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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 33  |  Issue : 4  |  Page : 1372-1378

Diagnostic and prognostic value of P53 and the human programmed cell death 4 genes in colorectal cancer


1 Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of General Surgery, Faculty of Medicine, Menoufia University, Menoufia, Egypt
3 Department of Clinical Oncology, Faculty of Medicine, Menoufia University, Menoufia, Egypt

Date of Submission24-Mar-2020
Date of Decision16-May-2020
Date of Acceptance31-May-2020
Date of Web Publication24-Dec-2020

Correspondence Address:
Suzy F Gohar
Department of Faculty Medicine- Lecturer, Clinical Oncology, Menoufia University
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_77_20

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  Abstract 


Objective
The aim was to study the human programmed cell death 4 (PDCD4) and p53 as diagnostic and prognostic markers in patients with colorectal cancer (CRC).
Background
CRC is one of the most common cancers. Novel molecules and pathways continue to emerge in the search for improved therapeutic strategies.
Patients and methods
A total of 120 patients diagnosed with CRC were included and classified based on stage to group 1 (early-stage patients) and group 2 (advanced-stage patients). Moreover, 60 patients diagnosed with benign colorectal polyps were included as a control group (group 3). P53 and PDCD4 antigen levels were measured at presentation, and their relations to patient demographics and clinical features were estimated.
Results
For PDCD4, area under the curve was 0.889 at cutoff more than 3 ng/ml, with a diagnostic sensitivity of 85.0% and specificity 93.33%, whereas for P53, area under the curve was 1.000 at cutoff greater than 295 pg/ml, with a sensitivity and specificity of 100%. PDCD4 was significantly elevated in group 1 patients compared with group 2 (mean ± SD: 6.9 ± 3.4 vs 1.6 ± 1), with P value less than 0.001. PDCD4 levels were significantly related to tumor grade in groups 1 and 2 (P = 0.041 and 0.011, respectively). P53 levels were higher among patients in group 1 than group 2 (mean ± SD: 2422 ± 692.5 vs 2392.7 ± 680.3, respectively). P53 levels had statistically significant relation with tumor grade in group 2 patients (P = 0.004).
Conclusion
Both P53 and PDCD4 had diagnostic value. Elevated PDCD4 levels had a positive prognostic value, whereas elevated P53 levels had a negative prognostic value.

Keywords: cancer, colorectal, markers, P53, programmed cell death 4


How to cite this article:
Abou-Elela DH, Abd-El azem WM, Abdel-Aziz TF, Elkhouly EA, Gohar SF. Diagnostic and prognostic value of P53 and the human programmed cell death 4 genes in colorectal cancer. Menoufia Med J 2020;33:1372-8

How to cite this URL:
Abou-Elela DH, Abd-El azem WM, Abdel-Aziz TF, Elkhouly EA, Gohar SF. Diagnostic and prognostic value of P53 and the human programmed cell death 4 genes in colorectal cancer. Menoufia Med J [serial online] 2020 [cited 2021 Apr 19];33:1372-8. Available from: http://www.mmj.eg.net/text.asp?2020/33/4/1372/304515




  Introduction Top


Colorectal cancer (CRC) is the fourth commonest cancer in males and the third in females all over the world[1].

In Egypt, it is one of the most common malignant neoplasms and the third leading cause of death, representing ∼6% of cancers[2].

CRC has led to the concept of adenoma-carcinoma sequence, which describes multistep carcinogenesis with the accumulation of multiple genetic alterations. Novel molecules and pathways continue to emerge in the search for improved targeted therapeutic strategies[1].

Tumor markers such as carcinoembryonic antigen (CEA) and CA19-9 have poor sensitivity and specificity for CRC diagnosis, particularly in the early stage[3]. Moreover, they are not effective in monitoring the surgical resection of CRC[4].

These facts have prompted the search for other noninvasive reliable markers for the disease.

The human programmed cell death 4 (PDCD4) gene is a tumor suppressor gene localized in chromosome 10q248. It is expressed in small duct epithelial cells of the normal mammary gland, normal human lung tissue, and senescent human fibroblasts[2]. It is commonly lost in lung cancer, and this loss was correlated with higher histological grade, disease stage, and poor prognosis[2]. PDCD4 expression is attenuated with progression in human tumors of the colon, prostate, and breast[5].

PDCD4 affects the transcription of specific genes by modulating the activities of certain transcription factors, such as p53[6].

P53 is the most frequently mutated gene in human cancer, leading to carcinogenesis. Accumulation of p53 protein has been shown by both direct sequencing and immunohistochemistry in a large panel of tumors, and both methods require tissue specimens[7].

In addition, a few studies had investigated the potential use of PDCD4 as a possible prognostic or clinically diagnostic factor, and most of them were performed in resected tumor tissues.

The aim of this study was to study the human PDCD4 and p53 as diagnostic and prognostic markers in patients with CRC.


  Patients and Methods Top


This case–control study was approved by ethical committee of faculty of medicine Menoufia University. The study included 120 patients diagnosed with CRC, who presented to clinical Oncology and General Surgery Departments in Menoufia University Hospitals from January 2015 to December 2016, in addition to 60 patients diagnosed with benign colorectal polyps as a control group.

Participants with co-morbid major physical illnesses (e.g., renal, hepatic, or heart diseases), poor performance status, or personal history of other cancers were excluded.

Patients with CRC were staged based on clinical TNM classification and were divided into two groups: group 1 included those with early disease (stage I and II) and group 2 included those with advanced disease (stage III and IV). Moreover, normal individuals as a control group was considered as group 3.

After consent, all participants were subjected to through history, clinical examination, and full investigations in the form of complete blood count, complete liver and kidney functions, and tumor markers in the form of CEA and CA19.9.

Patients with CRC were subjected to computed tomography (CT) of chest, abdomen, and pelvis; MRI pelvis (in case of rectal adenocarcinoma); and bone scan to exclude bone deposits.

Demographic data of the patients and tumor characteristics (site, regional node status, histological characteristics, and grade, sites, and number of metastases) were reported.

Sampling was done as follows: 10 ml of venous blood was withdrawn under complete aseptic conditions by sterile venipuncture. The sample was divided into three tubes as follows:tube 1:2 ml of whole blood was added to an EDTA-contained vacutainer to perform complete blood picture; tube 2:1.8 ml of blood was delivered into a tube containing 0.2 ml Na citrate, in which plasma was separated by centrifugation at 4000 rpm for 10 min, for prothrombin time estimation; and tube 3:4 ml of whole blood was added to plain vacutainer and was left to clot for 30 min at 37°C. The serum was separated by centrifugation. The separated serum was used for estimation of urea; creatinine; serum glutamic-oxaloacetic transaminase; serum glutamate-pyruvate transaminase; alkaline phosphatase; albumin, which was done on AU 480 autoanalyzer (Beckman Coulter, USA Beckman Coulter, Miami, FL, United States, Roche Egypt, Festival City Plot #14b01 Building 4th Floor, Cairo Governorate, Egypt); C-reactive protein, which was done on Heales turbidimetry analyzers QR-100 (Shenzhen Huisong Technology Development Co. Ltd, Shenzhen, China); and conventional tumor markers such as CEA and CA19-9, which were done on Cobas e 411 autoanalyzer (Roche Diagnostics GmbH, USA). The remaining of the blood was poured into a plain vacutainer, left to be clotted and centrifuged to separate serum, which was stored at −20°C for subsequent assay of PDCD4 and P53. They were measured using the commercially available enzyme-linked immunosorbent assay (ELISA) Kits. ELISA Kits were supplied by Sun Red Biotechnology Company, (No.6497, hutai Road,Baoshan District,Shanghai, China Shanghai, Shangha). China (www.sunredbio.com) For detection of serum PDCD4, PDCD4 was added to monoclonal antibody enzyme well, which was precoated with human PDCD4 monoclonal antibody. Then incubation was done, and then PDCD4 antibodies labeled with biotin and combined with streptavidin–horseradish peroxidase were added to form immune complex. Then incubation was carried out and washed again to remove the uncombined enzymes. Then chromogen solutions A and B were added. The color of the liquid changes into blue, and at the effect of acid, the color finally was yellow. The chromes of color and the concentration of the human PDCD4 of sample were positively correlated.

For detection of serum P53, it was added to monoclonal antibody enzyme well, which was precoated with P53 monoclonal antibody, and then incubated. Then P53antibodies labeled with biotin and combined with streptavidin–horseradish peroxidase were added to form immune complex. Then incubation was carried out and washed again to remove the uncombined enzymes. Then chromogen solutions A and B were added. The color of the liquid changed into blue, and at the effect of acid, the color finally was yellow. The chromes of color and the concentration of the human substance P53 of sample were positively correlated.

All patients with stage III and high-risk stage II colon cancer received adjuvant FOLFOX chemotherapy regimen, which is given on second day. The regimen is repeated for 12 cycles, every 2 weeks.

Patients with localized rectal carcinoma treated with concomitant chemotherapy and radiotherapy with capecitabine and were assessed by MRI pelvis and tumor markers (CEA and CA19.9).

In all cases of metastatic disease, FOLFOX regimen was the first-line regimen and evaluated by CTs and tumor markers every 3 months.

Patients who finished chemotherapy or concomitant chemo and radiotherapy were followed bytumor markers, CT and\or MRI, every 3 months and colonoscopy on annually.

Response assessment was done based on RECIST criteria. Follow-up was completed for all patients until August 31, 2018.

The progression-free survival (PFS) was defined as the time between the date of diagnosis and the date of progression (in the form of local recurrence, newly developed metastases for patients with localized disease, or increase in size and/or number of metastases in patients with stage IV disease) or last visit. The PFS was analyzed in relation to different prognostic factors among patients.

Data were fed to the computer and analyzed using IBM SPSS software package version 20.0. (IBM Corp., Armonk, New York, USA). Qualitative data were described using number and percentage. The Kolmogorov–Smirnov test was used to verify the normality of distribution. Quantitative data were described using range (minimum and maximum), mean, SD, and median. Significance of the obtained results was judged at the 5% level.


  Results Top


Comparison among the three studied groups is shown in [Table 1]. There was no significant difference between the studies groups regarding age and sex. However, tumor markers CEA and CA19.9, in addition to CRP and the studied markers P53 and PDCD4 levels, were significantly elevated among patient groups 1 and 2 compared with control group (group 3) (P < 0.001).
Table 1: Descriptive statistics among the different studied groups according to different parameters

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Assessment of the diagnostic performance of the studied markers in cases vs control using receiver operating characteristic curve analysis revealed that, for PDCD4, area under the curve (AUC) was 0.889 at cut off greater than 3 ng/ml, with a diagnostic sensitivity of 85.0%, specificity of 93.33%, positive predictive value (PPV) of 89.5%, and negative predictive value (NPV) was 90.3%, whereas for P53, AUC was 1.000 at cutoff more than 295 pg/ml, with sensitivity, specificity, PPV, and NPV were 100.0 [Figure 1].
Figure 1: Receiver operating characteristic curve for different parameters to predict early cases vs control.

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However, the assessment of the diagnostic performance of the studied markers in group 1 vs group 2 patients using receiver operating characteristic curve revealed that for PDCD4, AUC was 0.952 at cut off more than 2.2 ng/ml, with a diagnostic sensitivity of 95.0%, specificity 82.50%, PPV of 73.1%, and NPV of 79.1%, whereas for P53, AUC was 0.501 at cut off more than 1900 pg/ml, with sensitivity, specificity, PPV, and NPV of 80.0, 27.50, 35.6, and 73.3%, respectively [Figure 2].
Figure 2: Receiver operating characteristic curve for different parameters to predict late cases from early cases.

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There was no significant difference between groups 1 and 2 regarding tumor site and presenting symptom, whereas high-grade tumors were significantly more common in group 2, representing 35% of all patients (P = 0.027). PDCD4 level was significantly elevated in group 1 patients compared with group 2 (Mean ± SD6.9 ± 3.4 vs 1.6 ± 1), with P value less than 0.001 [Table 1].

The median PFS was significantly shorter among patients in group 2 compared with those in group 1 (7.1 vs 8.1 months, P = 0.048) [Table 2].
Table 2: Comparison between the groups 1 and 2 according to different disease features and progression-free survival

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There was a significant relation between PDCD4 levels and tumor grade in groups 1 and 2, where high levels are associated with well-differentiated tumors (P 0.041 and 0.011 in groups 1 and 2, respectively) [Table 3].
Table 3: Relation between PDCD4 and different parameters in each group

Click here to view


Although not statistically significant, P53 levels were higher among patients in group 1 than group 2 (Mean ± SD2422 ± 692.5 vs 2392.7 ± 680.3, respectively) [Table 1].

P53 levels had statistically significant relation with tumor grade in group 2 patients (P = 0.004). Relations between P53 level and other patient features were also determined [Table 4].
Table 4: Relation between P53 and different parameters in each group

Click here to view



  Discussion Top


Colon cancer is one of the most common malignant tumors all over the world[8].

Biomarkers for colon cancer are required for screening and early detection, because of their high sensitivity and the application advantages in guiding treatment[9].

The apoptotic marker PDCD4 is found to be downregulated in various types of carcinomas, including melanoma, hepatocarcinoma, and non-small cell lung cancer. However, roles of PDCD4 in colon cancer remain unclear[9].

The current study explored the diagnostic and prognostic value of the PDCD4 and its target P53 in patients with CRC compared with 60 apparently healthy controls (group 3).

Both markers were also investigated within the patients who were divided based on the stage into early-stage disease group (group 1), including 40 patients, and late-stage disease (group 2), including 80 patients.

There was no difference among the three studied groups regarding age and sex, indicating homogeneity of the groups.

CEA level was significantly elevated in patients with advanced-stage disease in comparison the early and control group, indicating its relation to the tumor burden and its well-known prognostic value.

The PDCD4 level was significantly higher in patients with early-stage disease (Mean ± SD 6.9 ± 3.4), whereas it was significantly lower in patients with advanced-stage disease (1.6 ± 1).

These results are similar to those of Abdel Maksoud et al.[2], who explained these conflicting results by considering that apoptosis is initiated in early disease stage (leading to increase PDCD4 levels) to limit the expansion of the tumor cell. The results of Ananthanarayanan et al.[10] who found that PDCD4 is upregulated on induction of apoptosis and downregulated in certain aggressive tumors like lung, breast, colon, brain, and prostate cancers should be considered.

Regarding P53, it was significantly elevated in patients with CRC compared with controls (2422 ± 692.5 and 2392.7 ± 680.3 vs 204.8 ± 43.6). This confirms the diagnostic value of P53.

Among patients with CRC, undifferentiated tumors were significantly more frequent in group 2 patients. TTP was significantly shorter in patients with group 2 (advanced-stage disease). These findings are expected as this group of patients had the most advanced disease.

In relation to different disease features, P53 antigen levels were significantly related to TTP as higher P53 is associated with shorter TTP.

In early-stage group (group 1 patients), PDCD4 was significantly elevated in female patients and in well-differentiated tumors. However, in late-stage disease, it was significantly elevated in older patients and well-differentiated tumors. These relations indicate the good prognostic value of high PDCD4 serum levels.

P53 among patients with early-stage disease showed that it was significantly high in younger patients, patients known to be smokers, and those who experienced disease progression. However, in advanced-stage patients, p53 was significantly high in patients with undifferentiated tumors.

These relations suggest the possible role of P53 in the process of carcinogenesis and tumor aggressiveness.


  Conclusion Top


Both markers P53 and PDCD4 had diagnostic value and can discriminate between patients with benign conditions vs those with cancer. However, PDCD4 only can discriminate early from late stages. Elevated PDCD4 levels had positive prognostic value, whereas elevated P53 levels had negative prognostic value.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Chang KH, Miller N, Kheirelseid EA, Ingoldsby H, Hennessy E, Curran CE, et al. MicroRNA-21 and PDCD4 expression in colorectal cancer. Eur J Surg Oncol 2011; 37:597–603.  Back to cited text no. 1
    
2.
Abdel Maksoud M, Afify M, Samy N, MosaT, Abo- ze M. Role of CXCL5 and PDCD-4 as prognostic biomarkers in patients with colorectal cancer. Int J Pharm Clin Res 2016; 8:90–94.  Back to cited text no. 2
    
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Crawford NP, Colliver DW, Galandiuk S. Tumor markers and colorectal cancer: utility in management. J Surg Oncol 2003; 84:239–248.  Back to cited text no. 3
    
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Smith RA, Cokkinides V, Eyre HJ. American Cancer Society guidelines for the early detection of cancer. CA Cancer J Clin 2004; 54:41–52.  Back to cited text no. 4
    
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Chen Y, Knosel T, Kristiansen G, Pietas A, Garber ME, Matsuhashi S, et al. Loss of PDCD-4 expression in human lung cancer correlates with tumour progression and prognosis. J Pathol 2003; 200:640–646.  Back to cited text no. 5
    
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Wedeken L, Ohnheiser J, Hirschi B, Wethkamp N, Klempnauer KH. Association of tumor suppressor protein Pdcd4 with ribosomes is mediated by protein-protein and protein-RNA interactions. Genes Cancer 2010; 1:293–301.  Back to cited text no. 6
    
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Nasif W, Mukhtar M, El-Emshaty H, Al-Bukhari T. Evaluation of circulating TP53 antigen in the sera of gastric cancer patients: as a tumor marker. Biomarkers Genom Med 2013; 5:175–180.  Back to cited text no. 7
    
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Stewart BW, Bray F, Forman D, Ohgaki H, Straif K, Ullrich A, et al. Cancer prevention as part of precision medicine: 'plenty to be done'. Carcinogenesis. 2016;37:2-9.  Back to cited text no. 8
    
9.
Yuan K, Yu X, Zhang Y, Sun Z, Wu K, Wang L, et al. Programmed cell death 4 (PDCD4) repression is involved with tumor cell differentiation and lymph node metastasis in patients with colon cancer. Int J Clin Exp Pathol 2016; 9:8617–8621.  Back to cited text no. 9
    
10.
Ananthanarayanan V, Deaton RJ, Yang XJ, Pins MR, Gann PH. Alteration of proliferation and apoptotic markers in normal and premalignant tissue associated with prostate cancer. BMC Cancer 2006; 6:73.  Back to cited text no. 10
    


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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