionalC65PositiveLowAlive10.807579313G > AMissenseexon4c.C374Tp.T125MNot-functionalC6537EC969T17577539G > AMissenseexon7c.C742Tp.R248WNot-functionalC65PositiveHighAlive4.8839EC971T17579311C > TSplice_Siteexon5c.375+1G >A———NegativeLowAlive5.2140EC973T17579415C > TNonsenseexon4c.G272Ap.W91X——NegativeLowDead0.8841EC975T17577058C > ANonsenseexon8c.G880Tp.E294X——PositiveLowAlive4.8842EC976T17577575A > GMissenseexon7c.T706Cp.Y236HNot-functionalC35PositiveLowDead1.8943EC978T17579722C > TSplice_Siteexon4c.75-1G >A———NegativeLowAlive5.2644EC981T17578370C > TSplice_Siteexon6c.559+1G >A———NegativeLowAlive4.9045EC982T17577105G > CMissenseexon8c.C833Gp.P278RNot-functionalC65PositiveLowAlive4.9046EC983T17574003G > ANonsenseexon10c.C1024Tp.R342X——NegativeLowAlive3.9547EC191T17578412A > GMissenseexon5c.T518Cp.V173APartially functionalC55PositiveLowDead4.1848EC984T17578466G > TMissenseexon5c.C464Ap.T155NNot-functionalC0PositiveLowDead1.3649EC985T17577511A > GMissenseexon7c.T770Cp.L257PNot-functionalC65NegativeLowAlive5.3050EC986T17578275G > ANonsenseexon6c.C574Tp.Q192X——NegativeLowDead3.2451EC987T17577018C > TSplice_Siteexon9c.919+1G >A———PositiveLowAlive4.9452EC990T17577539G > AMissenseexon7c.C742Tp.R248WNot-functionalC65NegativeLowDead1.6453EC991T17577503AGT​CTT​CCA​GT > AFrame_Shift_Delexon7c.768_777delp.T256fs——PositiveLowDead2.4255EC992T17577094G > AMissenseexon8c.C844Tp.R282WNot-functionalC65PositiveLowAlive4.9656EC994T17578394T > CMissenseexon5c.A536Gp.H179RNot-functionalC25PositiveLowAlive2.8158EC088T17578469CCGGG > CFrame_Shift_Delexon5c.457_460delp.P153fs——NegativeLowDead6.0759EC996T17578403C > AMissenseexon5c.G527Tp.C176FPartially functionalC65PositiveHighAlive4.9860EC630T17577153C > AMissenseexon8c.G785Tp.G262VNot-functionalC65PositiveHighDead2.7961EC997T17578406C > TMissenseexon5c.G524Ap.R175HNot-functionalC25PositiveLowAlive4.9862EC629T17577156C > TSplice_Siteexon9c.783-1G >A———PositiveLowDead2.4363EC1006T27577106G > TMissenseexon8c.C832Ap.P278TNot-functionalC35PositiveHighAlive5.047578475G > AMissenseexon5c.C455Tp.P152LNot-functionalC6564EC885T27577548C > TMissenseexon7c.G733Ap.G245SNot-functionalC55PositiveLowDead4.017576888T > ANonsenseexon9c.A958Tp.K320X——65EC1005T17578536T > CMissenseexon5c.A394Gp.K132ENot-functionalC55PositiveLowDead2.9267G008_T17578406C > TMissenseexon5c.G524Ap.R175HNot-functionalC25PositiveHighDead2.5368NR131225_2T17578290C > TSplice_Siteexon7c.560-1G >A———NegativeLowAlive5.2969EC080T17577143CAGT > Cnon_Frame_Delexon8c.792_794delp.264_265del——PositiveLowDead2.7070NR131225_3T17577082C > TMissenseexon8c.G856Ap.E286KNot-functionalC55PositiveHighAlive5.3271NR131225_5T17576870C > ANonsenseexon9c.G976Tp.E326X——NegativeLowDead0.9372NR131225_9T17578239C > ANonsenseexon6c.G610Tp.E204X——NegativeLowAlive5.6773NR131225_4T17577120C > TMissenseexon8c.G818Ap.R273HNot-functionalC25PositiveLowDead3.6175NR131225_1T17578403C > AMissenseexon5c.G527Tp.C176FPartially functionalC65PositiveHighDead3.7077NR131225_6T17577120C > AMissenseexon8c.G818Tp.R273LNot-functionalC65PositiveHighDead2.8378NR131121_10T17577565T > CMissenseexon7c.A716Gp.N239SNot-functionalC45PositiveHighDead0.6679NR131121_9T17577538C > TMissenseexon7c.G743Ap.R248QNot-functionalC35PositiveHighDead1.0081NR131121_8T17577120C > TMissenseexon8c.G818Ap.R273HNot-functionalC25PositiveLowDead4.0082NR131121_7T17579355A > TMissenseexon4c.T332Ap.L111QNot-functionalC65PositiveLowDead5.2783NR131121_6T17577142C > GMissenseexon8c.G796Cp.G266RNot-functionalC65PositiveHighDead1.0184NR131121_5T17577575A > CMissenseexon7c.T706Gp.Y236DNot-functionalC55PositiveLowAlive4.8085NR131121_4T17577117A > CMissenseexon8c.T821Gp.V274GNot-functionalC65PositiveHighDead0.5886NR131121_3T17577046C > ANonsenseexon8c.G892Tp.E298X——PositiveLowAlive5.5787NR131121_2T27578446T > AMissenseexon5c.A484Tp.I162FNot-functionalC0PositiveHighDead4.497578484G > TMissenseexon5c.C446Ap.S149YFunctionalC088NR131121_1T17577586A > TMissenseexon7c.T695Ap.I232NNot-functionalC35PositiveHighDead2.0389TW2352AF17578208T > CMissenseexon6c.A641Gp.H214RNot-functionalC0PositiveLowAlive7.8790TW2351BF17577085C > TMissenseexon8c.G853Ap.E285KNot-functionalC55PositiveHighAlive8.2891EC062T17578392C > ANonsenseexon5c.G538Tp.E180X——PositiveLowDead6.4992EC129T17578212G > ANonsenseexon6c.C637Tp.R213X——NegativeLowAlive10.3093EC127T17578239C > ANonsenseexon6c.G610Tp.E204X——NegativeLowDead8.4594MC16T17577609C > TSplice_Siteexon8c.673-1G >A———NegativeLowDead7.8095MC17T27578469C > AMissenseexon5c.G461Tp.G154VNot-functionalC65PositiveLowDead6.877579312C > TSilentexon4c.G375Ap.T125T——96MC13T17577090C > GMissenseexon8c.G848Cp.R283PNot-functionalC35PositiveHighDead4.2597MC7T27577106G > AMissenseexon8c.C832Tp.P278SNot-functionalC65PositiveHighDead0.757577538C > GMissenseexon7c.G743Cp.R248PNot-functionalC6598TW258AF17578406C > TMissenseexon5c.G524Ap.R175HNot-functionalC25PositiveHighDead0.6099FE0008T17577094G > AMissenseexon8c.C844Tp.R282WNot-functionalC65PositiveHighDead0.83100EC123T17573991C > ANonsenseexon10c.G1036Tp.E346X——PositiveLowDead4.06101G556T17579419AG > AFrame_Shift_Delexon4c.267delCp.P89fs——PositiveLowAlive2.65102G406T17577118C > AMissenseexon8c.G820Tp.V274FNot-functionalC45PositiveHighDead0.46103FG0067T27578449C > TMissenseexon5c.G481Ap.A161TPartially functionalC55PositiveHighAlive1.107579362A > CMissenseexon4c.T325Gp.F109VNot-functionalC0105G414T17578524G > ANonsenseexon5c.C406Tp.Q136X——NegativeLowAlive3.11106EC727T17577121G > AMissenseexon8c.C817Tp.R273CNot-functionalC65PositiveHighAlive3.14107EC720T17578275G > ANonsenseexon6c.C574Tp.Q192X——NegativeLowDead1.16109EC721T17578203C > TMissenseexon6c.G646Ap.V216MNot-functionalC15PositiveHighDead0.48110EC943T17578206T > CMissenseexon6c.A643Gp.S215GNot-functionalC0PositiveHighAlive3.79111EC121T17578271T > GMissenseexon6c.A578Cp.H193PNot-functionalC65PositiveHighDead1.00112EC854T27577124C > AMissenseexon8c.G814Tp.V272LNot-functionalC25PositiveHighAlive3.447578290C > GSplice_Siteexon7c.560-1G >C———113EC868T17577120C > TMissenseexon8c.G818Ap.R273HNot-functionalC25PositiveLowAlive3.44114EC686T27577124C > AMissenseexon8c.G814Tp.V272LNot-functionalC25PositiveLowAlive3.437578275G > ANonsenseexon6c.C574Tp.Q192X——118EC883T27577539G > AMissenseexon7c.C742Tp.R248WNot-functionalC65PositiveHighAlive3.497578192G > GGG​CAC​CAC​CAC​ACT​ATG​TCFrame_Shift_Insexon6c.656_657insGACATAGTGTGGTGGTGCCp.P219_Y220delinsPTX——119EC881T17577153C > AMissenseexon8c.G785Tp.G262VNot-functionalC65PositiveHighDead1.24120EC877T17578413C > AMissenseexon5c.G517Tp.V173LNot-functionalC25PositiveHighAlive3.51121EC880T17578443A > GMissenseexon5c.T487Cp.Y163HNot-functionalC65PositiveHighAlive3.47122EC122T17579485C > ANonsenseexon4c.G202Tp.E68X——NegativeLowAlive11.47124EC871T17577094G > AMissenseexon8c.C844Tp.R282WNot-functionalC65PositiveHighAlive3.51125EC874T17578211CGA > CFrame_Shift_Delexon6c.636_637delp.F212fs——NegativeLowAlive3.53128EC872T17577094G > AMissenseexon8c.C844Tp.R282WNot-functionalC65PositiveHighDead0.89129EC875T17574003G > ANonsenseexon10c.C1024Tp.R342X——PositiveLowDead1.99130EC869T17577106G > AMissenseexon8c.C832Tp.P278SNot-functionalC65PositiveHighDead1.53132G479T17578263G > ANonsenseexon6c.C586Tp.R196X——PositiveLowDead0.57133EC124T17577569A > TMissenseexon7c.T712Ap.C238SNot-functionalC65PositiveHighDead2.07134EC814T17579591C > ASplice_Siteexon5c.97-1G >T———NegativeLowAlive3.58135EC813T17578212G > ANonsenseexon6c.C637Tp.R213X——PositiveHighAlive3.58138EC822T17578190T > CMissenseexon6c.A659Gp.Y220CNot-functionalC65PositiveLowAlive3.95139EC818T27577094G > AMissenseexon8c.C844Tp.R282WNot-functionalC65PositiveLowAlive3.587578246CAA > CFrame_Shift_Delexon6c.601_602delp.L201fs——140EC821T17577538C > TMissenseexon7c.G743Ap.R248QNot-functionalC35PositiveHighAlive3.59142EC832T17577610T > CSplice_Siteexon8c.673-2A >G———NegativeLowDead0.25143EC851T17578212G > ANonsenseexon6c.C637Tp.R213X——NegativeLowAlive3.64144EC120T17577120C > TMissenseexon8c.G818Ap.R273HNot-functionalC25PositiveHighDead3.49145EC835T17578221TTC > TFrame_Shift_Delexon6c.626_627delp.R209fs——NegativeLowDead1.26146EC834T17579360G > GAFrame_Shift_Insexon4c.326dupTp.F109fs——NegativeLowAlive3.66147EC859T17577058C > ANonsenseexon8c.G880Tp.E294X——NegativeLowDead1.02149EC838T17577570C > TMissenseexon7c.G711Ap.M237INot-functionalC0PositiveHighAlive2.67150EC849T17578394T > CMissenseexon5c.A536Gp.H179RNot-functionalC25PositiveHighDead1.26151EC848T17578266TAAGATGCTG > Tnon_Frame_Delexon6c.574_582delp.192_194del——PositiveHighAlive3.73152EC847T17578263G > ANonsenseexon6c.C586Tp.R196X——NegativeLowAlive3.72153EC845T17578371C > TMissenseexon5c.G559Ap.G187SFunctionalC0NegativeLowAlive3.75154EC932T17579400G > GAAFrame_Shift_Insexon4c.286_287insTTp.S96fs——NegativeLowDead2.10155EC116T17577094G > AMissenseexon8c.C844Tp.R282WNot-functionalC65PositiveHighAlive11.53158EC933T17578440T > ANonsenseexon5c.A490Tp.K164X——NegativeLowDead3.20159EC941T17577609C > GSplice_Siteexon8c.673-1G >C———NegativeLowDead2.57160EC942T17577149AT > AFrame_Shift_Delexon8c.788delAp.N263fs——NegativeLowAlive3.78161EC945T17577082C > TMissenseexon8c.G856Ap.E286KNot-functionalC55PositiveHighAlive3.80162EC944T17577539G > AMissenseexon7c.C742Tp.R248WNot-functionalC65PositiveHighDead1.31165EC948T17576927C > TSplice_Siteexon10c.920-1G >A———NegativeLowAlive3.81166EC117T37577556C > AMissenseexon7c.G725Tp.C242FNot-functionalC65PositiveHighDead1.037577558G > ASilentexon7c.C723Tp.S241S——7578406C > TMissenseexon5c.G524Ap.R175HNot-functionalC25167EC146T17578395G > AMissenseexon5c.C535Tp.H179YPartially functionalC65PositiveLowDead3.25168EC940T17579591C > GSplice_Siteexon5c.97-1G >C———NegativeLowDead3.09171EC951T27577144A > CMissenseexon8c.T794Gp.L265RNot-functionalC65PositiveHighAlive3.857578446T > TGGCnon_Frame_Insexon5c.483_484insGCCp.I162delinsAI——172EC952T17579699C > TSplice_Siteexon4c.96+1G >A———NegativeLowAlive3.85173EC963T27576927C > TSplice_Siteexon10c.920-1G >A———NegativeLowAlive3.867578212G > ANonsenseexon6c.C637Tp.R213X——174EC958T17579470C > CGFrame_Shift_Insexon4c.216dupCp.V73fs——NegativeLowAlive3.86176EC961T27577121G > AMissenseexon8c.C817Tp.R273CNot-functionalC65PositiveHighAlive3.887578263G > ANonsenseexon6c.C586Tp.R196X——177EC962T17574029CG > CFrame_Shift_Delexon10c.997delCp.R333fs——PositiveLowAlive4.24178EC118T17578212G > ANonsenseexon6c.C637Tp.R213X——NegativeLowDead3.05180EC960T27578242C > AMissenseexon6c.G607Tp.V203LPartially functionalC0PositiveHighDead1.987576865A > CNonsenseexon9c.T981Gp.Y327X——182EC955T17578249AT > AFrame_Shift_Delexon6c.599delAp.N200fs——NegativeLowDead1.20184EC802T17578368CA > CSplice_Site InDelexon6c.559+2T > -———NegativeLowDead1.48185EC793T17577610T > GSplice_Siteexon8c.673-2A >C———NegativeLowAlive3.95186EC789T17576927C > ASplice_Siteexon10c.920-1G >T———NegativeLowAlive3.95187EC797T17578176C > TSplice_Siteexon7c.672+1G >A———NegativeLowAlive4.33188EC798T17576928T > CSplice_Siteexon10c.920-2A >G———NegativeLowAlive3.96189EC115T27577105G > AMissenseexon8c.C833Tp.P278LNot-functionalC65PositiveHighDead1.487578538T > AMissenseexon5c.A392Tp.N131INot-functionalC65191EC792T17574029CG > CFrame_Shift_Delexon10c.997delCp.R333fs——PositiveLowDead3.21192EC791T37578215A > CMissenseexon6c.T634Gp.F212VPartially functionalC0PositiveHighDead1.257577022G > ANonsenseexon8c.C916Tp.R306X——7579699C > ASplice_Siteexon4c.96+1G >T———193EC790T17578419C > ANonsenseexon5c.G511Tp.E171X——NegativeLowDead1.78194EC794T27578406C > TMissenseexon5c.G524Ap.R175HNot-functionalC25PositiveHighDead1.747578370CCA​TCG​CTA​TCT​GAG​CAG > CFrame_Shift_Delexon5c.543_559delp.R181fs——195EC783T17577121G > CMissenseexon8c.C817Gp.R273GNot-functionalC65PositiveHighAlive3.97197EC795T17578388C > GMissenseexon5c.G542Cp.R181PNot-functionalC65PositiveHighDead1.51198EC787T17577547C > TMissenseexon7c.G734Ap.G245DNot-functionalC65PositiveHighDead0.84199G521T17579311C > ASplice_Siteexon5c.375+1G >T———NegativeLowAlive3.63200EC113T27577518T > AMissenseexon7c.A763Tp.I255FNot-functionalC15PositiveLowAlive11.547578212G > ANonsenseexon6c.C637Tp.R213X——201EC780T17577093CG > CFrame_Shift_Delexon8c.844delCp.R282fs——NegativeLowDead1.96202EC786T17578271T > CMissenseexon6c.A578Gp.H193RNot-functionalC65PositiveHighAlive4.05203EC781T17578524G > CMissenseexon5c.C406Gp.Q136ENot-functionalC25PositiveHighDead1.33205EC779T17577120C > TMissenseexon8c.G818Ap.R273HNot-functionalC25PositiveHighAlive4.42206EC766T17577035TG > TFrame_Shift_Delexon8c.902delCp.P301fs——PositiveLowDead1.61207EC769T17577106G > TMissenseexon8c.C832Ap.P278TNot-functionalC35PositiveHighAlive4.46208EC807T17578454G > AMissenseexon5c.C476Tp.A159VNot-functionalC0PositiveHighAlive4.11209EC900T17579414C > TNonsenseexon4c.G273Ap.W91X——NegativeLowAlive4.11210EC898T17578212G > ANonsenseexon6c.C637Tp.R213X——NegativeLowAlive4.13212EC904T17577142C > TMissenseexon8c.G796Ap.G266RNot-functionalC65PositiveHighAlive4.12214EC908T17574018G > AMissenseexon10c.C1009Tp.R337CNot-functionalC45PositiveHighDead1.88215EC907T17577124C > AMissenseexon8c.G814Tp.V272LNot-functionalC25PositiveHighDead15.80217EC902T17577094G > CMissenseexon8c.C844Gp.R282GNot-functionalC65PositiveLowAlive4.15218EC906T17578437G > ANonsenseexon5c.C493Tp.Q165X——Not detectedNot detectedAlive4.51222TW094BF27576857A > CMissenseexon9c.T989Gp.L330RNot-functionalC65PositiveLowDead2.747578176C > ASplice_Siteexon7c.672 + 1G >T———223G042_T17578427T > CMissenseexon5c.A503Gp.H168RNot-functionalC25NegativeLowAlive4.16226EC909T17578177C > TSilentexon6c.G672Ap.E224E——NegativeLowAlive3.12227EC916T17578395G > AMissenseexon5c.C535Tp.H179YPartially functionalC65PositiveLowAlive4.18229EC913T17578249AT > AFrame_Shift_Delexon6c.599delAp.N200fs——NegativeLowDead3.05231EC920T17578442T > CMissenseexon5c.A488Gp.Y163CNot-functionalC65PositiveHighDead2.15232EC918T17578406C > TMissenseexon5c.G524Ap.R175HNot-functionalC25PositiveHighDead2.21233TW890BF17577121G > AMissenseexon8c.C817Tp.R273CNot-functionalC65PositiveLowDead2.35236EC921T17579592T > ASplice_Siteexon5c.97-2A >T———NegativeLowDead2.00238EC924T17577121G > AMissenseexon8c.C817Tp.R273CNot-functionalC65PositiveHighDead3.30240EC684T17578223CT > CFrame_Shift_Delexon6c.625delAp.R209fs——NegativeLowDead3.84241EC682T17577152AC > AFrame_Shift_Delexon8c.785delGp.G262fs——NegativeLowAlive4.42243EC680T17579419AG > AFrame_Shift_Delexon4c.267delCp.P89fs——NegativeLowAlive4.42244EC055T17577124C > TMissenseexon8c.G814Ap.V272MNot-functionalC15PositiveHighDead0.88245EC681T17577610T > ASplice_Siteexon8c.673-2A >T———NegativeLowAlive4.44247EC676T17579311C > TSplice_Siteexon5c.375+1G >A———NegativeLowDead0.98248EC677T17576851A > GSplice_Siteexon10c.993+2T >C———NegativeLowAlive4.44249EC672T17576851A > GSplice_Siteexon10c.993+2T >C———NegativeLowDead1.83250EC666T17578212G > ANonsenseexon6c.C637Tp.R213X——NegativeLowDead1.54251EC660T17579358C > AMissenseexon4c.G329Tp.R110LNot-functionalC25PositiveHighAlive4.50253EC659T17578479G > AMissenseexon5c.C451Tp.P151SNot-functionalC65PositiveHighAlive4.87254EC1002T17577094G > AMissenseexon8c.C844Tp.R282WNot-functionalC65PositiveHighAlive4.65258EC651T17578413C > AMissenseexon5c.G517Tp.V173LNot-functionalC25PositiveHighAlive4.53259EC645T17579528C > TNonsenseexon4c.G159Ap.W53X——NegativeLowAlive4.55260EC928T27577141C > TMissenseexon8c.G797Ap.G266ENot-functionalC65PositiveLowAlive4.587578263G > ANonsenseexon6c.C586Tp.R196X——261EC926T17578265A > ATFrame_Shift_Insexon6c.583dupAp.I195fs——NegativeLowAlive4.96262EC929T27579355A > GMissenseexon4c.T332Cp.L111PNot-functionalC65PositiveHighDead1.547579354C > ASilentexon4c.G333Tp.L111L——263EC927T17578175A > GSplice_Siteexon7c.672+2T >C———NegativeLowDead1.99265EC038T17579335TC > TFrame_Shift_Delexon4c.351delGp.G117fs——NegativeLowDead1.47266EC930T17578265A > TMissenseexon6c.T584Ap.I195NNot-functionalC65PositiveHighAlive4.91268EC1000T27577105G > CMissenseexon8c.C833Gp.P278RNot-functionalC65PositiveLowAlive4.647578406C > TMissenseexon5c.G524Ap.R175HNot-functionalC25269EC999T17578469C > AMissenseexon5c.G461Tp.G154VNot-functionalC65PositiveLowAlive4.64270EC1001T27578413C > TMissenseexon5c.G517Ap.V173MNot-functionalC15PositiveHighDead1.927578263G > ANonsenseexon6c.C586Tp.R196X——271G017_T17578212G > ANonsenseexon6c.C637Tp.R213X——NegativeLowAlive4.70272EC894T17577094G > AMissenseexon8c.C844Tp.R282WNot-functionalC65PositiveHighAlive4.68274EC895T17578265A > GMissenseexon6c.T584Cp.I195TNot-functionalC55PositiveHighDead2.11275EC890T17578245G > GCFrame_Shift_Insexon6c.603dupGp.R202fs——NegativeLowAlive4.69277EC027T17578526C > AMissenseexon5c.G404Tp.C135FNot-functionalC65PositiveLowAlive11.89278TW258BF17577609C > TSplice_Siteexon8c.673-1G >A———Not detectedNot detectedDead1.39279EC114T17577144A > CMissenseexon8c.T794Gp.L265RNot-functionalC65Not detectedNot detectedAlive10.78280EC099T17578384G > TNonsenseexon5c.C546Ap.C182X——Not detectedNot detectedDead1.76281TW1375BF17578263G > ANonsenseexon6c.C586Tp.R196X——Not detectedNot detectedAlive9.72283EC107T17579388T > TGFrame_Shift_Insexon4c.298dupCp.Q100fs——Not detectedNot detectedDead3.06285EC188T17579315G > GCFrame_Shift_Insexon4c.371dupGp.C124fs——Not detectedNot detectedDead1.93288EC081T17578265A > GMissenseexon6c.T584Cp.I195TNot-functionalC55Not detectedNot detectedDead2.62290EC145T17578413C > TMissenseexon5c.G517Ap.V173MNot-functionalC15Not detectedNot detectedAlive2.88291EC131T27578413C > AMissenseexon5c.G517Tp.V173LNot-functionalC25Not detectedNot detectedAlive12.357574000C > ANonsenseexon10c.G1027Tp.E343X——292TW470BF17577544A > TMissenseexon7c.T737Ap.M246KNot-functionalC65Not detectedNot detectedAlive15.36293EC086T17579372GC > GFrame_Shift_Delexon4c.314delGp.G105fs——Not detectedNot detectedAlive11.00295EC126T17579470CG > CFrame_Shift_Delexon4c.216delCp.P72fs——Not detectedNot detectedAlive11.37296EC040T27577568C > GMissenseexon7c.G713Cp.C238SNot-functionalC65Not detectedNot detectedAlive8.247577022G > ANonsenseexon8c.C916Tp.R306X——297TW1371BF17578483GGAAT > GFrame_Shift_Delexon5c.443_446delp.D148fs——Not detectedNot detectedDead2.22298TW1371AF17578271T > AMissenseexon6c.A578Tp.H193LNot-functionalC65Not detectedNot detectedDead0.92299MC34T17577538C > AMissenseexon7c.G743Tp.R248LNot-functionalC65Not detectedNot detectedDead7.54301CE2T17578205CTA > CFrame_Shift_Delexon6c.642_643delp.H214fs——Not detectedNot detectedAlive13.37302MC29T17579345CA > CFrame_Shift_Delexon4c.341delTp.L114fs——Not detectedNot detectedDead4.26303EC034T17577539G > AMissenseexon7c.C742Tp.R248WNot-functionalC65Not detectedNot detectedDead6.17305TW2309BF17577022G > ANonsenseexon8c.C916Tp.R306X——Not detectedNot detectedAlive11.74306EC128T17578406C > TMissenseexon5c.G524Ap.R175HNot-functionalC25Not detectedNot detectedDead4.00307EC192T17578395G > AMissenseexon5c.C535Tp.H179YPartially functionalC65Not detectedNot detectedDead2.78310MC26T17578265A > GMissenseexon6c.T584Cp.I195TNot-functionalC55Not detectedNot detectedDead1.66311EC060T17578179C > ANonsenseexon6c.G670Tp.E224X——Not detectedNot detectedAlive6.87312TW829AF17579315G > GCFrame_Shift_Insexon4c.371dupGp.C124fs——Not detectedNot detectedAlive14.09313TW1099AF27578208T > CMissenseexon6c.A641Gp.H214RNot-functionalC0Not detectedNot detectedAlive11.027579485C > ANonsenseexon4c.G202Tp.E68X——314EC094T27577539G > AMissenseexon7c.C742Tp.R248WNot-functionalC65Not detectedNot detectedDead5.707576928T > ASplice_Siteexon10c.920-2A >T———316EC012T17579315G > GCFrame_Shift_Insexon4c.371dupGp.C124fs——Not detectedNot detectedDead1.36Notably, we found the frequency of nine protein mutations of p53 protein, including four hotspot mutations (p.R175, p.R248, p.R273, and p.R282) and five nonhotspot mutations (p.V173, p.H179, p.R196, p.R213, and p.P278) [21, 22], were more than five, respectively. Hotspot mutations comprised 29.0% (42/145) of all p53 missense mutations (Figure 4(d)).


## 3.3. Correlation of p53 Protein Expression with TA Class and Align GVGD Classifications

To speculate on the effect of the protein function of theTP53 missense mutation sites, we submitted queries to the IARC TP53 Database (https://p53.iarc.fr) [23–25]. According to TA classification, 145 TP53 missense mutation sites were divided into three categories: functional (4/145, 2.7%), partially functional (11/145, 7.6%), and not-functional (130/145, 89.7%) based on the TA class of the protein function (Table 4). We further observed that four of the 145 sites (in 135 patients) with missense mutations didn’t affect the function of the wild-p53 protein. However, only one of the four sites was a single mutation, and its p53 protein expression was negative. The other three sites were multiple mutations, and their p53 protein expression was positively detected (two were high, and one was low). Among the 130 sites (126 patients) with not-functional (108 patients with p53 protein expression tests), 105 patients were positive p53 protein expression (105/108, 97.2%), including 74 patients with p53 protein high expression (74/108, 68.5%). The p53 protein expression level is consistent with the predicted function based on the mutation sites. In the Align GVGD classification, we did not observe a significant difference in the positive rate of p53 protein in each group.


## 3.4. Association betweenTP53 Mutation and Protein Expression with Clinicopathological Parameters and Survival in ESCC

In the first cohort, we did not find any remarkable association between theTP53 mutation and these clinicopathological characteristics in ESCC (Table 2). Although there was no statistically significant difference in survival time when comparing the patients with and without TP53 mutations (Figure 5(a)), surprisingly, the results showed that the patients who had high protein expression of p53 exhibited significantly worse survival than those with low expression when the population narrow down to 276 patients (p=0.002, Figure 5(b)). The median survival time for 108 patients with high protein expression of p53 and for 168 patients who had low expression was 2.79 ± 0.63 and 5.27 ± 0.52 years, respectively. In addition, no significant difference was observed in the survival time of different mutation types of TP53 (p>0.05, Figure 5(c)), and no significant difference was observed in survival time between the hotspot and nonhotspot in TP53 missense mutations patients (p>0.05, Figure 5(d)).Figure 5
Kaplan–Meier curves survival analysis of ESCC patients with differentTP53 mutations and p53 protein expression. (a) OS of TP53 mutation and wild-type in 316 patients with sequencing data. (b) OS of p53 protein high and low expression in 276 patients who have TP53 mutation and p53 protein expression. (c) OS of different types of mutation in 316 patients with sequencing data. (d) OS of nonhotspot and hotspot mutation in 135 patients who have missense mutation. (e) OS of different groups of TP53 mutation status/p53 protein expression in 276 patients who have TP53 mutation and p53 protein expression. (f) OS of p53 protein high and low expression in 6,028 patients who have p53 expression in TMAs.
(a)(b)(c)(d)(e)(f)To better understand the factors which contribute to the association betweenTP53 mutations/high p53 expression in patients and survival, we compared the mutation types of p53 patients with ESCC. The high expression of the p53 protein was 66.9% among the 121 patients with TP53 gene missense mutations and only 5.6% in the 36 cases with nonsense mutations. Other types of mutations in the 55 patients (including frame shift, silent, and splice) were 3.6%. Apparently, missense mutation was most likely the cause of p53 protein mutation, leading to the high expression of p53 protein. Other mutation types were possible causes of low expression of the p53 protein. Surprisingly, in the 64 cases of TP53wild-type, high expression of p53 protein accounted for 35.9%, while low expression was 64.1% (41 patients, of which 26 were not expressed at all) (Figure 4(e) and Table 5).Table 5
Association betweenTP53 mutation and p53 protein expression in 276 ESCC patients.
p53 protein expressionTP53 MutationTP53wild-type, n = 64Missense,n = 121Nonsense,n = 36Others,n = 55Totalp53 high/low expressionp53 high expression81228523p53 low expression40345312741p53 positive/negative expressionp53 expression (+)117111013838p53 expression (−)425457426Note. When a missense mutation occurs, patients with multiple mutations are preferentially included in the missense mutation group. When a nonsense mutation occurs, the remaining patients are preferentially included in the nonsense mutation group.


## 3.5. A New Classification for Evaluating the Association betweenTP53 Mutation/p53 Protein and Survival

To address the controversial role ofTP53 mutation in ESCC, in this study, we established a new classification for evaluating TP53-related survival in ESCC by combining TP53 mutations and p53 protein expression analysis. The results clearly showed that 276 ESCC patients were divided into four groups as follows: TP53 mutation/p53 high expression (85/276, 30.8%), TP53 mutation/p53 low expression (127/276, 46.0%), TP53 wild-type/p53 high expression (23/276, 8.3%), TP53 wild-type/p53 low expression (41/276, 14.9%) (Table 5). Furthermore, the total survival time of the four groups was significantly different (p<0.01), among which the TP53 mutation/p53 high expression group had the worst survival time, followed by the TP53 wild-type/p53 high expression group (Figure 5(e)).


## 3.6. Association between p53 Protein Expression and Clinicopathological Changes in Cohort with Large-Scale ESCC Patients

To further validate our discovery of the association between the p53 expression-related prognosis and clinicopathological changes, we assessed additional cohorts to make an analysis (Table6). The positive expression of p53 protein was observed in 3,562 (59.1%) ESCC patients, of which 1,819 patients (51.1%) showed high expression of p53 protein. Moreover, the high expression of p53 protein in high-incidence areas was more common than in low-incidence areas with a rate and p value of 31.5% vs. 27.7%, p=0.003. High expression of p53 protein was closely correlated with poor tumor differentiation (p<0.001). The frequency of high expression of p53 protein in the early ESCC (0 + I stage) was 1.27-fold higher than that in the advanced ESCC (38.2% vs. 30.0%, p=0.034). Furthermore, the rate of positive cancer embolus in high-expression groups was 1.31-fold higher than in low-expression groups (5.1% vs. 3.9%, p=0.028).Table 6
Association between p53 protein expression and clinicopathologic features in patients with ESCC in tissue microarray.
CharacteristicsAll patients,p53 protein expressionp valueLow expression,High expression,n = 6028n = 4209(%)n = 1819(%)Sex0.525Female2107148270.362529.7Male3921272769.5119430.5Age at diagnosis0.492≤602917204970.286829.8>603111216069.495130.6High/low incidence area0.003Low2119153172.358827.7High3909267868.5123131.5Cigarette smoking0.393Negative3160221770.294329.8Positive2731188869.184330.9Alcohol consumption0.901Negative4103285769.6124630.4Positive1791125069.854130.2Family history0.615Negative3875270369.8117230.2Positive2037140869.162930.9Location0.667Cervical + upper95467871.127628.9Middle4092284269.5125030.5Lower87961369.726630.3Mix513364.71835.3Differentiation<0.001Well differentiated50138576.811623.2Moderate differentiated3303235071.195328.9Poor differentiated1935128066.165533.9Pathological T stage0.335Tis + T120513465.47134.6T21466102269.744430.3T34242297870.2126429.8T41157565.24034.8Pathological N stage0.494N03354235470.2100029.8N12674185569.481930.6Pathological M stage0.930M05814405969.8175530.2M121415070.16429.9UICC stage (6th)0.1440 + I1448961.85538.2II3691260070.4109129.6III1979137069.260930.8IV21415070.16429.9Cancer embolus0.028Negative5772404670.1172629.9Positive25616363.79336.3Type of treatment0.596Surgical5006349969.9150730.1Surgical + chemo49335271.414128.6Surgical + radio39526968.112631.9Surgical + chemo + radio1348966.44533.6


## 3.7. Independent Prognosis Marker Role of p53 Protein on Survival Analysis in 6,028 ESCC Patients

To determine whether a high expression level of the p53 protein could be used as a prognosis marker in patients with ESCC, we conducted the analysis using the p53 expression in 6,028 ESCC patients. In this cohort, the analysis showed median OS time in patients with p53 high expression and with low was 2.71 ± 0.09 and 3.08 ± 0.06 years, respectively. The presence of high expression of p53 protein was significantly associated with decreased OS (p<0.001, Figure 5(f)).


## 3.8. Cox Univariate and Multivariate Regression Analyses

Cox proportional-hazard models were chosen for the univariate and multivariate analyses. The univariate Cox regression analysis demonstrated that the sex, age, high/low incidence area, cigarette smoking, alcohol consumption, location, differentiation, T stage, N stage, M stage, UICC stage, cancer embolus, and p53 protein high/low expression were dependent prognostic factors (p<0.05, Figures 6(a), 6(b), and 7). In multivariate Cox regression analysis, as compared with the low expression of p53 protein, the patients with high expression (HR = 1.134, 95% CI 1.065 to 1.207) were associated with decreased survival after adjustment for the age, high/low incidence area, cigarette smoking, differentiation, and T and N stages as independent prognostic factors (p<0.05, Figure 6(c)).Figure 6
Forest plot demonstrating univariate and multivariate analysis of factors that influence OS in 6,028 ESCC patients. The results of the univariate analysis of factors that influence OS in 6,028 ESCC patients are shown in (a) and (b). The results of the multivariate analysis of factors that influence OS in 6,028 ESCC patients are shown in (c).
(a)(b)(c)Figure 7
Kaplan–Meier curves analysis estimating OS following stratifying 6,028 patients with ESCC according to sex (a), age at diagnosis (b), high/low incidence area (c), cigarette smoking (d), alcohol consumption (e), family history (f), location (g), differentiation (h), T stage (i), N stage (j), M stage (k), UICC stage (l), cancer embolus (m), and treatment (n), respectively.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)(k)(l)(m)(n)


## 3.9. Stratified Survival Analysis by within Independent Prognostic Factors

We performed a stratified analysis to eliminate the influence of the independent prognostic factors on evaluating the expression of the p53 protein on prognosis. When patients were stratified according to high/low incidence area, cigarette smoking, and N stages, the consistent trend of OS was observed in different stratification (Figures8(a)–8(f)). When stratified according to patients’ age at diagnosis, degree of differentiation, and T stage, we only found that patients the high expression of p53 protein in >60 (p<0.001, Figure 8(g)), medium and low differentiation (p<0.001, Figures 8(h) and 8(i)), and T3 (p<0.001, Figure 8(j)) had shorter OS than those with low expression.Figure 8
Kaplan–Meier curves survival analysis of ESCC patients with high/low p53 protein expression after stratification based on independent prognostic factors. OS of patients with p53 protein high vs. low expression in patients with high-incidence area (a) and low-incidence area (b). OS of patients with p53 protein high vs. low expression in patients with (c) and without cigarette smoking (d). OS of patients with p53 protein high vs. low expression in patients with N0 (e) and N1 stage (f). OS of patients with p53 protein high vs. low expression in patients >60 years (g). OS of patients with p53 protein high vs. low expression in patients with moderate (h) and poor differentiation (i). OS of patients with p53 protein high vs. low expression in patients with T3 stage (j).
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)


## 4. Discussion

We have conducted a two-large ESCC cohort analysis to evaluate the association betweenTP53 mutation statuses/the expression of the p53 protein and clinicopathological features and prognosis in ESCC patients. Most importantly, our results further confirmed that the expression of the p53 protein can reflect the prognosis of ESCC patients by using a large number of tissue samples, and high p53 expression indicates a poor prognosis. However, the mutation of the TP53 gene has no obvious correlation with prognosis, which is controversial in previous reports.With the rapid development of sequencing technology, the prognostic value ofTP53 has been confirmed in a variety of tumors, and the mutation of TP53 indicates a poor prognosis across different types of human cancers. In this study, despite the high frequency of TP53 mutations, there is no obvious evidence showing its association with clinicopathological features and prognosis in patients with ESCC. Previously, other colleagues showed that TP53 status was correlated with tumor invasion depth, TNM stage, lymph node metastasis, distant metastasis, and differentiation degree [15]. Meanwhile, the survival time of TP53 gene mutation and p53 protein overexpression was shorter than the control group in ESCC [15]. However, Zhao et al. showed that the high expression of p53 protein was an independent prognostic factor for survival, while the mutation of the TP53 gene was unrelated to prognosis [26]. The conflicting results might be caused by the following reasons: of the limited number of patients, insufficient clinical follow-up, different experimental techniques, mutation sites on different target exons, and variable factors.We further found that the frequency of theTP53 mutation was 76.3% in ESCC, which was between 66.7% and 82.7% of the frequency of the TP53 mutation in EC by NGS, as previously reported [27–30]. Cui et al. published WGS results for 508 cases of ESCC, which also showed a similar TP53 mutation frequency of 74.8% [30]. The cause might be that the study population and histopathological type were different. Our study confirmed that the main mutation type of TP53 in ESCC patients is a missense mutation, which may play a pivotal role in tumorigenesis because the p53 protein usually has positive expression, or even higher expression, owing to the accumulation of a nonfunctional protein that loss activity as a tumor suppressor, and some of which exert trans-dominant repression over the wild-type counterpart [31, 32]. C > T/G > A Transition is the predominant mutation of TP53 gene mutations in our study, and it is a typical marker associated with betel quid chewing, tobacco use, and alcohol drinking in oral squamous cell carcinoma in Taiwanese [33]. C > A/G > T transversion is a typical feature of carcinogen exposure associated with cigarette smoking, which is the most risk factor for ESCC and lung cancer. This transversion occurred at the sites of adduct formation for the metabolites of benzo (a) pyrene, a major tobacco carcinogen (codons 157, 248, and 273) [34, 35].To our knowledge, this is the largest sample-scale study that has examined p53 protein expression by IHC. Our study herein showed that the expression of p53 protein was associated with high/low incidence area, degree of differentiation, and cancer embolus, and revealed that high expression of p53 protein is an independent prognostic factor rather thanTP53 gene mutation, with 1.134-fold mortality risk. In previous studies, the correlation between p53 protein expression, clinicopathological features, and prognosis was significantly different [36–40]. To date, only 13 of the 30 articles have suggested that p53 protein was an adverse factor for prognosis, and the rest of the reports suggested that p53 expression had no effect on prognosis [39]. Wang et al. revealed that a more advanced TNM stage, positive lymph node metastasis, and distant metastasis were associated with p53 protein expression [41]. Although there were no significant differences between p53 expression and T stage, N stage, and M stage in our finding, our further validation of 6,028 patients demonstrated a significantly strong correlation between the expression of p53 protein and clinical phenotype and prognosis. The judgment for the p53 protein expression is different because of the variety of using antibodies and experimental methods used in IHC. In addition to the source of the tumors and the existence of tumor heterogeneity, the difference in evaluation criteria is the most critical factors leading to inconsistent conclusions in previous studies [36, 37, 40].Our study showed that the expression of p53 protein in the high-incidence areas of EC was significantly higher than in low. The existence of high/low incidence areas of EC is one of the prominent epidemiological features of ESCC, suggesting that environmental factors play an important role in the pathogenesis of ESCC. In high-incidence areas of EC, exposure to environmental carcinogens (such as nitrite and mold-contaminated foods) may lead to carcinogenesis of the esophageal squamous epithelium.TP53 is one of the most susceptible genes to environmental carcinogens in the process of tumor induction. Aflatoxin B1 (AFB1) are potent carcinogens, which induces an arginine to serine at a single base substitution at the third base of codon 249 in TP53 (G > T transversion) [42, 43]. N-nitroso compounds (NOC) are an alkylating agent and cause guanine alkylation to generate O6-alkylguanine, which results in C > T/G > A transition during DNA replication when paired with thymine [44–46]. The DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) specifically repairs O6-alkylguanine adducts in DNA. However, AGT mutations occurred more frequently in patients in high-incidence areas of esophageal cancer than in the normal population [47].In ESCC, it is not feasible to determine the presence ofTP53 gene mutations through the loss of p53 protein expression. When the TP53 gene is mutated, it will cause different changes in proteins. Nonsense, frameshift, silent, and splice mutations usually cause protein truncation and make the synthesis of normal p53 protein interfere in order to cause LOF, and p53 protein is not expressed either [21]. The situation became complicated when a missense mutation occurred because the p53 protein can be caused by various missense mutations. Some will still have the function of wild-type p53 protein, and some will only retain part of the function and acquire gain GOF, which is associated with malignancy, invasion, and metastasis [48]. Due to the broad existence of LOF and GOF of the TP53 mutation, false-positive or false-negative levels of p53 protein will be detected by using IHC in clinical applications, which might not be a reliable method for evaluating the function of the TP53 mutation [49]. Our observation confirmed that immunohistochemical detection of p53 protein expression could more effectively reflect the relationship with prognosis and clinicopathologic features than the mutation status of the TP53 gene in ESCC, which was further supported by studies with a larger sample.The limitation of this study is the representativeness and accuracy of the application of TMA-based IHC showing the expression of p53 protein with extreme heterogeneity. To conclude, laboratory techniques need to be used to detect p53 protein in TMAs, and the criterion of IHC evaluation must be constant. Limited tissue sampling cannot effectively reflect the real status of the whole section because of tumor heterogeneity. Our research simultaneously detected p53 protein in a part of the original pathological sections made TMAs to reduce potential limits, and used the same scoring criteria for judgment. The results are no different from those in TMAs.In conclusion,TP53 is the most mutated gene in ESCC, showing great potential to be a diagnostic biomarker in treating cancer. However, the role of TP53 mutation and expression of p53 in ESCC still remains unclear. Our study, involving two large-scale cohorts, was the first to conduct genomic profiling and expression of the p53 protein, while demonstrating the association with clinical phenotypes and prognosis. Our findings show that the expression of p53 protein is more effective in predicting clinicopathological features and prognosis, is a valid biomarker of an unfavorable prognosis, and will contribute to clinical diagnosis, prognosis prediction, and targeted therapy.



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