|
Advertisement |
|
|
||
 |
|
|||||
|
|||||
|
 |
||||||
|
||||||
|
||||||
|
© 2003 American Society for Clinical Oncology Expression Level of Valosin-Containing Protein Is Strongly Associated With Progression and Prognosis of Gastric CarcinomaFrom the Departments of Surgery and Clinical Oncology, and Pathology, Osaka University Graduate School of Medicine, Osaka, Japan. Address reprint requests to Yasuhiko Tomita, MD, Department of Pathology (C3), Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita Osaka 565-0871, Japan; email: yt{at}molpath.med.osaka-u.ac.jp.
Purpose: Valosin-containing protein (VCP; also known as p97) was shown to be associated with antiapoptotic function and metastasis via activation of nuclear factor kappa-B signaling pathway. In this study, association of VCP expression with recurrence of gastric carcinoma (GC), in which lymphatic vessels are the main route of spread, was examined. Patients and Methods: VCP expression in 330 patients with GC (242 males and 88 females) with ages ranging from 26 to 81 years (median, 60 years) was analyzed by immunohistochemistry, in which staining intensity in tumor cells was categorized as weaker (level 1) or equal to or stronger (level 2) than that in endothelial cells. Results: Ninety-four (28.7%) patient cases showed level 1 and 233 patient cases (71.3%) showed level 2 VCP expression. Patients with level 2 expression showed higher rates of large tumor size (P < .0001), undifferentiated histologic subtype (P < .05), presence of vascular and lymphatic invasion (P < .0001 for both), presence of lymph node metastasis (P < .0001), deep tumor invasion (P < .0001), and poorer disease-free and overall survivals (P < .0001 for both) compared with those with level 1 VCP expression. Multivariate analysis revealed VCP expression level as an independent prognosticator for disease-free and overall survival. VCP level was an indicator for disease-free and overall survival in the early (pT1; P < .01 and P < .05, respectively) and advanced (pT2–4; P < .05 for both) group of pathologic tumor-node-metastasis system classification. Conclusion: The prognostic significance of VCP expression level in GC was demonstrated.
GASTRIC CARCINOMA (GC) is one of the most common causes of cancer-related death worldwide, especially in Japan and other Asian countries.1,2 The annual incidence rate of GC in Japan is about 40 per 100,000 population, and as a cancer-related cause of death, GC is ranked second.1 Introduction in Japan during the last decades of a nationwide survey system for GC that uses x-ray and endoscopic examination has enabled detection of GC at an early stage, which has resulted in improved prognosis of GC.2 However, systemic metastasis within a few years after surgery occurs occasionally, even in patients who undergo curative resection.3 In such patients, there might be occult micrometastases at the time of surgery, from which cancer cells begin to proliferate and disseminate to other organs.4–6 In this respect, an assessment of the metastatic potential in GC is important to establish appropriate therapeutic modalities. Previous studies showed several clinicopathologic factors to be prognosticators for GC. Among them, depth of cancer invasion in the stomach and lymph node metastasis are the main factors for tumor recurrence;5,7 these two factors, together with distant metastasis, were included in the pathologic tumor-node-metastasis system staging for GC.8 Because prognoses of patients with advanced disease (stage II and III) are heterogeneous, a strategy to establish appropriate therapeutic modalities for each patient has not been formulated to date.
Recently, we identified that the gene encoding valosin-containing protein (VCP; also known as p97) is associated with metastasis of the murine osteosarcoma cell line using an mRNA subtraction technique.9 VCP, one of the superfamily of adenosine triphosphatases associated with various cellular activities, is known to be involved in the ubiquitin-dependent proteasome degradation pathway of inhibitor kappa-B-alpha, an inhibitor of nuclear factor kappa-B (NF Metastasis via lymphatic vessels is the main route for GC recurrence and spread.4–6,12 To verify whether VCP expression level could be used for prediction of recurrence and prognosis in such tumors, VCP expression was analyzed in 330 patients with GC and its correlation with clinicopathologic features and patient prognosis was evaluated.
Patients A total of 364 patients underwent curative resections for GC at the Gastroenterological Surgery Division, Osaka University Hospital (Osaka, Japan) during the period from December 1993 to April 1999. Curative resection was defined as the complete resection of gastric lesions, with no tumorous lesions remaining postoperatively. Thirty-four of these patients were excluded from the present analysis because of inadequate histologic specimens in 26 patients and missing clinical information in eight patients. The remaining 330 patients were selected for this study. There were 242 males and 88 females, with ages ranging from 26 to 81 years (median, 60 years). Surgery techniques used were total gastrectomy in 98 patients, proximal gastrectomy in 24 patients, distal gastrectomy in 207 patients, partial gastrectomy in one patient, and resection of recurrent GC in the remnant stomach in three patients. Resected stomach was macroscopically examined to determine location and size of the tumor. Tumors were located in the following areas: upper third in 86 patients, middle third in 135 patients, and lower third in 109 patients. Size of main tumor ranged from 2 to 185 mm (mean, 43.8 mm). Samples obtained from the gastric lesions and dissected lymph nodes were fixed in 10% formalin and routinely processed for paraffin embedding. Histologic sections cut at 4 µm were stained with hematoxylin and eosin and immunoperoxidase procedures (avidin-biotin complex method). Histologic sections were reviewed by one of the authors (Y.H.) to define the extent and mode of cancer invasion in the stomach, lymph node metastasis, and histologic subtype of GC on the basis of the criteria of the Japanese Research Society for Gastric Cancer.13 Tumor stages were classified according to the p tumor-node-metastasis classification.8 After surgery, all patients received laboratory examinations including routine peripheral-blood cell counts and serum carcinoembryonic antigen level at 1- to 6-month intervals, chest roentgenogram, ultrasonography of liver, computerized tomographic scan of abdomen, and endoscopic examination of the rest of the stomach at 6- to 12-month intervals. Adjuvant chemotherapy was performed in 43 patients (preoperative in three patients, during surgery in one patient, postoperative in 28 patients, both pre- and postoperative in five patients, and both during surgery and the postoperative period in six patients) with a high risk for tumor recurrence (ie, presence of lymph node metastasis, large tumor size [diameter larger than 10 cm], and tumor invasion to the serosa). Chemotherapeutic protocols were as follows: fluorouracil (FU) or its derivative alone in six patients; cisplatinum (CDDP) or its derivative alone in four patients; FU and CDDP in 16 patients; FU and mitomycin (MM) in two patients, CDDP and MM in one patient; FU, CDDP, and MM in one patient; FU, CDDP, and doxorubicin (DXR) or its analogues in one patient; FU, MM, and DXR in one patient; FU, CDDP, and methotrexate (MTX) in one patient; FU, CDDP, DXR, and MM in four patients; FU, CDDP, MTX, and epirubicin (EPI) in four patients; FU, CDDP, MM, and EPI in one patient; and FU, CDDP, DXR, MM, and EPI in one patient. Follow-up period for survivors ranged from 1 to 96 months (median, 52.0 months).
Immunohistochemistry Strong correlation between VCP expression at mRNA level, determined by reverse transcriptase polymerase chain reaction or in situ hybridization, and protein level, determined by immunohistochemistry, was described previously.11,14
Statistical Analysis
Histologic Findings Histologically, 70 tumors were well differentiated, 81 tumors were moderately differentiated adenocarcinomas, two tumors were papillary carcinomas, and one tumor was a medullary carcinoma. These 154 patient cases were categorized as differentiated carcinomas. The remaining 130 patient cases with poorly differentiated adenocarcinomas, 38 with signet ring cell carcinomas, and eight with mucinous carcinomas were categorized as having undifferentiated carcinomas. Tumor cells invaded to mucosa or submucosa (pT1) in 174 patients, muscularis propria or subserosa (pT2) in 101 patients, serosa (pT3) in 43 patients, and adjacent organs (pT4) in 12 patients. Two to 101 lymph nodes (median, 23 lymph nodes) per patient were analyzed for the presence or absence of lymph node metastasis. Two hundred fourteen patients were node-negative (pN0), 65 patients had one to six positive nodes (pN1), 31 patients had seven to 15 positive nodes (pN2), and 17 had more than 15 positive nodes (pN3).
Patient Outcome
VCP Expression in GC
Uni- and Multivariate Analysis of Prognostic Factors in GC Patients The association between VCP expression and clinical factors is listed in Table 1  . In comparison to GC with level 1 VCP expression, level 2 GCs showed higher rates in the following categories: proximal location (16.9% in level 1 and 30.5% in level 2), large size (diameter larger than 50 mm; 19.1% in level 1 and 45.9% in level 2), undifferentiated histologic subtype (41.5% in level 1 and 54.1% in level 2), infiltrative growth (20.2% in level 1 and 42.1% in level 2), presence of vascular invasion (6.4% in level 1 and 34.8% in level 2), presence of lymphatic invasion (21.3% in level 1 and 70.4% in level 2), presence of lymph node metastasis (4.3% in level 1 and 46.8% in level 2), and deep invasion (pT2–4; 14.9% of level 1 and 60.5% in level 2).
Patients with level 1 GC had better 5-year survival rates than those with level 2 GC (disease-free survival, 97.8% v 67.4%, P < .0001; overall survival, 97.8% v 69.0%, P < .001; Table 2 ; Fig 2). Univariate analysis revealed that location and size of tumor, histologic differentiation, depth of tumor invasion, pattern of tumor growth, vascular invasion, lymphatic invasion, and lymph node metastasis were significant factors for both disease-free and overall survival (Table 2). Age of patients significantly affected overall survival but not disease-free survival.
Multivariate analysis with factors proven to be significant in the univariate analysis revealed that VCP expression level, tumor size, depth of tumor invasion, pattern of tumor growth, and lymph node metastasis were independent prognostic factors for disease-free and overall survival (Table 3 ). Age of patients was an independent prognosticator for overall survival but not for disease-free survival.
Prognostic Significance of VCP Expression in pT Classification Prognostic significance of VCP expression was analyzed further in patients with GC according to the pT classification.8 There was a significant difference in disease-free and overall survival between patients with VCP-expression level 1 and 2 in both early (pT1; P < .01 and P < .05, respectively) and advanced groups (pT2–4; P < .05 for both; Fig 3 ). In addition, patients with level 2 VCP expression showed lymph node metastasis more frequently than those with level 1 expression in both groups (P < .05 and P < .001, respectively; Table 4).
The conventional tumor-node-metastasis staging system is useful for prediction of disease-free and overall survival of patients with GC,8,17,18 and is used worldwide. However, there are several points to be addressed for adequate stratification of treatment for patients with early (pT1) and advanced (pT2–4) GC. For patients with early GC, recently introduced reduction surgery for GC, such as endoscopic mucosal resection (EMR), is beneficial because it enables tumor excision without significant influence on patient’s general condition.19 In contrast, existence of lymph node metastasis at the time of surgery, which was reportedly found in 4% to 5% of patients (with even early-stage GC), is problematic for the application of EMR to patients with early GC.20 The prognoses of patients with advanced GC are heterogeneous, indicating that prediction of tumor metastasis or recurrence is important to establish appropriate therapeutic modalities for these patients.7 Therefore, several biologic indices, such as aberrant expression or mutation of tumor-suppressor gene p5321,22 and adhesion molecule E-cadherin,23 DNA replication error identified as microsatellite instability,24 or DNA ploidy pattern,25 have been proposed as additional prognosticators to reinforce the tumor-node-metastasis staging system. However, prognostic significance of combined use of these factors has been controversial among previous reports.26 This study was undertaken to clarify whether VCP expression level could be a new prognostic factor for GC. The patient characteristics such as sex, age, and 5-year survival rates in the present series were similar to those in the previous reports from Japan.2,6 In Western countries, where mass screening for GC is not performed, most patients with GC are detected at an advanced stage (pT2–4) and show poorer prognosis than those in Japan. Although prognosis of GC in each stage is similar between Japan and Western countries, results obtained from this study on early and advanced GC are applicable to GC in other countries.
VCP is involved in the regulation of activation of NF In this study, VCP expression level was examined by immunohistochemical analysis. Clear correlation in VCP expression between mRNA and protein level has been reported by us in patients with hepatocellular carcinomas and Muller et al14 in murine nontumoral tissue using reverse transcriptase polymerase chain reaction and immunohistochemistry, and in situ hybridization and immunohistochemistry, respectively, indicating the reliability of immunohistochemistry for evaluation of VCP expression. Among the clinicopathologic factors examined, a significant association was observed among VCP expression and tumor size, depth of invasion, histologic differentiation, vascular and lymphatic invasion, and lymph node metastasis, indicating the close association between VCP expression and growth and invasiveness of GC. These findings are consistent with our previous reports showing the relationship of VCP overexpression with increased metastatic potential of tumor cells in the experimental metastasis model,9 and increased recurrence rate and poor prognosis of hepatocellular carcinomas in clinical analysis.11 The present uni- and multivariate analyses revealed that VCP expression level was the independent prognosticator for GC recurrence and patient survival. In addition, VCP expression level proved to be a prognosticator for recurrence of GC in patients at both the early (pT1) and advanced (pT2–4) group of pT classifications: 5-year disease-free survival rate in patients with VCP level 1 and 2 was 100% and 90.4% in the pT1 group, and 85.7% and 51.8% in the pT2–4 group, respectively. The pathologic tumor-node-metastasis classification, with the aid of VCP expression level evaluation, is a useful tool for prediction of prognosis of patients with GC.
According to these results, analysis of VCP expression in clinical decision making seems more justified by the result of VCP versus disease-free survival (Fig 3 In conclusion, VCP expression as determined by immunohistochemistry could be used as a new prognosticator for GC. Stratification of GC patients on the basis of the stage of disease and VCP expression level would be a useful tool for predicting tumor recurrence and patient prognosis. This system might provide a novel way to explore effective treatment modalities for GC.
1. Parkin DM, Whelan SL, Ferlay J, et al (eds): Cancer Incidence in Five Continents (vol VII). Lyon, France, International Agency for Research on Cancer, 1997, pp 332–445 2. Lambert R, Guilloux A, Oshima A, et al: Incidence and mortality from stomach cancer in Japan, Slovenia and the USA. Int J Cancer 97:811–818, 2002[CrossRef][Medline] 3. Roder JD, Stein HJ, Bottcher K, et al: Surgical therapy for gastric cancer. J Infus Chemother 5:97–103, 1995[Medline] 4. Maehara Y, Oshiro T, Endo K, et al: Clinical significance of occult micrometastasis lymph nodes from patients with early gastric cancer who died of recurrence. Surgery 119:397–402, 1996[CrossRef][Medline] 5. Okada Y, Fujiwara Y, Yamamoto H, et al: Genetic detection of lymph node micrometastases in patients with gastric carcinoma by multiple-marker reverse transcriptase-polymerase chain reaction assay. Cancer 92:2056–2064, 2001[CrossRef][Medline] 6. Fukagawa T, Sasako M, Mann GB, et al: Immunohistochemically detected micrometastases of the lymph nodes in patients with gastric carcinoma. Cancer 92:753–760, 2001[CrossRef][Medline] 7. Karpeh MJ, Brennan MF: Progress in the management of gastric cancer. Curr Opin Gen Surg:125–130, 1994 8. Sobin LH, Wittekind CH: TNM Classification of Malignant Tumours (ed 5). New York, NY, John Wiley & Sons, 1997, pp 59–62 9. Asai T, Tomita Y, Nakatsuka S, et al: VCP (p97) regulates NF-kappa-B signaling pathway, which is important for metastasis of osteosarcoma cell line. Jpn J Cancer Res 93:296–304, 2002[CrossRef][Medline]
10. Dai RM, Chen E, Longo DL, et al: Involvement of valosin-containing protein, an ATPase co-purified with I-kappa-B-alpha and 26 S proteasome, in ubiquitin-proteasome-mediated degradation of I-kappa-B-alpha. J Biol Chem 273:3562–3573, 1998
11. Yamamoto S, Tomita Y, Nakamori S, et al: Elevated expression of VCP (p97) in hepatocellular carcinoma correlates with increased incidence of tumor recurrence. J Clin Oncol 21:447–451, 2003 12. Stacker SA, Achen MG, Jussila L, et al: Lymphangiogenesis and cancer metastasis. Nat Rev Cancer 2:573–583, 2002[CrossRef][Medline] 13. Japanese Gastric Cancer Association: Japanese Classification of Gastric Carcinoma: 2nd English edition. Gastric Cancer 1:10–24, 1998[Medline]
14. Muller JM, Meyer HH, Ruhrberg C, et al: The mouse p97 (CDC48) gene: Genomic structure, definition of transcriptional regulatory sequences, gene expression, and characterization of a pseudogene. J Biol Chem 274:10154–10162, 1999 15. Kaplan EL, Meier P: Non-parametric estimation for incomplete observations. J Am Stat Assoc 53:457–481, 1958[CrossRef] 16. Cox DR: Regression models and life tables. J R Stat Soc 34:187–220, 1972 17. Kikuchi S, Sakakibara Y, Sakuramoto S, et al: Recent results in the surgical treatment of gastric cancer according to the Japanese and TNM classification. Anticancer Res 21:3589–3593, 2001[Medline] 18. D’Ugo D, Pacelli F, Persiani R, et al: Impact of the latest TNM classification for gastric cancer: Retrospective analysis on 94 D2 gastrectomies. World J Surg 26:672–677, 2002[Medline] 19. Fujino MA, Morozumi A, Kojima Y, et al: Gastric carcinoma, an endoscopically curable disease. Bildgebung 61:38–40, 1994 (suppl 1) 20. Sano T, Sasako M, Kinoshita T, et al: Recurrence of early gastric cancer: Follow-up of 1475 patients and review of the Japanese literature. Cancer 72:3174–3178, 1993[CrossRef][Medline] 21. Martin HM, Filipe MI, Morris RW, et al: p53 expression and prognosis in gastric carcinoma. Int J Cancer 50:859–862, 1992[Medline] 22. Lim BH, Soong R, Grieu F, et al: p53 accumulation and mutation are prognostic indicators of poor survival in human gastric carcinoma. Int J Cancer 69:200–204, 1996[CrossRef][Medline] 23. Shiozaki H, Tahara H, Oka H, et al: Expression of immunoreactive E-cadherin adhesion molecules in human cancers. Am J Pathol 139:17–23, 1991[Abstract]
24. Oliveira C, Seruca R, Seixas M, et al: The clinicopathological features of gastric carcinomas with microsatellite instability may be mediated by mutations of different "target genes": A study of the TGF-beta RII, IGFII R, and BAX genes. Am J Pathol 153:1211–1219, 1998 25. Russo A, Bazan V, Migliavacca M, et al: DNA aneuploidy and high proliferative activity but not K-ras-2 mutations as independent predictors of clinical outcome in operable gastric carcinoma: Results of a 5-year Gruppo Oncologico dell’Italia Meridonale (GDIM) prospective study. Cancer 92:294–302, 2001[CrossRef][Medline] 26. Fiocca R, Luinetti O, Villani L, et al: Molecular mechanisms involved in the pathogenesis of gastric carcinoma: Interactions between genetic alterations, cellular phenotype and cancer histotype. Hepatogastroenterology 48:1523–1530, 2001[Medline] 27. Mayo MW, Baldwin AS: The transcription factor NF-kappa-B: Control of oncogenesis and cancer therapy resistance. Biochim Biophys Acta 1470:M55–M62, 2000[Medline]
28. Andela VB, Schwarz EM, Puzas JE, et al: Tumor metastasis and the reciprocal regulation of prometastatic and antimetastatic factors by nuclear factor kappa B. Cancer Res 60:6557–6562, 2000
29. Sasaki N, Morisaki T, Hashizume K, et al: Nuclear factor-kappa B p65 (RelA) transcription factor is constitutively activated in human gastric carcinoma tissue. Clin Cancer Res 7:4136–4142, 2001
30. Macdonald JS, Smalley SR, Benedetti J, et al: Chemoradiotherapy after surgery compared with surgery alone for adenocarcinoma of the stomach or gastroesophageal junction. N Engl J Med 345:725–730, 2001 Submitted December 17, 2002; accepted April 14, 2003.
This article has been cited by other articles:
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
|||||||||||
|
|||||||||||
|
|
Copyright © 2003 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
|
TOP
ABSTRACT
INTRODUCTION
B).
2 test and Fisher’s exact test were used to analyze the association between VCP expression measured by immunohistochemistry and clinicopathologic features of GC. Kaplan-Meier methods with the log-rank test were used to calculate survival rates and differences in survival curves.
