A comparison of patterns of care of nonsmall cell lung carcinoma patients in a younger and Medigap commercially insured cohortкод для вставкиСкачать
1917 Expression of p53 Protein and Resistance to Preoperative Chemotherapy in Locally Advanced Gastric Carcinoma Stefano Cascinu, M.D.1,2 Francesco Graziano, M.D.1,2 Elena Del Ferro, Sc.D.1,2 Maria P. Staccioli, Sc.D.3 Marco Ligi, Sc.D.1,2 Andrea Carnevali, M.D.3 Pietro Muretto, M.D.3 Giuseppina Catalano, M.D.2 1 Section of Experimental Oncology, Azienda Ospedaliera S. Salvatore, Pesaro, Italy. 2 Division of Medical Oncology, Department of Hematology/Oncology, Azienda Ospedaliera S. Salvatore, Pesaro, Italy. 3 Department of Pathology; Azienda Ospedaliera S. Salvatore, Pesaro. Italy. BACKGROUND. Inactivation of the p53 gene has been reported to be associated with resistance to chemotherapy. The authors evaluated the significance of p53 status to the clinical outcomes of patients with locally advanced, unresectable gastric carcinoma (LAGC) who received chemotherapy. METHODS. Thirty chemotherapy-naive patients with LAGC received weekly administration of cisplatin 40 mg/m2, epi-doxorubicin 35 mg/m2, 5-fluorouracil 500 mg/m2, 6S-leucovorin 250 mg/m2, and glutathione 1500 mg/m2. After eight administrations of these agents, patients were assessed for response. Biopsy specimens of primary tumors were analyzed for p53 status using monoclonal antibody Bp53–12. RESULTS. Characteristics of patients were as follows: The median age was 66 years (range, 44 –70 years); 18 were males and 12 were females. Eastern Cooperative Oncology Group performance status was 0 for 14 patients and 1 for 16. Histology was intestinal for 13 patients; for 17, it was diffuse. The site of the primary tumor was the cardia in 8 patients, the body of the stomach in 13, and the antrum in 9. The response rate (assessed with CT scan and endoscopy) for patients with p53 negative tumors was significantly higher than for those with overexpression of p53 (71% vs. 12%, P ⫽ 0.004). CONCLUSIONS. p53 status analyzed before chemotherapy seems to be associated with response to treatment in patients with LAGC. This may provide a useful guide to selecting neoadjuvant chemotherapy for these patients. Cancer 1998;83: 1917–22. © 1998 American Cancer Society. KEYWORDS: p53, locally advanced gastric carcinoma, chemotherapy. A Address for reprints: Stefano Cascinu, M.D., Section of Experimental Oncology, Department of Hematology/Oncology, Azienda Ospedaliera S. Salvatore. v. Lombroso, 61100 Pesaro. Italy. Received December 23, 1997; revision received April 9, 1998; accepted April 9, 1998. © 1998 American Cancer Society lthough gastric carcinoma is declining in incidence, it remains a significant cause of mortality.1 Many patients present with locally advanced disease; and in more than two-thirds of cases, local extension prevents curative resection. Unfortunately, resectability is one of the main prognostic factors in patients with gastric carcinoma, and survival is longer when tumors are completely removed.2 The use of neoadjuvant chemotherapy is an attractive idea for increasing curative resection. Although this approach determined about 50 – 60% of clinical responses and allowed radical surgery in 40 –50% of patients with previously unresectable tumors,3– 8 chemotherapeutic regimens currently administered to gastric carcinoma patients have substantial toxicities, so that identification of responsive patients appears worthwhile to avoid the side effects associated with chemotherapy in unresponsive patients. Currently, the use of clinical parameters cannot accurately predict which patients may be best served by preoperative chemotherapy. 1918 CANCER November 1, 1998 / Volume 83 / Number 9 TABLE 1 Patient Characteristics Characteristics No. of patients Total no. of patients Age (yrs) Median Range Gender Male/female Site of primary tumor Cardia Body of stomach Antrum Histologic type Intestinal Diffuse ECOG performance status 0 1 30 66 44–70 18/12 8 13 9 13 17 14 16 ECOG: Eastern Cooperative Oncology Group. The p53 protein is a pleiotropic molecule with numerous functions. One of these functions involves the pathway of programmed cell death induced by DNA-damaging chemotherapeutic drugs.9 It has been demonstrated that p53-dependent apoptosis modulates the cytotoxic effects of common antitumor agents such as 5-fluorouracil, doxorubicin, and cisplatin.10 Cells lacking wild-type p53 are resistant to these agents, and p53 assay could be used to predict the response of cancer to chemotherapy.11,12 This association was recently found in nonsmall cell lung carcinoma and ovarian carcinoma.13,14 Nuclear overexpression of p53 protein determined by immunohistochemistry (IHC) occurs in approximately 50% of gastric carcinomas and is an independent prognostic factor for relapse and survival.15–18 To test the hypothesis that p53 alteration could lead to resistance to cytotoxic therapy and also predict responsiveness in gastric carcinoma patients, we examined the p53 status of patients with locally advanced, unresectable gastric carcinoma who received preoperative chemotherapy including cisplatin, 5-fluorouracil (5-FU), and epi-doxorubicin (epi-ADR). PATIENTS AND METHODS Patients’ Characteristics and Treatment Table 1 shows the characteristics of the 30 patients included in this study. All patients had locally advanced, unresectable gastric carcinoma confirmed pathologically. In 14 patients, the diagnosis was based on a computed tomography (CT) scan that evaluated for tumor size, invasion of adjacent structures, or advanced locoregional lymph node involvement, whereas in the other 16 patients locally advanced disease was confirmed by laparotomy. The chemotherapeutic regimen consisted of a 1-day weekly administration of cisplatin (CDDP) 40 mg/m2 as a 30-minute infusion in 250 mL of normal saline solution, 5-FU 500 mg/m2 as a 15-minute infusion in 100 mL of normal saline solution, and epi-ADR 35 mg/m2 by intravenous bolus. 6S-stereoisomer of leucovorin was administered at a dose of 250 mg/m2 diluted in 250 mL of normal saline solution in a 4-hour infusion concurrent with hydration. Glutathione was given at a dose of 1.5 g/m2 in 100 mL of normal saline over 15 minutes immediately before each CDDP administration. Standard intravenous hydration was used: 2 hours before initiation of the CDDP infusion, patients were hydrated with 1500 mL of 0.9% sodium chloride, to which 20 mEq of potassium chloride and 15 mEq of magnesium sulfate were added. Posthydration was continued for 2 hours with 1000 mL of normal saline solution. From the day after to the day before each chemotherapy administration, filgastrim was administered by subcutaneous injection at a dose of 5 g/kg. Evaluation of response was performed after eight weekly treatments. Patients were required to have CT scan and endoscopic evaluation with biopsy if tumor was visible. Partial response (PR) was defined as evidence on both CT scan and endoscopy of a ⬎50% reduction in the visible tumor or complete disappearance of tumor but positive histology on biopsy of the previously involved area. Complete response (CR) was defined as a normal-appearing stomach on CT scan with a complete resolution of the endoscopically visible tumor and a negative biopsy of the original site of the tumor. Tumor Specimen Selection For each patient, three endoscopic samples were collected and examined. Tumor sampling was supervised by a pathologist. Sections of the samples were examined by conventional histology and were used for immunohistochemistry. Immunohistochemistry Formalin fixed, paraffin embedded tissue sections from the tumors were analyzed immunohistochemically for altered patterns of p53 expression, using a standard avidin-biotin technique. Sections (4 m thick) were deparaffinized in xylene, rehydrated in a graded ethanol series, and incubated in 3% hydrogen peroxide for 20 minutes. Specimens were placed in a plastic Coplin jar containing citric buffer and heated p53 in Locally Advanced Gastric Carcinoma/Cascinu et al. 1919 FIGURE 1. Positive (A) and negative (B) immunohistochemical staining with monoclonal antibody Bp53–12 are shown. 4 ⫻ 2.5 minutes in a microwave processor at 95°C. After the microwave processing, sections were left in the Coplin jar at room temperature for 30 minutes. Specimens were covered with normal goat serum for 15 minutes to reduce nonspecific staining and incubated with a 1:100 dilution of primary antibody Bp53–12 (Biogenex, San Ramon, CA) at room temperature for 2 hours. The sections were washed with Tris-buffered saline, incubated with a 1:100 dilution of biotinylated goat antimouse immunoglobulin G at room temperature for 30 minutes, and then covered with a 1:100 dilution of streptavidin-biotin-peroxidase complex at room temperature for 30 minutes. The antibody was localized with 3,3⬘-diaminobenzidine tetrahydrochloride (DAB). Tissue sections were counterstained with light hematoxylin, dehydrated with ethanol, and mounted under a coverslip.19 Immunohistochemical staining of tumors with this antibody shows primarily a nuclear localization of p53 protein. Bp53–12 reacts with both wild-type and mutant p53 protein. The staining results were interpreted independently by one investigator who was unaware of the clinical outcome. In each case, the entire section was systemically examined on high-power fields (⫻40) for p53 immunoreactivity. Among all immunoreactive nuclei, only those clearly immunostained were recorded as being p53 positive. The level of immunoreactivity was expressed as the percentage of p53 positive cancer cell nuclei. For analysis, tumors were classified into p53 immunoreactivity categories of low level (negative or ⬍20% positive nuclei) and high level (⬎20% positive nuclei). The patients were cross-classified by p53 expression and by clinical responses to chemotherapy. Fisher’s exact test was used to evaluate the statistical significance. P ⬍ 0.05 was considered significant.20 RESULTS Adequate pathologic material for this study was available in at least 2 of the 3 endoscopic samples from all of the 30 patients entered into the study of preoperative chemotherapy. A high level of p53 immunoreactivity was seen in 16 of 30 patients (53%) (Fig. 1). Intratumor heterogeneity of p53 expression was systemically assessed in the different endoscopic samples, without finding variations able to modify the interpretation of p53 status in each tumor. Correlation of p53 status with other histologic and 1920 CANCER November 1, 1998 / Volume 83 / Number 9 TABLE 2 p53 Protein Expression and Clinicopathologic Findings DISCUSSION p53 Characteristics Age (yrs) ⬍60 ⬎60 Gender Male Female ECOG performance status 0 I Histology Diffuse Intestinal Site of primary Cardia Body of stomach Antrum Tumor size (cm) ⬍5 ⬎5 Clinical outcome Response No response Positive Negative % positive P value 5 11 4 10 55 55 ns 10 6 8 6 55 50 ns 7 9 7 7 50 56 ns 9 7 8 6 52 53 ns 5 6 5 3 7 4 62 46 55 9 7 8 6 52 53 ns 2 14 10 4 16 77 0.004 ns ECOG: Eastern Cooperative Oncology Group; ns: not statistically significant. clinical variables was reported in Table 2. No association was seen among p53 immunoreactivity and any of variables analyzed, apart from clinical response. Among the 30 patients who received chemotherapy and had an assessment of p53 immunoreactivity, 12 patients achieved an objective response (3 CRs) (40%), 13 had stable disease, and 5 experienced disease progression during therapy. Ten of 12 patients who showed a clinical response had p53 negative tumors, whereas 14 of 18 nonresponders had p53 positive tumors. The 3 patients who achieved a CR had the lowest levels of p53 (⬍5%, ⬍10%, and ⬍10%, respectively). Eleven patients (36%) underwent radical surgery. Nine of them are alive and disease free at a median follow-up of 20 months. Two relapsed (peritoneum and liver) and died 8 and 10 months after surgery, respectively. In these two patients, p53 expression was 20% and 50%, respectively. In nine of the patients who underwent surgery, p53 expression status was assessed in the resection specimens; in two cases it was not performed because of the pathologic CR status of the patients. A high level of p53 immunoreactivity (ⱖ50%) was seen in 7 patients, whereas in the other 2 cases it was considered lower than the cutoff. Preoperative chemotherapy seems to be a logical approach to improving surgical resectability that is one of the main prognostic factors in patients with gastric carcinoma.2 However, only 40 –50% of patients take advantage of this therapeutic modality, whereas approximately 30% of patients experience moderate-tosevere toxic effects.5– 8 Understanding the molecular genetic features that determine response or resistance to chemotherapy should permit the selection of the most suitable patients for neoadjuvant therapy. This would avoid exposing some patients to ineffective treatment and could tailor chemotherapy treatment to those cancer patients most likely to respond. In vitro studies indicate that p53-dependent apoptosis modulates the cytotoxicity of chemotherapeutic agents such as 5-FU, doxorubicin, and CDDP, and that the absence of wild-type p53 function results in cellular resistance to chemotherapy in several cell lines, including gastric carcinoma.10 –12,21 Overexpression of p53 protein as the result of an altered gene has been demonstrated in 40 – 60% of primary gastric carcinomas.15–18 Hamada et al. reported that p53 status seemed to predict response to preoperative chemotherapy in three patients with gastric carcinoma.22 Similar data were also reported by Nakata et al. for 15 patients who received preoperative chemotherapy with 5-FU and CDDP. Four of the six patients who responded to chemotherapy were p53 negative, whereas all of nonresponders overexpressed p53 protein.23 The current report extends previous works by exploring the correlation between p53 expression in tumors and clinical treatment response to chemotherapy in locally advanced gastric carcinoma. The results of this study show clearly that the absence of p53 immunostaining before chemotherapy correlates with clinical response to neoadjuvant chemotherapy in this clinical setting. Another finding seems to confirm indirectly that inactivation of the p53 gene contributes to enhanced cellular resistance to chemotherapy. In seven of nine resected specimens, determination of p53 levels demonstrated higher values than endoscopic specimens and all of them were higher than the cutoff value, suggesting that chemotherapy kills mainly p53 negative cells. However, some caution is required in the interpretation of these data, because some factors could result in misinterpretation. Immunohistochemical staining does not distinguish p53 expression that is abnormal because of p53 gene mutations from expression that is abnormal because of deregulated expres- p53 in Locally Advanced Gastric Carcinoma/Cascinu et al. sion of a structurally normal p53 protein.24 Although molecular studies have confirmed point mutations in the gene to be the most usual cause of p53 accumulation,25,26 other events can also result in overexpression of the protein (i.e., changes in the cellular environment resulting from DNA-damaging events).27 Nevertheless, p53 protein accumulation, whether it is dependent on gene alteration or not, could be the main prognostic factor affecting response to chemotherapy, as recently found for ovarian carcinoma.14 In fact, as stabilization of a mutant form of p53 abrogates the normal function of the protein as an inducer of apoptosis, overexpression of a wild-type p53 could increase the repair efficiency of the cell, thus reducing the cytotoxic effects of the DNA-damaging agents.14 The choice of Bp53–12 antibody was unlikely to influence results of p53 analysis. In fact, although for gastric carcinoma there are no data, Baas et al. showed that six different antibodies (including Bp53–12) gave the same results in colorectal carcinoma.28 A critical point in this study could be the choice of a higher cutoff value (20%) than that generally used to analyze the prognostic implications of p53 overexpression in gastric carcinoma (10%).17,29 In our opinion, because the aim of preoperative chemotherapy for patients with gastric carcinoma is to reduce tumor size in order to increase the chance of a curative resection, focally positive areas that result from a small subclone of cells with mutations and are potentially unresponsive to chemotherapy should not preclude the effectiveness of a preoperative chemotherapeutic program. However, a similar cutoff value was used by Hamada et al. to assess the role of p53 expression as a determinant of chemosensitivity in gastric and colorectal carcinoma.22 Furthermore, for other tumors, such as nonsmall cell lung carcinoma and bladder carcinoma, in which the correlation between p53 overexpression and preoperative chemotherapy was investigated, the cutoff value was similarly defined as 20% of positive cells.13,30 In conclusion, the results of the current study are consistent with the idea that p53 is a direct determinant of the chemosensitivity of gastric carcinoma. These data may have clinical relevance for implications in the treatment of locally advanced gastric carcinoma. Additional studies are needed to identify other cell cycle regulators that act in concert with or independently from p53 (such as Rb and cyclin-dependent kinase) and may contribute to the modulation of clinical response to chemotherapy. 1921 REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. Parkin DM, Laara E, Muir CS. Estimates of the worldwide frequency of sixteen majors cancers in 1980. Int J Cancer 1988;41:184 –7. Roder JD, Bottcher K, Siewert JR, Busch R, Hermanek P, Meyer HJ. Prognostic factors in gastric carcinoma. Cancer 1993;72:2089 –97. Wilke H, Preusser P, Fink U, Achterrath W, Meyer HJ, Stahl M, et al. New developments in the treatment of gastric carcinoma. Semin Oncol 1990;17(Suppl 2):61–70. Ajani JA, Roth JA, Ryan MB, Putnam JB, Pazdur R, Levin B, et al. Intensive preoperative chemotherapy with colony-stimulating factor for resectable adenocarcinoma of the esophagus or gastroesophageal junction. J Clin Oncol 1993;11: 22– 8. 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