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Ann Thorac Surg 1999;68:2021-2024
© 1999 The Society of Thoracic Surgeons

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Original Articles: General Thoracic

P53 gene protein overexpression predicts results of trimodality therapy in esophageal cancer patients

^a Division of Thoracic Surgery and Department of Medicine (Gastroenterology Division), University of Maryland School of Medicine and Veterans Affairs Hospital, Greenebaum Cancer Center, Baltimore, Maryland, USA

Address reprint requests to Dr Krasna, Division of Thoracic Surgery, University of Maryland Medical Center, 22 South Greene St, Room N4W94, Baltimore, MD 21201
e-mail: mkrasna{at}surgery1.ab.umd.edu

Presented at the Thirty-fifth Annual Meeting of The Society of Thoracic Surgeons, San Antonio, TX, Jan 25–27, 1999.

Abstract

Background. P53 protein overexpression in esophageal cancer and its correlation with response and survival after chemoradiation was retrospectively investigated.

Methods. Pretreatment and resection specimens were stained by automatic p53 immunohistochemical staining technique.

Results. P53 was expressed in 84.0% of esophagoscopy (EGD) biopsies; 71.4% of patients with metastasis of thoracoscopy/laparoscopy lymph nodes (TS/LS LN) identified by hematoxylin/eosin (H/E) were p53 (+); 14.2% of patients with negative TS/LS LN by H/E were p53 (+). Eleven out of 18 patients with p53 (+) in pretreatment EGD remained p53 (+) after chemoradiation; 38.8% of these patients had a pathological complete response (pCR). The median survival of this group was 15 months. Of 4 patients with p53 (-) pretreatment EGD, all of those were still p53 (-) after chemoradiation; 75% of these patients had pCR. The median survival was 30 months. In patients with p53 (+) TS/LS LN, 23% had a pCR after chemoradiation with a median survival of 16 months. In patients with p53 (-) TS/LS LN, 50.0% had a pCR with a median survival of 31.5 months.

Conclusions. P53 protein overexpression in pretreatment EGD and TS/LS LN may predict response to chemoradiation and survival in esophageal cancer patients.

Esophageal cancer is the fastest growing malignancy in the United States with 10,000 to 11,000 deaths per year [1]. The results of single modality treatment using surgery, chemotherapy, or radiotherapy alone have been poor because of a high rate of local recurrence and distant metastasis [2–5]. This is probably due to the prevalence of advanced esophageal cancer at the time of diagnosis. Even though a small number of esophageal cancer patients survive longer than 5 years after initial surgical treatment, over 60% of patients still die of metastasis and local recurrence in one series [3]. It is therefore imperative to investigate combined modality therapy in the treatment of esophageal cancer. In recent years, neoadjuvant preoperative chemotherapy, radiation therapy, and combined chemoradiation have been added to the treatment of this disease in order to enhance local control, increase resectability rate, and improve disease-free survival [5]. Although trimodality therapy (chemoradiation followed by surgical resection) to treat esophageal cancer has been reported, about 50% of patients who undergo chemoradiation have no response or only partial response [6–8].

The survival of patients without a response was worse than that of patients with a pathological complete response (pCR). All these patients still suffer from the toxicity of chemoradiation and higher risk of postoperative morbidity and mortality. If we could predict esophageal cancer patients’ response to chemoradiation from pretreatment specimens, their morbidity from chemoradiation might be spared.

P53 gene is an important gene in repairing cellular DNA and triggering apoptosis after cellular DNA is injured [9]. It has been suggested that it might play an important role during chemoradiation [10–12]. In recent years, several investigators have found that p53 gene protein is one of the biomarkers that correlate with the prognosis of various cancers [13–16]. This retrospective study was designed to investigate the prevalence and changes of p53 gene protein expression before and after chemoradiation in esophageal cancer patients, and its correlation with chemoradiation response and survival.

Material and methods

Immunohistochemical staining technique
Five thick sections were cut from formalin fixed and paraffin embedded specimen blocks of 37 esophageal cancer patients, which included all available pretreatment esophagoscopy (EGD) biopsy, thoracoscopic/laparoscopic lymph node staging biopsies (TS/LS LN), and postchemoradiation esophagectomy specimens. Standard programmed automatic immunohistochemical (IHC) staining technique for p53 protein was performed using a VENTANA ES IHC staining machine (Ventana Medical Systems, Tucson, AZ). Positive and negative controls from lung tumor tissue for each run (40 slides) were used for quality control. The antibody used in this series was Do-7, M7001 with a dilution of 1:50 (Dako, Denmark). The slides were interpreted and scored separately by three experienced pathologists as 1 (< 10%), 2 (10% to 60%) and 3 (> 60%) for p53 positive staining cell distribution, and 1 (weak), 2 (moderate), and 3 (strong) for intensity of p53 staining. Slides were reviewed by three independent pathologists.

Clinical data
Thirty-seven esophageal cancer patients underwent surgery with or without neoadjuvant chemoradiation. There were 31 male and 6 female patients with a mean age of 60 (ranged from 41 to 75). Thirty-two patients had trimodality therapy consisting of preoperative chemoradiation and surgery; the first 5 patients had surgery alone. All patients underwent clinical staging by computed tomography and esophageal ultrasound (CT/EUS) and minimally invasive staging by thoracoscopy/laparoscopy (TS/LS). In this series, 25 patients had pretreatment EGD specimens available for p53 staining examination. Of those 25 patients, 22 patients had trimodality therapy. Thirty-five patients had TS/LS LN staging biopsy specimens available for p53 staining. (The other 2 patients’ blocks from TS/LS were not available.) Thirty-one trimodality therapy patients had postchemoradiation resection specimens available for p53 staining.

The chemoradiation regimen used was CDDP/100 mg/m² on day 1, and 5-FU/1,000 mg/m² on days 1 through 4, in weeks 1 and 5. This was combined with concurrent radiation 50.4 Gy in 1.8 Gy fraction, 5 days a week for 5 weeks. The radiation field was modified according to thoracoscopy/laparoscopy TNM staging, as previously described [17]. All of the patients underwent esophagectomy through a right thoracotomy and abdominal incision (Ivor–Lewis). The follow-up period was 4 to 56 months. Statistical analysis using student t test and ² test was performed. Actuarial survival was performed using Kaplan–Meier life tables method with a computerized (SAS) program.

Results

Of 25 esophageal cancer patients with available pretreatment EGD specimens, p53 protein was expressed in 84.0% (21 of 25) of EGD specimens with a rate of 80.0% (8 of 10) in adenocarcinoma and 86.6% (13 of 15) in squamous cell carcinoma. Sixteen of 37 (43.2%) patients had LN metastasis by H/E. At the time of TS/LS LN biopsy, 14 of 16 patients had available specimens; 71.4% (10 of 14) of these LN were p53 (+). The remaining 21 patients were HE (-) in the TS/LS LN biopsies; 14.2% of these patients’ TS/LS LN (3 of 21) were also p53 (+).

Twenty-two patients treated by trimodality had available pretreatment EGD specimens for p53 staining. Eighteen of these 22 patients were p53 (+) in the pretreatment EGD specimen (Table 1). After chemoradiation, 11 of 18 (61.1%) patients remained HE (+) and p53 (+). Seven patients had a pathological complete response (pCR) with a pCR rate of 38.8%. The median survival of this group was 15 months. The actuarial 3-year survival was 15.0%. Four patients were HE (+) but p53 (-) in their pretreatment EGD specimens. After chemoradiation, only 1 patient remained HE (+), but all of these patients still remained p53 (-). Three patients had pCR with a pCR rate of 75.0% (3/4). The median survival was 30 months and actuarial 3-year survival was 50.0% in this group. There was an apparent difference in pCR rate and survival between those two groups of patients but it was not statistically significant.

Comment

Since the results of single modality therapy such as surgery, chemotherapy, and radiation for esophageal cancer has been poor [2–5], the effect of neoadjuvant therapy for esophageal cancer has been investigated in many recent clinical trials [5–8]. Compared with therapy including chemotherapy or radiation followed by resection, trimodality therapy consisting of chemoradiation followed by surgery seems to be more promising in esophageal cancer patients [6–8]. It has been reported that a pathological complete response can be achieved in about 20% to 50% of esophageal cancer patients after chemoradiation. Those patients with a pCR have been proven to have a much better survival rate than the patients without a complete pathological response. It is therefore imperative to separate the responders from the nonresponders so that we can spare the nonresponders unnecessary toxicity of chemoradiation and avoid the high risk of postoperative morbidity and mortality.

The p53 gene has been shown to be the most widely mutated gene in many kinds of human cancer including esophageal cancer. It is one of the important genes in normal cells responsible for repairing damaged cells’ DNA or triggering apoptosis when the damaged cells’ DNA cannot be repaired [9]. When a tumor has a mutated p53 gene, it likely has a poorer prognosis and risk of early metastasis [12–16]. The ability of chemoradiation to control tumor growth is mainly through damaging the tumor cells’ DNA. It is therefore presumed that the p53 gene may play an important role during chemoradiation.

Our results show that overexpression of p53 protein correlates with a poorer response to chemoradiation and worse survival in esophageal cancer patients treated by trimodality therapy. Of 18 patients with overexpression of p53 protein in pretreatment EGD specimens, only 38.8% of the patients had a pCR after chemoradiation. The median survival in this group was only 15 months. However, in 4 patients who were p53 negative in pretreatment EGD specimens, 75.0% of the patients had a pCR after chemoradiation and their median survival was 30 months. The difference in pCR rate and survival was impressive but not statistically significant due to the small number of P53 (-) patients. When, we looked at patients with p53 (+) LN, 13 of 31 patients were p53 (+) with HE (+) or (-) in their pretreatment TS/LS LN biopsy specimens. After chemoradiation, only 3 of these 13 patients had a pCR with a pCR rate of 23.1% (3 of 13). The median survival in this group was 16 months. The other 18 patients were p53 (-) in their TS/LS LN biopsy specimens before any treatment. After chemoradiation, 9 patients became pCR with a pCR rate of 50.0%. The median survival of those patients was 31.5 months. This difference in pCR rate and survival between the groups was only marginally significant because of the small number of patients in each group.

It has been demonstrated that patients with mutated p53 gene, proven by polymerase chain reaction and sequencing, had much poorer survival than those with wild type p53 in lung and esophageal cancers [12, 18]. The correlation between overexpression of p53 protein with chemoradiation response and poor prognosis in many reports including esophageal cancer is quite controversial [19–22]. Although 70% to 80% of esophageal cancer patients have p53 gene protein overexpression, not all of them have mutated p53 gene [12, 23]. It was reported by Coggi and associates that 53% of esophageal adenocarcinoma patients had mutated p53 gene by PCR and sequencing and 57% had p53 protein overexpression by immunohistochemical staining. However, there were 38% discordant cases in this series [23]. The same findings have been reported in lung cancer patients with a discordant rate of 40% to 70% [24]. In order to clarify whether overexpression of p53 gene protein can be an important biomarker to predict the response of chemoradiation, analysis of fresh specimens from trimodality therapy patients using pCR and sequencing technique is mandatory. For this purpose, we are now collecting fresh tissue from the patients treated with esophageal cancer and will study the gene sequence in our p53 (+) patients.

Since about 70% to 80% of esophageal cancer patients have p53 gene protein overexpression in pretreatment specimens [11, 22, 23], it may be possible to use p53 to detect micrometastasis in TS/LS LN. The significance of this finding has been already reported in lung cancer patients [25]. In the current series of 37 esophageal cancer patients, 84.0% of pretreatment patients’ EGD had p3 protein overexpression; 43.2% of patients had LN metastasis by H/E in TS/LS LN biopsy. Although the remaining 21 patients were HE (-) in the TS/LS LN biopsies, 14.2% of them were p53 (+). It is not yet clear whether this truly represents micrometastasis, although, in terms of prognosis, this subset of patients did have a less favorable outcome. Since some lymphocytes may be p53 staining positive [26], the significance of p53 staining for detecting micrometastasis in negative lymph nodes needs to be clarified by other biomarkers such as CK-18 and Ber-Ep4.

We anticipate that, in the future, prognostic groups can be identified using molecular biomarkers with IHC staining of patients’ pretreatment EGD and TS/LS LN. It is apparent that the presence of these biomarkers in LN will also be an important prognosticator. Pretreatment LN staging would then allow us to further prognosticate patients based on biomarkers for poor outcome. In this way, poor-risk patients might be spared the morbidity of combined modality therapy. Alternatively, this approach can perhaps identify responders posttreatment and allocate which patients should go onto surgical resection. The ultimate role of trimodality therapy in esophageal cancer will only be clarified by large prospective, randomized trials such as the Intergroup Cancer of the Esophagus Trial (ICE-T): CALGB 9781. This study also includes an optional correlative science comparison that will investigate the relationship between biomarkers, response, and survival.

In conclusion, p53 had a high rate of expression in pretreatment EGD biopsies of esophageal cancer patients. Chemoradiation apparently does not change the p53 expression in the residual tumor. P53 gene protein expression in preoperative EGD and LN biopsy specimens may be a useful predictor for the effect of chemoradiation and survival. Presence of p53 in HE (-) TS/LS LN samples deserves to be further investigated with other biomarkers for possible occult micrometastases.

Acknowledgments

This study was supported in part by UICC–ICRETT grant 577, CA85069, and the Office of Medical Research, Department of Veterans Affairs. Thanks to Ms Carol Schuder for preparation of the manuscript, and to Ms Julie Schuetz and Dr Timothy Chen for analysis of the survival data.

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