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Right arrow Esophagus - cancer

Ann Thorac Surg 2005;80:1007-1016
© 2005 The Society of Thoracic Surgeons

Original article: General thoracic

Deregulated P21WAF1 Overexpression Impacts Survival of Surgically Resected Esophageal Squamous Cell Carcinoma Patients

Yih-Gang Goan, MD a , b , c , Hon-Ki Hsu, MD a , Huang-Chou Chang, MD a , Yi-Ping Chou, MD a , Ke-Hsin Chiang, MD d , Jiin-Tsuey Cheng, PhD c , *

a Division of Thoracic Surgery, Department of Surgery, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan, Republic of China
b Department of Surgery, National Yang-Ming University, Taipei, Taiwan, Republic of China
c Department of Biological Sciences, National Sun Yat-Sen University, Kaohsiung Taiwan, Republic of China
d Union Pathologic and Laboratory Center, Kaohsiung, Taiwan, Republic of China

Accepted for publication March 4, 2005.

* Address reprint requests to Dr Cheng, Department of Biological Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan 80424, Republic of China (Email: tusya{at}mail.nsysu.edu.tw).


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Comment
 Southern Thoracic Surgical...
 Acknowledgments
 References
 
BACKGROUND: Prognostic effects of cell cycle regulation associated tumor suppressor genes for esophageal squamous cell carcinoma patients are controversial. Clarifying the effects of these genes is beneficial for optimizing patient’s outcome.

METHODS: Forty esophageal squamous cell carcinoma patients were included in the study. Tissue samples were analyzed for cell proliferation, DNA content, mutation of p53 gene, and expression of p21 and p53 proteins. Prognostic effects of these parameters were assessed by multivariate analysis.

RESULTS: Seventy-five percent of tumors exhibited aneuploid DNA content. Significantly higher S-phase fractions were detected in tumor samples (p < 0.001). The p53 gene mutated in 52.5% (21 of 40) of tumor samples, p53 immunostaining was detected in 62.5% (25 of 40) of tumor tissues and 50% of tumors over expressed p21. Overexpression of p21 protein did not correlate with p53 gene status, but significantly correlated with abnormal DNA content (p = 0.028). Advanced pTNM stage, lymph node metastasis and p21 overexpression conferred survival disadvantages in univariate analysis (p = 0.013, 0.045 and 0.017, respectively). A Cox multivariable analysis revealed pTNM stage (IIB/III/IV vs. I/IIA; p = 0.024) associated with p21 overexpression (positive vs. negative; p = 0.035) as independent prognostic factors in esophageal squamous cell carcinomas. Surprisingly, p21 overexpression significantly compromised the survival of patients with mutated p53 gene (p = 0.035). However, no significant dismal effect of p21 overexpression can be seen in patients with wild-type p53 gene (p = 0.175).

CONCLUSIONS: Overexpression of p21 correlates with chromosomal instability and serves as an adverse prognostic predictor for patients with esophageal squamous cell carcinoma. Its adverse effect is more prominent when the p53 gene is mutated.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Comment
 Southern Thoracic Surgical...
 Acknowledgments
 References
 
Esophageal squamous cell carcinoma (ESCC) is one of the most tedious malignancies in digestive system. In spite of the progressed surgical techniques and peri-operative management in the pass decades, the 5-year survival rate after surgical treatment is less than 20% for ESCC patients [1, 2. Therefore, it is necessary to identify additional indicators besides the conventional staging system to better assess the malignant potential of ESCC cases.

Human cancer arises via a multistep process involving the activation of oncogenes and inactivation of tumor suppressor genes. Accumulation of genetic alterations derange the regulation of cell growth and differentiation, and play a key role in the development of human cancers including esophageal squamous cell carcinoma [3].

The p53 and p21 WAF1/CIP1 are the two cell cycle negative regulators that draw the most attention and have been investigated in a variety of human cancers. This is not only due to their role in regulating G1-S transition of cell cycle but also their function in regulation of programmed cell death [4]. The p53 functions as a cellular genome guidance. When cells are exposed to DNA damaging agents, wild-type p53 protein accumulates and convenes several downstream target genes to induce G1 arrest and allows ample time for cells to repair their damaged DNA before entering cell cycle [5, 6. If DNA damage is so dramatic that cells could not repair, p53 then activates its target gene BAX, which elicits programmed death of damaged cells. Another p53 target gene, p21 WAF1/CIP1 , encodes a cyclin-dependent kinase inhibitor p21, which is one of the downstream effecters of the cell cycle arrest function of p53 gene, and its expression is directly up-regulated by wild-type but not mutant p53 at transcriptional level [6, 7. However, recent in vitro and in vivo data also suggested that p21 WAF1/CIP1 gene can be regulated via a p53-independent pathway [7, 8. This might explain why several reports address the frequent p53 mutation in ESCC, while the correlation between p53 gene mutation and p21 protein expression in ESCC are controversial and need further clarification [9–16.

The presence of aneuploid DNA content is the indication of genetic instability and is a critical event for cell progressing thru the initial stages of carcinogenesis [17, 18. In ESCC, nuclear DNA content was reported to be useful in predicting the prognosis of cancer [19, 20. This study intends to investigate the relationship between DNA content and the genetic status of cell cycle associated genes p53 and p21 in ESCC. Hopefully by correlation these molecular factors with patients clinicopathological feature, we could evaluate the predicting value of these factors on patients’ outcome as well as clarify the role of p53 gene status and p21 expression on the prognosis of ESCC patients.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Comment
 Southern Thoracic Surgical...
 Acknowledgments
 References
 
Patients
Between March 2000 and February 2003, 121 consecutive ESCC patients were referred to Kaohsiung Veterans General Hospital. Seventy-two of them were treated by esophagectomies. Primary esophageal squamous cell carcinoma tissue samples were prospectively collected at the time of surgery and all patients gave informed consent before surgery. Excluding patients receiving radiation therapy or chemotherapy before surgery, forty patients underwent curative esophagectomy via a combined cervical, thoracic, and abdominal approach with lymph node dissection were enrolled in this study. Patients’ data were available for age, gender, pathologic result, and tumor stage according to the TNM system on the basis of AJCC classification [21]. After discharge, all patients were regularly followed up at outpatient clinic every 3 months for the first 3 years and then every 6 months subsequently. Four patients died of postoperative complications, leaving 36 patients for survival analysis.

Tissues of Esophageal Squamous Cell Carcinoma
Several small pieces of fresh tumor tissue samples were dissected from the main tumor part of each surgically removed specimen and frozen immediately in liquid nitrogen, then stored at –80°C for future analysis. The remaining part of the specimen was then fixed in formalin solution and sent for histological examination. The paraffin-embedded sections from the specimens, which were diagnosed as having ESCC, were used for immunostaining of p53, p21 protein with p53 and p21 antibodies respectively. The corresponding frozen samples were used for the analysis of p53 gene mutation.

Reverse Transcriptase Polymerase Chain Reaction and DNA Sequencing
Total RNA was extracted from frozen specimens using Trizol reagent (Life Technologies, Gaithersburg, MD) according to manufacturer’s instructions. First-strand cDNA was synthesized from 3 µg of total RNA using the Advantage RT for PCR kit (Clontech Laboratories, Palo Alto, CA) in a 25 µL of reaction volume. Then, 1 µL of the first strand cDNA was mixed with 5 µL of 10x polymerase chain reaction (PCR) buffer, 4 µL of 2.5 mM dNTP, 1.4 µL of 10 µM p53 forward primer (5'-CACGACGGTGACACGCTTCC was used for amplification of exons 2 to 4 and 5'-CGTGCAACTCACAGACTTGG was used for amplification of exons 5 to 11), 1.4 µL of 10 µM p53 reverse primer (5'-GCTACGGTTTCCGTCTGGGC was used for amplification of exons 2 to 4 and 5'-TCAGTCTGAGTCAGGCCCTT was used for amplification of exons 5 to 11), 1 µL of Taq polymerase (Promega, Madison, WI) and ddH2O to 50 µL final volume. The mixture was denatured at 94°C for 5 minutes and followed by 35 cycles of PCR reaction with denaturing at 94°C for 45 seconds; annealing at 57.2°C (exons 2 to 4) or 60°C (exons 5 to 11) for 1 minute; and extension at 72°C for 1 minute. The amplification procedure was carried out completely after further incubation at 72°C for 10 minutes. The sizes of PCR products were 440 base pairs and 863 base pairs for exons 2 to 4 and exons 5 to 11, respectively. After purification, the PCR products were sequenced at the sequencing facility laboratory of the National Sun Yat-Sen University. The PCR amplification primers also served as sequencing primers. To confirm the results, all detected mutations were double confirmed with cDNA synthesized from another batch of RNA isolation.

Immunohistochemistry
Avidin-biotin-peroxidase method was used for immunohistochemistry. Briefly, paraffin-embedded tissue blocks were sectioned in 5-µm slices and placed on poly-L-lysine coated slides. Then the sections were de-waxed in xylene, rinsed in phosphate buffered saline (PBS) solution, blocked with 3% hydrogen peroxide and antigen retrieval in boiling boric acid solution. Diluted (1:100) NCL-DO7 anti-p53 monoclonal antibody (Novocastra, Newcastle upon Tyne, UK) and anti-p21WAF1/CIP1 antibody (Santa Cruz Biotechnology, Inc, Santa Cruz, CA) were applied on to the specimens and incubated at room temperature for 30 minutes. After washing with PBS solution, biotnylated secondary antibody and streptavidin-horseradish peroxidase were applied to the specimens in succession according to the manufacturer’s instruction (LSAB Plus Kit, DAKO, Denmark). Finally, peroxidase substrate diaminobenzidine (Zymed, South San Francisco, CA) was used for developing signals and Mayer’s hematoxylin for counter-staining. The 1% BSA was used as negative control. The results of staining were evaluated independently by two physicians under a standard microscope. Any differences in interpretation were resolved by consensus. We adopted the criteria by Casey and colleagues [16] as more than 10% positive nuclear staining was considered as positive p53 staining. For p21protien, we adopted the criteria by Sarbia and colleagues [14] as more than 50% positive nuclear staining of tumor cells was considered as overexpressed p21 protein (Fig 1).



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  		Fig 1. Immunohistochemistry of p21 and p53 in esophageal squamous cell carcinomas. Nucleus stained p21 protein was seen (a) heterogeneously in tumor component (original magnification x100) and (b) only in the para-basal layer of normal esophageal mucosa (original magnification x100). The p53 protein expressed (c) prominently in nuclei of cancer cells (original magnification x200), but not in (d) normal mucosa (original magnification x100).

 
Flow Cytometry Analysis
Before flow cytometric analysis, all selected paraffin blocks were re-checked by H&E stain. A pepsinization technique modified from that of Hedley and colleagues [[22] was used. Briefly, 6 50-µm thick sections were obtained from paraffin blocks. After de-paraffinization and subsequent rehydration, tissues were digested in 0.5% pepsin solution (Sigma Chemical Co., St. Louis, MO) at 37°C for 90 minutes. Then, the suspension was filtered through a 60-µm nylon mesh and centrifuged. Sediments were resuspended in 1 mL of Hedley buffer (0.04M Tris, 5mM/L MgCl2, 0.3% NP-40 and 0.005 mg/mL Propidion iodide adjusted to pH = 7.0) with RNase (1 mg/mL) and incubated at 37°C in the dark. Then the suspension was filtered again through a 40-µm mesh before performing measurements on FACSort flow cytometer (Becton Dickinson Immunocytometry System, Mountain View, CA). At least 10,000 nuclei per sample were counted [23]. The cell cycle fractions were analyzed using MultiCycle AV software version 5.0 of Phoenix Flow System, Inc. If excessive tissue debris were detected (> 20%) or the coefficient of variation of G0/G1 peak was greater than 7%, another attempt was made to process the sample [24]. Aneuploidy DNA content was defined as a clearly distinct additional G0/G1 peak with a small G2/M peak (Fig 2). An experienced staff member of the Department of Surgery performed all of these cytometric measurements independently with no knowledge of the histopathologic diagnosis or other results of this study.



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  		Fig 2. Patterns of DNA histogram. Diploid samples were regarded as those for which the histogram showed a single defined G0/G1 peak on the DNA histogram (A). The aneuploid samples were regarded as those for which the histograms presented a second G0/G1 peak in an unexpected location (B) or a clearly distinct additional G0/G1 peak with a small G2/M peak (C).

 
Statistical Analysis
The {chi}2 test was used for analysis in the case of the two-by-two table. Overall survival was constructed by the Kaplan-Meier method and evaluated by a log-rank test. Survival time was defined as the time between date of operation and date of death. The telephone inquiry and the medical records confirmed the date of death. Independent prognostic factors for survival were analyzed using a Cox proportional hazard model in a forward stepwise procedure, using a SPSS statistical software program package (SPSS version 10.0 for Windows, SPSS Inc, Chicago, IL). A 0.10 level of probability was the significant value used for adding and deleting a co-variable from the model. Statistical analysis was regarded as statistically significant if p was less than 0.05.


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Comment
 Southern Thoracic Surgical...
 Acknowledgments
 References
 
There were 38 male and 2 female patients enrolled for the analysis. The mean age of these 40 patients was 60.3 years old (range from 33 to 81 years old). At the end of the follow-up period (April 1, 2004), 8 (20.0%) of 40 patients were still alive. The follow-up time ranged from 4 to 48 months (median, 15 months). All 40 patients were squamous cell carcinoma. The demographic data of patients are shown in Table 1. Among them, one was stage I, 11 were stage IIA, 7 were IIB, 20 were III and one was stage IV disease. There were 2 patients with T1 classification, 10 with T2 classification, 24 for T3 classification, and 4 were T4 classification. Lymph node metastases were detected in 26 of 40 patients (65.0%). The M1 tumor was due to distant lymph node metastases.


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  		Table 1. Characteristics of Clinicopathologic Parameters, Status of p53 Gene Mutation, p53 and p21 Immunoreactivities, and DNA Content in Esophageal Squamous Cell Carcinoma Patients
 
The PCR products of p53 gene were amplified in all tumor tissue samples. Twenty-three mutations were found in 21 (52.5%) of the 40 tested specimens. Mutations clustered significantly in exon 5 (13 of 23) of the p53 gene. Mutation hot spots located at codons 131, 151, and 193. The prevalent p53 mutational spectrum was A:T to G:C transition (6 of 23, 26%) and G:C to T:A transeversion (5 of 23, 21.7%) in our series. All of these mutations were point mutations and most of them were mis-sense mutations except two silent mutations (unpublished data). The correlations between clinicopathological parameters and p53 gene status are presented in [Table 1. No significant associations between p53 mutation and clinicopathological parameters were detected in our patients.

The p53 protein was detected in 25 of the 40 ESCCs (62.5%) by immunohistochemistry. All immunostaining of p53 protein was confined to the nuclei of tumor cells (Fig 1). Also, no significant correlation could be found between p53 protein expression and gender, depth of tumor invasion, lymph-node involvement and pTNM stage (Table 1). The positive rate of p53 protein expression was higher than that of p53 gene mutations (21 cases; 52.5%). Cross-tabulation for association between p53 protein expression and gene mutational status was analyzed using a {chi}2-test. There was a significant relationship between p53 gene mutations and p53 protein immunoreactivities (p = 0.011). The sensitivity rate of immunohistochemical detection relative to p53 gene mutations was 80.9% (17 of 21). The specificity rate of immunostaining relative to p53 gene mutations was 57.9% (11 of 19) (Table 2).


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  		Table 2. Correlation Between Genetic p53 Status and IHC Results of p53 and p21
 
Overexpression of p21 protein was seen in 50% (20 of 40) of tumor samples (Table 1). Nucleus stained p21 protein was seen heterogeneously in tumor sections and only in the para-basal layer of normal esophageal mucosa (Fig 1). The existence of overexpressed p21 protein significantly correlated with abnormal DNA content (p = 0.028). The p21 expression status did not correlate with the clinicopathological parameters of patients. However, patients with overexpressed p21 protein were more likely with lymph node metastasis (p = 0.025) (Table 1). In evaluating the relationship between p21 expression and p53 gene mutation, p21 immunoreactivities did not correlate with the mutational status of p53 gene (p = 0.752). However, significant correlation was noticed between p21 immunoreactivity and the results of p53 immunostaining (p = 0.022; Table 2).

In flow cytometry analysis, 10,600 to 26,100 nuclei per specimen (mean, 15,800) were analyzed and the mean coefficient of variation was 5.1% (range, 3.1%–7.0%) in our study. The complete results of flow cytometry analysis were demonstrated in Table 3. Aneuploid DNA content was seen in 30 (75%) of 40 tumor specimens and all tissues from non-tumor part were diploid DNA histogram. The average S-phase fractions of tumor tissues were significantly higher than that of non-tumor part (p < 0.001). Tumors with aneuploid DNA content had significantly higher S-phase fractions than diploid tumor samples (p < 0.001). Even in tumors with diploid DNA content, the S-phase fraction of tumor tissues were also significantly higher than nontumor specimens (p < 0.001). The presence of abnormal DNA content didn’t correlate with clinicopathologic parameters of ESCC patients but it significantly correlates with the expression of p21 protein (p = 0.028, Table 1). Meanwhile, patients with positive metastatic lymph node usually had abnormal DNA content in tumor cells (p = 0.006, Table 1).


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  		Table 3. Flow Cytometric Cell Cycle Analysis and DNA Content of Tumor and Nontumor Tissues
 
The 1-year survival rate was 70%, 42% and 24.2% for stage IIA, IIB and III respectively. No one-year survival was noted for stage IV disease. As no patients exhibited 5-year survival, the three-year survival rate was 42.2%, 28.6% and 12.2% for stage IIA, IIB and III, respectively. The overall one-year and three-year survival rates were 41% and 16%, respectively. In a univariate survival analysis for the entire cohort, all available clinicopathologic factors did not show significant survival effect for esophageal squamous cell carcinoma patients except the pTNM stage (p = 0.001) and lymph node status (p=0.045). The abnormality of DNA content (p = 0.171) and p53 mutational status (p = 0.488) also was not significant in predicting survival for ESCC patients. The expression of p53 protein showed marginal significance in predicting patients’ survival (p = 0.051). However, p21 overexpression was significantly associated with a poorer outcome (p = 0.017) (Fig 3). In patients with positive lymph node metastasis, overexpression of p21 demonstrated a more compromised survival as compared with p21-negative patients (p = 0.009).



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  		Fig 3. Kaplan-Meier survival estimation of surgically treated esophageal squamous cell carcinoma (ESCC) patients related to possible predictors. (A) Significant prognostic effect of pTNM stage for ESCC patients (p = 0.001). (B) The p21 overexpression significantly impacts on ESCC patients survival (p = 0.017, 250 and 467 days of median survival for positive and negative p21 overexpression respectively). (C) The p53 mutation status (p = 0.488), and (D) p53 immunohistochemistry (IHC) stain (p = 0.051) did not predict outcome of ESCC patients.

 
In determining the role of p53 gene status on the prognostic effect of p21 overexpression, the uncontrolled p21 expression exhibited a significant survival disadvantageous effect on ESCC patients harbor mutant p53 (p = 0.035). As when wild-type p53 gene exists, the expression of p21 is presumably under the control of p53 gene, the p21 overexpression did not exhibit significant effects on patients’ survival (p = 0.175). Noticeably, the dismal effect of p21 expression on patient’s survival will not be seen if immunohistochemistry was used to predict the functional status of p53 gene (p = 0.121) (Fig 4).



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  		Fig 4. Kaplan-Meier survival estimation of p21 overexpression adjusted by p53 gene mutation status. (A) Overexpression of p21 significantly impacts patients’ survival when the p53 gene mutated (p = 0.035). The median survival was 314 days and 930 days for positive and negative p21 overexpression, respectively. (B) However, this dismal effect cannot be observed in p53 immunohistochemistry (IHC). (C) Under wild-type p53 gene control, p21 expression plays no significant effect on patients’ survival (p = 0.175).

 
In Cox multivariable regression analysis, only significant prognostic factors in univariate analysis such as pTNM stage, lymph node metastasis and p21 expression status were included for analysis. In that analysis, p21 overexpression (positive vs negative; p = 0.035, HR = 2.301, 95% CI = 1.061–4.989) together with pTNM stage (IIB/III/IV vs I/IIA; p = 0.024, HR = 3.096, 95% CI = 1.156–8.291) was shown to have independent prognostic impact on survival of ESCC patients (Table 4).


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  		Table 4. Univariate and Multivariate Analyses for Factors Predicting the Outcome of ESCC Patients
 

   Comment
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Comment
Southern Thoracic Surgical...
 Acknowledgments
 References
 
Carcinogenesis is a molecular genetic process cumulating from a long process of preneoplasia. Progression of neoplastic transformation also requires high rates of cell turnover [25]. Recent work from Watanabe and colleagues [26] has demonstrated a significant relationship between DNA ploidy and multiples genetic alterations in ESCCs. Previous reports addressed that early esophageal malignant changes are already associated with alterations in DNA content [20]. Under continuous stress of genomic disability, multiple aneuploid subclones will evolve, then progress to neoplasm [27]. In our present series, an aneuploidy DNA pattern was detected in 75% of ESCC specimens. The average proliferating rate of tumor tissues was significantly higher than that of non-tumor parts. The highest average proliferating ability was noticed in tumors bearing aneuploid DNA content. Even with diploid DNA content, tumor tissues harbor significantly higher percentage of S-phase cells than non-tumor tissues. These results are compatible with the concept that accelerated cellular proliferation is needed for the transforming process, and the subsequent accumulation of genetic alterations which then resolved in the carcinogenesis of ESCC.

Although it has been advocated as an independent prognostic indicator of tumor behavior, the clinicopathologic significance of DNA aneuploidy remains controversial. In our series, the existence of aneuploid DNA content does not correlate with patients’ survival. This result is consistent with the results of Blant and coworkers [20], but discordant with the observations by Watanabe and colleagues [26] and Bottger and associates [28], who reported a significant correlation between DNA content and survival rates. Several previous reports had suggested that DNA content evaluation could detect early neoplastic changes [29]. Although others reported a significant aneuploidy pattern could be seen in macroscopically tumor-free esophageal specimens [25], all our samples from non-tumor part of ESCC patients were diploid. The aneuploid DNA contents were only detected in ESCC tumor samples. This discordance could be explained by the fact that we double checked the paraffin-block section microscopically for the existing of submucosal tumor infiltration before analyzing DNA content.

Unlike p53 gene, mutations and deletions of p21WAF1/CIP1 gene are rare in human cancers [30]. The degree of p21 immunoreactivity is assumed to reflect the degree of expression of normal p21 gene and serves as a useful tool for assessing p21 function. In normal esophageal squamous epithelium, p21 only expressed in cells located at para-basal layer. However, p21 staining was quite variable both between and within tumors. Although p21 has been reported as a useful molecular prognostic factor in many human tumors, studies concerning p21 as a prognostic factor in ESCC have produced mixed results [11, 31, 32. This might be because there is no uniform definition of what constitutes a positive p21 stain. Therefore, the degree of p21 expression varied from no expression to moderate and strong in carcinoma cells [13]. In present study, we adopted the criteria of ≥50% nuclear staining as a positive stain for p21 overexpression used by other reports [14, 15. Using this criterion, p21 was overexpressed in 50% of our ESCC specimens. In univariate as well as multivariable analysis, p21 overexpression together with pTNM stage was shown to have independent prognostic impact on survival of ESCC patients. The adverse effect of p21 overexpression seen in this study is consistent with the observation of Sarbia and coworkers [14], but contradict the result of Shimada and colleagues [15]. In our series, the higher potential of lymph node metastases in p21 overexpressed ESCC might contribute to the significant dismal outcome of p21 overexpressed ESCC patients.

As initially discovered in experimental models, p21WAF1/CIP1 expression is thought to be a specific down-stream effecter of wild-type p53 gene induced cell cycle arrest [6]. However, several recent studies demonstrated the existence of p53-independent expression of p21 in varieties of malignant tissues including ESCC [8, 33. Accordingly, the relationship between p21 expression and the status of p53 gene might not be as simple as intuitive. In our present results, overexpression of p21 does not correlate with the sequence status, thereby the functional state of p53 gene. This is agreed with the findings of Seta and colleagues [12] and Rigberg and coworkers [33], but unlike other reports [13, 34. The conflicts might result from many researchers having evaluated the relationship of p21 expression to p53 status by p53 immunostaining instead of sequencing p53 gene. As p53 immunoreactivity might not be synonymous with p53 gene mutation, since other factors such as the inactivation of the enzymatic pathway responsible for p53 degradation or prolonged half-life of wild-type p53 might resolve through binding with other cellular proteins [34, 35. As presented in our work, even though the p53 immunoreactivity was significantly correlating with p53 gene alteration, the p53 immunostaining could not detect around 20% (4 of 21) of specimens harbor p53 mutation. On the other hand, approximately 40% (8 of 19) of tumors with wild-type p53 sequences will be mis-interpreted as having p53 mutation.

Furthermore, in analysis of p21 protein expression in relation to p53 gene status, overexpression of p21 protein was observed in 10 of 21 (48%) of ESCCs with mutated p53 gene that suggests the induction of p21 might via a p53 independent pathway in this subset of tumors. Recent data from cDNA array hybridization analysis and assessing the function of p21 protein under DNA damage model have demonstrated that p21 protein might not only function in cell cycle arrest but also involve in inhibition of DNA repair and mitotic control [36–38. In addition, several reports also propose that the p21 protein correlates with the differentiation status of ESCC at the early stages of esophageal squamous cell carcinogenesis [39]. Accordingly, the counterintuitive adverse effect of p21 expression on ESCC patients especially when p53 gene mutated might result from the uncontrolled p21 overexpression in cancer cells which inhibits DNA repair as well as impairs mitosis control. This might increase the chance for genetic instable cells to re-enter the cell cycle with the consequences of tumor progression [36]. This might be the possible explanation of why p21 overexpression showed impact on this subset of ESCC patients’ survival.

Although from a limited number of patients, our results of 70%, 42% and 24.2% 1-year survival rate for stage IIA, IIB, and III diseases as well as the 42.2%, 28.6%, and 12.2% 3-year survival rate for stage IIA, IIB, and III diseases, respectively, were comparable with the recent results reported by Kukreja and associates [40]. However, low overall 3-year survival rate of 16% was noticed in this series. This poorer outcome might result from the fact that we analyzed a small number of population with a relative high percentage of late stage disease in this series.

In conclusion, the effect of p21 on tumor phenotype is still unclear. The significance of p21 expression is just beginning to be elucidated. Besides acting as an effecter in p53-induced cell cycle arrest, p21 might have other unknown important molecular biologic functions in carcinogenesis. In our present study, overexpression of p21 significantly associated with the existence of abnormal DNA content in esophageal squamous cell carcinoma. We were surprised to find a compromised survival in ESCC patients with overexpressed p21 in tumor cells. This compromised effect became more significant when p53 gene is mutated. Accordingly, the flow cytometric measurement of DNA content might be a good tool for detecting early malignant changes in ESCC carcinogenesis. Meanwhile, detection of p21 immunoreactivity and p53 mutation status along with conventional TNM system could afford clinicians the opportunity to define a new subgroup of esophageal squamous cell carcinoma patients who would benefit from tailoring more aggressively individualized adjuvant therapy after surgical resection of ESCCs.

Of course, our data are preliminary inasmuch as we have analyzed a relatively small number of patients. Conclusions concerning the importance of p21 overexpression in ESCCs patient from the results of a limited number of patients should be taken with precaution. However, it remains an impressive finding of our study. Detections of p21 expression condition, together with analyzing p53 status and DNA content, will supplement the insufficiency of standard histopathologic examinations and the traditional staging system in predicting ESCC patients’ outcome. To confirm the value of p21 overexpression as a prognostic and therapeutic marker, further prospective studies with larger series and uniform methodologies are needed.


   Southern Thoracic Surgical Association: Fifty-Second Annual Meeting
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Comment
 Southern Thoracic Surgical...
Acknowledgments
 References
 
The Fifty-Second Annual Meeting of the Southern Thoracic Surgical Association (STSA) will be held November 10–12, 2005, in Orlando, Florida.

The Postgraduate Course will be held the morning of Thursday, November 10, 2005, and will provide in-depth coverage of cardiothoracic surgical topics selected primarily as a means to enhance and broaden the knowledge of practicing thoracic and cardiac surgeons.

Manuscripts accepted for the Resident Competition must be submitted to the STSA headquarters office no later than September 16, 2005. The Resident Award will be based on abstract, presentation, and manuscript.

Applications for membership should be completed by September 15, 2005, and forwarded to Richard L. Prager, MD, Membership Committee Chairman, Southern Thoracic Surgical Association, 633 N Saint Clair St, Suite 2320, Chicago, IL 60611-3658.


Please visit the STSA (http://www.stsa.org) or CTSNet (http://www.ctsnet.org) websites for detailed information on submitting abstracts. All abstracts must be submitted electronically for consideration.

 


   Acknowledgments
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Comment
 Southern Thoracic Surgical...
 Acknowledgments
References
 
We express our appreciation to Yan-Jing Huang for helping with technical assistance. This research was supported by grants NSC-91-2134-B-075B-002 and VGHKS 92-101 (to Yih-Gang Goan).


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Comment
 Southern Thoracic Surgical...
 Acknowledgments
 References
 

  1. Medical Research Council Esophageal Cancer Working Group Surgical resection with or without preoperative chemotherapy in esophageal cancera randomized controlled trial. Lancet 2002;359:1727-1733.[Medline]
  2. Malaisrie SC, Untch B, Aranha GV, et al. Neoadjuvant chemotherapy for locally advanced esophageal cancerexperience at a single institution. Arch Surg 2004;139:352-358.[Free Full Text]
  3. Watanabe M, Kuwano H, Tanaka S, et al. Flow cytometric DNA analysis is useful in detecting multiple genetic alterations in squamous cell carcinoma of the esophagus Cancer 1999;85:2322-2328.[Medline]
  4. Hollstein MC, Shomer B, Greenblatt M, et al. Somatic point mutation in the p53 gene of human tumors and cell linesupdated compilation. Nucleic Acid Res 1996;24:141-146.[Abstract/Free Full Text]
  5. Yin Y, Tainsky MA, Bischoff FZ, et al. Wild-type p53 restores cell cycle control and inhibits gene amplification in cells with mutant p53 alleles Cell 1992;70:937-948.[Medline]
  6. El-Deiry WS, Tokino T, Velculescu VE, et al. EAF1, a potential mediator of p53 tumor suppression Cell 1993;75:817-825.[Medline]
  7. Harper JW, Adami GR, Wei N, et al. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases Cell 1993;75:805-816.[Medline]
  8. Michieli P, Chedid M, Lin D, et al. Induction of WAF1/CIP1 by a p53-indepent pathway Cancer Res 1994;54:3391-3395.[Abstract/Free Full Text]
  9. Casson AG, Tammemagi M, Eskandarian S, et al. p53 alterations in oesophageal cancerassociation with clinicopathological features, risk factors, and survival. Mol Pathol 1998;51:71-79.[Abstract]
  10. Susumu K, Yoshio K, Masato E, et al. The p53 gene mutation is of prognostic value in esophageal squamous cell carcinoma patients in unified stages of curability Am J Surg 1999;177:497-502.[Medline]
  11. Lam KY, Law S, Tin L, et al. The clinicopathological significance of p21 and p53 expression in esophageal squamous cell carcinomaan analysis of 153 patients. Am J Gastroenterol 1999;94:2060-2068.[Medline]
  12. Seta T, Imazeki F, Yokosuka O, et al. Expression of p53 and p21 WAF1/Cip1 proteins in gastric and esophageal cancerscomparison with mutations of the p53 gene. Dig Dis Sci 1998;43:279-289.[Medline]
  13. Ohashi K, Nemot T, Eishi Y, et al. Expression of the cyclin dependent kinase inhibitor p21WAF1/CIP1 in esophageal squamous cell carcinomas Virch Arch 1997;430:389-395.
  14. Sarbia M, Stahl M, Hausen AZ, et al. Expression of p21WAF1 predict outcome of esophageal cancer patients treated by surgery alone or by combined therapy modalities Clin Cancer Res 1998;4:2615-2623.[Abstract]
  15. Shimada Y, Imamura M, Watanabe G, et al. Prognostic factors of esophageal squamous cell carcinoma from the perspective of molecular biology Br J Cancer 1999;80:1281-1288.[Medline]
  16. Casey G, Lopez ME, Ramos JC, et al. DNA sequence analysis of exon 2 through 11 and immunohistochemical staining are required to detect all known p53 proliferations in human malignancies Oncogene 1996;13:1971-1981.[Medline]
  17. Lengauer C, Kinzler K, Vogelstein B. Genetic instabilities in human cancers Nature 1998;396:643-649.[Medline]
  18. Shackney S, Shankey T. Common patterns of genetic evolution in human solid tumors Cytometry 1997;29:1-27.[Medline]
  19. Kuwano H, Sumiyoshi K, Nozoe T, et al. The prognostic significance of the cytophotometric DNA content and it relationship with the argyrophilic nucleolar organizer regions (AgNOR) and proliferating cell nuclear antigen (PCNA) in esophageal cancer Eur J Surg Oncol 1995;21:368-373.[Medline]
  20. Blant SA, Ballini JP, Caron CTL, et al. Evolution of DNA ploidy during squamous cell carcinogenesis in the esophagus Dis Esophagus 2001;14:178-184.[Medline]
  21. AJCC cancer staging manual Digestive system, esophagus. 5th ed. Philadelphia, PA: Lippincott-Raven; 1997. pp. 65-68.
  22. Hedley DW, Friedlander ML, Taylor IW. Method for analysis of cellular DNA content of paraffin embedded pathological material using flow cytometry J Histochem Cytochem 1983;31:1333-1335.[Abstract]
  23. Brown RD, Linden MD, Mackowiak P, et al. The effect of number of histogram events on reproducibility and variation of flow cytometric proliferation measurement Am J Clin Pathol 1995;105:696-704.
  24. Shankey TV, Rabinovich PS, Bagwell B. Guidelines for implementation of clinical DNA cytometry Cytometry 1993;14:472-477.[Medline]
  25. Wang LS, Wu LH, Chang CJ, et al. Flow-cytometric DNA content analysis of esophageal carcinomacomparison between tumor and sequential non-tumor mucosa. Scand Cardiovasc J 1998;32:205-212.[Medline]
  26. Watanabe J, Kuwano H, Tanaka S, et al. Flow cytometric DNA analysis is useful in detecting multiple genetic alterations in squamous cell carcinoma of esophagus Cancer 1999;85:2322-2328.
  27. Fahn Hu, Wang LS, Huang BS, et al. Tumor recurrence in long-term survivors following treatment of carcinoma of the esophagus Ann Thorac Surg 1994;57:677-681.[Abstract]
  28. Bottger T, Storkel S, Stockle M, et al. DNA image cytometry. A prognostic tool in squamous cell carcinoma of the esophagus? Cancer 1991;67:2290-2294.[Medline]
  29. Nowak R. A new test gives early warning of a growing killer Science 1994;264:1847-1848.[Free Full Text]
  30. Shiohara M, El-Deiry WS, Wada M, et al. Absence of WAF1 mutations in a variety of human malignancies Blood 1994;84:3781-3784.[Abstract/Free Full Text]
  31. Nita ME, Nagawa H, Tominaga O, et al. p21WAF1/CIP1 expression is a prognostic marker in curatively resected esophageal squamous cell carcinoma, but not p27Kip1, p53, or Rb Ann Surg Oncol 1999;6:481-488.[Medline]
  32. Guner D, Sturm I, Hemmatt P, et al. Multigene analysis of Rb pathway and apoptosis control in esophageal squamous cell carcinoma identifies patients with good prognosis Int J Cancer 2003;103:445-454.[Medline]
  33. Rigberg DA, Frances SK, Blinman TA, et al. p21 expression in increased by irradiation in esophageal squamous cell carcinoma J Surg Res 1998;76:137-142.[Medline]
  34. Bennett WP, Hollstein MC, Metcalf RA, et al. P53 mutation and protein accumulation during multistage human esophageal carcinogenesis Cancer Res 1992;52:6092-6097.[Abstract/Free Full Text]
  35. Wynford-Thomas D. p53 in tumor pathologycan we trust immunocytochemistry?. J Pathol 1992;166:329-330.[Medline]
  36. Chang BD, Watanabe K, Broude EV, et al. Effect of p21WAF1/CIP1/Sdi1 on cellular gene expressionimplications for carcinogenesis, senescence, and age-related diseases. Proc Natl Acad Sci USA 2000;97:4291-4296.[Abstract/Free Full Text]
  37. Pan ZQ, Reardon JT, Li L, et al. Inhibition of nucleotide excision repair by the cyclin-dependent kinase inhibitor p21 J Biol Chem 1995;270:22008-22016.[Abstract/Free Full Text]
  38. Chang BD, Broude EV, Fang J, et al. p21Waf1/Cip1/Sdi1-induced growth arrest is associated with depletion of mitosis-control proteins and leads to abnormal mitosis and endoreduplication in recovering cells Oncogene 2000;19:2165-2170.[Medline]
  39. Shirakawa Y, Naomoto Y, Kimura M, et al. Topological analysis of p21WAE1/CIP1 expression in esophageal squamous dysplasia Clin Cancer Res 2000;6:541-550.[Abstract/Free Full Text]
  40. Kukreja J, Jaklitsh MT. Selective use of neoadjuvant therapy Semin Thorac Cardiovasc Surg 2003;15:187-196.[Medline]



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Is p21 an oncogene?
Mol. Cancer Ther., June 1, 2006; 5(6): 1385 - 1386.
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