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Abstract
Aims Colorectal cancer (CRC) is the third most common malignancy worldwide. Accurate pathological diagnosis and predictive abilities for treatment response and prognosis are crucial for patients with CRC. This study aims to analyse the expressions of p21 and EGFR in CRC and their relationships with clinicopathological characteristics and prognosis to enhance diagnostic and prognostic evaluations.
Methods This study conducted a retrospective analysis of p21 and EGFR expressions in 12 319 Chinese patients with CRC using immunohistochemistry. The relationships between these expressions and clinicopathological characteristics and survival outcomes were explored through statistical and survival analyses.
Results Differential expressions of p21 and EGFR in CRC were closely related to clinicopathological characteristics and significantly impacted overall survival (OS). p21 expression was associated with the primary tumour site, mucinous subtype, lymphovascular invasion, perineural invasion, circumferential resection margin, T stage, N stage, tumour, node, metastases (TNM) stage, and mismatch repair status. EGFR expression was related to mucinous subtype, tumour differentiation, lymphovascular invasion, perineural invasion, tumour size, T stage, N stage, TNM stage and BRAF gene mutation. p21 and EGFR expressions were positively correlated (r=0.11). High p21 expression correlated with favourable OS, whereas high EGFR expression predicted poorer OS. A prognostic nomogram incorporating these biomarkers and clinical variables demonstrated robust predictive power for patient survival rates.
Conclusion p21 and EGFR serve as potential indicators for pathological diagnosis, risk stratification, and predicting treatment efficacy and prognosis in patients with CRC. The study’s findings provide valuable references for personalised treatment and prognosis evaluation in clinical practice.
- COLORECTAL CANCER
- EGFR
- Colorectal Neoplasms
- Pathology, Surgical
Data availability statement
Data are available upon reasonable request.
This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/.
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WHAT IS ALREADY KNOWN ON THIS TOPIC
Colorectal cancer (CRC) is a prevalent malignancy globally, necessitating accurate pathological diagnosis and prognosis prediction. p21 regulates the cell cycle and immunosurveillance, while EGFR plays a crucial role in cell proliferation, invasion and angiogenesis, with its overexpression linked to tumour growth and poor prognosis. The specific implications of these biomarkers in CRC, particularly regarding clinicopathological characteristics and prognosis in large patient cohorts, required further investigation.
WHAT THIS STUDY ADDS
This study, involving 12 319 Chinese patients with CRC, reveals that differential expressions of p21 and EGFR are significantly associated with clinicopathological features and overall survival. A positive correlation between p21 and EGFR was also identified. The study demonstrates that a prognostic nomogram incorporating these biomarkers and clinical variables has strong predictive power for patient survival rates.
HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY
The findings highlight the potential of p21 and EGFR as biomarkers for CRC diagnosis, risk stratification and prognosis. These biomarkers can guide personalised treatment strategies, improving patient outcomes, and emphasise the importance of integrating molecular and clinicopathological data in CRC management.
Introduction
Colorectal cancer (CRC) is the third leading malignancy on a global scale,with than 1.9 million new cases annually and an alarming increase in both incidence and mortality.1 CRC commonly occurs in middle-aged people, with about 60–65% of cases being sporadic and 35–40% attributed to genetic factors.2 Surgical treatment is the primary option for early-stage CRC, and the advent of molecularly targeted and immunotherapeutic agents has significantly enhanced clinical outcomes.3 However, early symptoms of CRC are often subtle, and the disease is usually advanced by the time symptoms like bloody stools and abdominal pain appear, with no standard methods to predict treatment efficacy and prognosis.4 Therefore, providing precise pathological diagnosis and improving predictive abilities for treatment response and prognosis in patients with CRC are crucial.
The academic inquiry of cancer development has entered the stages of molecular biology and genetics. p21 and EGFR, key molecules in cell regulation, have received significant scholarly focus due to their implications in tumour genesis and progression. p21 is a cyclin-dependent kinase inhibitor with immunosurveillance functions in senescent cells.5 EGFR, a transmembrane tyrosine kinase receptor, plays a crucial role in signalling pathways and is involved in cell proliferation, invasion and angiogenesis.6
This study focuses on the clinicopathological significance and prognostic analysis of p21 and EGFR in CRC, aiming to provide references for pathological diagnosis and prognosis in patients with CRC.
Materials and methods
Patient selection
This study conducted a retrospective analysis of pathological diagnostic reports and immunohistochemistry reports from 12 319 patients with confirmed postoperative pathology of CRC at the Fudan University Shanghai Cancer Center in China between January 2008 and December 2020. These data were retrieved from the hospital’s case archive and formatted for analysis. Inclusion criteria: (1) patients aged 18 years and above; and (2) patients with CRC as the primary site of the tumour, including those with metastases. Exclusion criteria: (1) patients with primary tumours originating from other organs that have metastasised to the colon; and (2) patients with autoimmune diseases. Tumour, node, metastases (TNM) staging was performed using the eighth edition of the American Joint Committee on Cancer staging standards.
Patients were followed up post-surgery through regular clinic visits or phone calls until September 2022, with a follow-up duration of 0.6–281 months and a median follow-up time of 47.6 months. Overall survival (OS) was used as a prognostic indicator.
Pathological evaluation
Pathological slides were reviewed by two physicians. Immunohistochemistry was scored based on the intensity of staining and the percentage of positive cells, classified as negative, weakly positive and positive. Clear absence of discernible staining is indicative of negativity. Faint, focal light brown staining observed in some cells represents weak positivity, while widespread, brown-yellow staining seen throughout the cells signifies positivity. Samples that failed the analysis due to insufficient or low-quality material were excluded.
Statistical analysis
Data were analysed using SPSS (V.25.0). Differences in counts data between groups were compared using the χ2 test. Correlation analysis for ordered categorical variables was conducted using Spearman’s correlation analysis. Kaplan-Meier survival curves and log-rank tests were used for prognostic analysis. On the basis of univariate and multivariate Cox regression analyses, a nomogram prediction model was constructed using R (V.4.2.1). All statistical tests were two sided, and p<0.05 was considered statistically significant.
Results
Expression levels and distribution of p21 and EGFR in patients with CRC
Staining intensity varies in correlation with the expression levels of tumour cells. Based on the depth of the immunostaining colour and the proportion of positive cells, the expression levels of p21 and EGFR were categorised as negative, weakly positive and positive. Representative histological images and the distribution of p21 and EGFR expression are shown in figure 1. p21 staining is primarily located in the nuclei, whereas EGFR staining mainly occurs on the cell membrane. In the negative group, no discernible staining is observed; in the weakly positive group, some cells exhibit faint, focal light brown staining; and in the positive group, widespread brown-yellow staining is generally visible. In our study, p21 expression was negative in 33% of CRC samples, weakly positive in 9% of the samples and positive in 58% of the samples. For EGFR protein, the proportions were 40% negative, 21% weakly positive and 39% positive.
Representative histological images (A) and distribution analysis (B) of p21 and EGFR in patients with colorectal cancer.
Correlation of p21 and EGFR expressions with clinicopathological characteristics
This study collected 12 319 colorectal cancer specimens, including 4926 males and 7393 females, aged 18–95 years, with an average age of 59.46±12.08 years. The results showed that the differential expressions of p21 and EGFR are closely related to the clinicopathological characteristics of CRC (table 1).
Relationship between p21, EGFR expression and clinicopathological characteristics of colorectal cancer
The differential expression of p21 was associated with primary tumour site, mucinous subtype, lymphovascular invasion, perineural invasion, circumferential resection margin, T stage, N stage, TNM stage and mismatch repair of patients with CRC (p<0.05), but not with age, gender, tumour differentiation, tumour size, M stage, microsatellite instability, KRAS gene, NRAS gene and BRAF gene mutation (p>0.05).
The differential expression of EGFR was related to mucinous subtype, tumour differentiation, lymphovascular invasion, perineural invasion, tumour size, T stage, N stage, TNM stage and BRAF gene mutation (p<0.05), but not with age, gender, primary tumour site, circumferential resection margin, M stage, mismatch repair, microsatellite instability, KRAS gene and NRAS gene mutation (p>0.05).
Expression concordance between p21 and EGFR in CRC
In this group, 916 patients showed co-negativity, and 2105 patients co-positivity for p21 and EGFR. Spearman’s correlation analysis revealed a positive correlation between p21 and EGFR expression (r=0.11) with statistical significance (p<0.001) (table 2).
Spearman’s correlation analysis of p21 and EGFR levels
Survival outcomes associated with p21 and EGFR expressions
Postoperative follow-up classified patients into low expression (negative expression) and high expression (weakly positive and positive) groups for survival analysis. Results indicated that p21 and EGFR expression differences significantly impacted patient OS in CRC. Kaplan-Meier analysis showed that high p21 expression was associated with significantly higher OS compared with low expression (p=0.004), whereas high EGFR expression was associated with lower OS (p=0.009) (figure 2), suggesting p21 and EGFR as potential prognostic indicators.
Correlation of p21 (A) and EGFR (B) expression levels with overall survival in patients with colorectal cancer.
Cox regression analysis of prognostic factors in CRC
Univariate Cox proportional hazards regression model identified significant factors affecting prognosis of patients with CRC, including age, mucinous subtype, tumour differentiation, lymphovascular invasion, perineural invasion, circumferential resection margin, tumour size, T stage, N stage, M stage, TNM stage, p21 expression and EGFR expression (p<0.05). Multivariate analysis revealed independent risk factors: age ≥50 years, poor tumour differentiation, lymphovascular invasion, perineural invasion, positive circumferential resection margin, tumour size ≥4 cm, advanced N stage, advanced M stage, advanced TNM stage and low p21 expression (p<0.05) (table 3), whereas high EGFR expression was not an independent prognostic factor.
Univariate and multivariate Cox regression analyses for CRC prognostics
Nomogram model for predicting survival of patients with CRC
Based on Cox regression analysis, a nomogram prediction model for survival rates of patients with CRC was constructed, integrating various prognostic factors. The total score obtained by adding individual scores predicts the 1-year, 3-year and 5-year survival rates for patients with CRC (figure 3). The model, combining different pathological parameters, offers better predictive performance and clinical applicability, translating regression equations into a visual format for patient assessment.
Nomogram model for the survival rates of patients with colorectal cancer.
Discussion
The incidence of CRC is influenced by diet, social environment, genetics and other factors. The disease is typically asymptomatic in its early stages, while later stages may present with bloody or pus-filled stools, diarrhoea, constipation and other adverse symptoms. Current treatments are limited for patients with large tumours, severe local infiltration or widespread metastasis.7 Molecular testing plays a crucial role in tumour pathological diagnosis, risk stratification, treatment monitoring and prognosis prediction.
p21 and EGFR play important roles in the occurrence and development of cancer. Their expression and function have significant clinical relevance in the diagnosis, prognosis assessment and selection of treatment strategies for cancer. Currently, targeted therapies for EGFR mainly include tyrosine kinase inhibitors (TKIs) and monoclonal antibodies. EGFR-TKIs act primarily on the intracellular tyrosine kinase domain of the EGFR receptor, blocking its kinase activity and thereby inhibiting downstream signal transduction. EGFR-TKIs include drugs from several generations: the first generation includes gefitinib, erlotinib and icotinib; the second generation includes afatinib and dacomitinib; and the third generation includes osimertinib and amivantamab. EGFR monoclonal antibodies, by binding to the extracellular domain of the EGFR receptor and preventing its natural ligands from binding, can also effectively block the EGFR-mediated signalling pathway. Commonly used EGFR monoclonal antibodies include cetuximab, necitumumab, panitumumab and nimotuzumab. In contrast, targeted therapies for p21 remains limited. The known p21 inhibitor UC2288 is synthesised based on the chemical structure model of sorafenib. Targeting other genes in the p21 cascade to induce the expression of p21 is seen as a potential strategy to inhibit tumour growth and metastasis.
Protein p21, expressed by the CDKN1A gene, is a cyclin-dependent kinase inhibitor that can inhibit the activity of cyclin-dependent kinases and proliferating cell nuclear antigen.8 The classic tumour suppressor protein P53 plays a key transcriptional regulatory role in the cell cycle checkpoint, apoptosis and senescence, promoting the expression of p21 by binding to two sites upstream of the p21 promoter. High levels of p21 result from P53 or mitogen stimulation, leading to the formation of the Rb–E2F protein complex and downregulation of numerous cell cycle-related proteins, causing cell cycle arrest in the G1 phase until damaged DNA is repaired.9 p21 also acts as an immunosurveillance ‘scout’, promoting the clearance of senescent cells to ensure homeostasis.5 Clinical studies have reported that high p21 expression is associated with better prognosis in cancers such as urothelial carcinoma, breast cancer, bladder cancer, oesophageal cancer and ovarian cancer.10–14 Research in CRC mouse models found that p21 deficiency in Th1 cells promotes tumour growth, suggesting p21’s vital role in regulating T cell effector functions and preventing DNA damage accumulation in highly proliferative effector CD4+ T cells, with low p21 expression in tumour-infiltrating CD4+ T cells correlated with shorter survival in patients with CRC.15 Research found that RNA-binding protein PUMILIO promotes cancer cell growth by suppressing p21 expression in CRC.16 Consistent with these findings, this study shows that patients with CRC with high p21 expression have significantly higher OS compared with those with low expression. The expression differences in p21 are associated with primary tumour site, mucinous subtype, lymphovascular invasion, perineural invasion, circumferential resection margin, T stage, N stage, TNM stage and mismatch repair. Cox multivariate regression analysis identifies low p21 expression as an independent risk factor affecting patient prognosis.
Current research widely suggests that overexpression and hyperactivation of EGFR are responsible for tumour cell growth, apoptosis resistance, angiogenesis and metastasis.17 EGFR tyrosine kinase activity is influenced by various carcinogenic factors, including EGFR gene mutations, increased gene copy number and overexpression of the EGFR protein. When activated, EGFR triggers a cascade in downstream signalling pathways, including the MAPK, JAK/STAT and PI3K/Akt pathways.18 Reports from European Society for Medical Oncology in 2023 summarised survival data from previous clinical studies using EGFR inhibitors for the treatment of patients with RAS/BRAF wild-type metastatic colorectal cancer, indicating that about one-third of patients significantly benefit from EGFR inhibitor treatment.19–22 This study shows that patients with high EGFR expression have significantly lower OS compared with those with low expression, consistent with previous studies. The analysis indicates that EGFR expression differences correlate with mucinous subtype, tumour differentiation, lymphovascular invasion, perineural invasion, tumour size, T stage, N stage, TNM stage and BRAF gene mutation, with high EGFR expression identified as a risk factor affecting prognosis in univariate Cox regression analysis.
In addition, research indicated that the expression of p21 in human bronchial epithelial cells depends on the activation of EGFR.23 When EGFR inhibitors were used on the cells, p21 expression was significantly reduced. Through a statistical analysis of 426 patients with liver cancer, EGFR mutation was found to play a significant role in the regulation of p21, exhibiting a positive RNA level correlation between the two.24 This is consistent with the findings of this study where a significant positive correlation exists between p21 and EGFR expression in patients with CRC. However, the specific mechanisms behind this correlation in CRC require further research.
This article discusses the clinicopathological significance of p21 and EGFR in CRC and their prognostic value, revealing that their differential expression is closely related to clinicopathological characteristics and is significantly correlated with OS, offering predictive value for patient treatment outcomes and prognosis. A nomogram prediction model incorporating various factors improves predictive performance and clinical utility. The elderly, mucinous subtype positive, tumour poorly differentiated, lymphovascular invasion positive, perineural invasion positive, circumferential resection margin positive, tumour size ≥4 cm, advanced TNM staging, low p21 expression and high EGFR expression in patients with CRC are associated with higher total scores in the nomogram prognostic model, resulting in lower survival rates.
In conclusion, p21 and EGFR are involved in the pathophysiological process of CRC development, providing indications for clinicopathological diagnosis and risk stratification, and may serve as potential indicators for predicting patient treatment efficacy and prognosis.
Data availability statement
Data are available upon reasonable request.
Ethics statements
Patient consent for publication
Ethics approval
This study involves human participants and was approved by the Ethics Committee of Fudan University Shanghai Cancer Center (IRB approval no. 050432-4-2108*).Written informed consent was obtained from the patients for publication and any accompanying images.
Footnotes
Handling editor Deepa T Patil.
YF, MM and LG contributed equally.
Contributors YF designed the study, contributed to data analysis and interpretation, and approved the final version of the manuscript to be published. MM participated in data collection and contributed to the initial drafting of the manuscript. LG was involved in data collection and analysis, and contributed to the preparation of materials. MX was responsible for the acquisition and analysis of data. YY and DH both provided critical revisions. WS is responsible for the content as guarantor. All authors have read and agreed to the published version of the manuscript.
Funding This work was supported by the National Natural Science Foundation of China (82273370, 81972249, 82002543), Shanghai Clinical Science and Technology Innovation Project of Municipal Hospital (SHDC12020102), Shanghai Science and Technology Development Fund (19MC1911000), Natural Science Foundation of Shanghai (23ZR1421300) and Shanghai Municipal Key Clinical Specialty (shslczdzk01301).
Competing interests None declared.
Provenance and peer review Not commissioned; externally peer reviewed.