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Keywords:

  • caveolin-1;
  • esophageal carcinoma;
  • lymph node metastasis;
  • prognosis

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. STATISTICAL ANALYSIS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

BACKGROUND

Caveolin-1 plays a regulatory role in several signaling pathways. Recently caveolin-1 was identified as a metastasis-related gene in prostate carcinoma. However, the clinical impact of caveolin-1 expression in esophageal carcinoma remains unknown.

METHODS

One hundred thirty surgical specimens of esophageal squamous cell carcinoma were immunohistochemically assessed for caveolin-1 expression by polyclonal antibody. Caveolin-1 immunoreactivity was correlated with patients' clinicopathologic parameters and outcome.

RESULTS

Positive caveolin-1 immunostaining was detected in 58 patients (44.6%). Positive caveolin-1 immunostaining correlated positively with pathologic stage (P = 0.029), pN (P = 0.023), and pM (P = 0.018). The overall survival rate was worse in patients with caveolin-1-positive tumors than in patients with caveolin-1-negative tumors (P = 0.0215). Univariate analyses identified caveolin-1 positivity (P = 0.0238), pT (P = 0.0002), pN (P < 0.0001), pM (P = 0.0002), lymphatic invasion (P = 0.0021), and positive surgical margin (P < 0.0001) as negative prognostic predictors. Multivariate analyses indicated that pT (P = 0.0296), pN (P = 0.0003), and a positive surgical margin (P = 0.0452) were independent prognostic factors.

CONCLUSIONS

Over-expression of caveolin-1 is associated with lymph node metastasis and a worse prognosis after surgery in esophageal squamous cell carcinoma. Cancer 2002;94:929–33. © 2002 American Cancer Society.

DOI 10.1002/cncr.10329

Esophageal carcinoma remains a tumor with a poor prognosis. Advances in surgical technique and perioperative management have improved survival to some extent. However, the overall 5-year survival rate generally remains less than 50%, despite the use of multimodality therapy.1–3

Lymph node metastasis is the principal negative prognostic factor in this disease.2, 4 Therefore, it is important to determine whether lymph node metastases have occurred when developing a therapeutic strategy. However, it is difficult to determine whether lymph node metastasis has occurred preoperatively, in spite of new imaging techniques, such as positron emission tomography.5, 6 Thus, the identification of a marker that predicts the lymph node metastasis, and hence prognosis, is highly desirable.

Caveolin is a 21 to 24 kDa molecule and is a principal component of caveolae membranes. Caveolin participates in vesicular trafficking events and transduction processes. Caveolin-1 plays a regulatory role in several signaling pathways, such as Src family tyrosine kinase, epidermal growth factor receptor, Neu/HER2 (c-erbB2), protein kinase C, transforming growth factor β/SMAD, and the Wnt/beta-catenin/lef-1 pathway.7–9 It has been reported that caveolin-1 is a metastasis-related gene and is an independent predictor for time to disease progression in prostate carcinoma.10 Caveolin-1 expression also is increased in primary and metastatic breast carcinoma.11 However, it is not known whether the expression of this antigen in other organs is related to tumor progression and prognosis; at present, the physiologic consequences of caveolin-1 over-expression remain controversial.

In the current study, we correlated caveolin-1 immunoreactivity in archival material obtained from 130 surgical specimens of esophageal squamous cell carcinoma (ESCC) with clinical and histopathologic factors obtained by a retrospective review of patient records.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. STATISTICAL ANALYSIS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Patients and Esophageal Specimens

All complete surgical specimens of ESCC resected from 1989 to 1999 from patients with no evidence of metastasis to other organs and without prior anticancer treatment were examined. Cases of in-hospital death were excluded. Surgical specimens from 130 patients who had undergone radical esophagectomy at the Department of Surgical Oncology of Hokkaido University, Hokkaido Gastroenterology Hospital, and Teine Keijinkai Hospital were included in the current study, and findings were correlated with the patients' clinical records. One of the deepest sections from each tumor was selected for evaluation. The specimens were examined histologically after staining with hematoxylin and eosin, and the clinicopathologic stage was determined according to the TNM classification system of the International Union Against Cancer.12

Immunohistochemistry

Surgical specimens were fixed in 10% formalin solution and embedded by routine methods in paraffin for sectioning at a thickness of 4 μm. Sections were deparaffinized in xylene and rehydrated through a series of graded ethanol. Endogenous peroxidase was blocked with 3% hydrogen peroxide for 10 minutes. Sections were washed twice in phosphate-buffered saline (PBS) and incubated with 10% normal goat serum (Histofine SAB-PO kit; Nichirei, Tokyo, Japan) for 30 minutes. Primary antibody (anti-caveolin-1 rabbit polyclonal antibody; Santa Cruz Biotechnology, Santa Cruz, CA) was applied in a 1:400 dilution in PBS, and the sections were incubated overnight at 4 °C. After three additional washes, sections were incubated with polyvalent biotinylated goat anti-rabbit antibody for 30 minutes at room temperature. Sections were washed three times in PBS and incubated with streptavidin-conjugated peroxidase for 30 minutes. After three additional washes, reaction product was visualized by incubation with 3,3′-diaminobenzidine tetrahydrochloride (Histofine SAB-PO kit) for approximately 15 minutes, followed by washing in distilled water. Sections then were counterstained in hematoxylin for 1 minute and mounted in Permount (Micro-slides; Muto-Glass, Tokyo, Japan). Sections of smooth muscle cells or endothelium known to be abundant in caveolin-1 were used as positive controls. Nonimmune purified rabbit serum was used for the primary antibody in negative controls. The number of stained cells per 1000 cells was determined under an Olympus microscope (Olympus Optical Co., Ltd., Tokyo, Japan) in three visual fields at a magnification of ×200. When the total number of cancer cells was less than 1000, all the cells were counted. When over 50% of all cancer cell cytoplasm was stained, the tumor was considered to be caveolin-1 positive. This cut-off value (50%) was adopted from a previous report.12 The current study was performed in a retrospective manner, but all specimens were evaluated by three investigators (Y. H., M. M., and T. I.) who were blinded to the patients' clinical information.

STATISTICAL ANALYSIS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. STATISTICAL ANALYSIS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Either the chi square test or Fisher exact test were used to analyze the correlation between caveolin-1 expression and the patients' parameters, including histopathologic findings. The cumulative survival rate was calculated by the Kaplan-Meier method, and statistical significance was analyzed by the log rank test. Univariate and multivariate analyses were performed using the Cox proportional hazard regression model; P < 0.05 was considered statistically significant in all analyses, which were performed with the Statview J version 4.5 (SAS Institute, Inc., Cary, NC) software package.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. STATISTICAL ANALYSIS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Patient Factors

Specimens from 130 patients were included in the current study (113 males and 17 females). More patients underwent surgery in the second half of the study period (85 patients) than in the first half (45 patients). The median patient age was 63 years (range, 38 to 82 years). A relatively large number of patients had early stage disease (81 patients, 62%). Sixty-six patients (51%) had lymph node metastases, and 22 patients (17%) had distant nodal metastases. The study population had the following performance status (PS): PS0, 114 patients; PS1, 15 patients; and PS2, 1 patient. The median follow-up period was 29 months (range, 2 to 114 months).

Expression of Caveolin-1

Immunoreactivity was observed at the cell membrane and in the cytoplasm as evidenced by the presence of stained granular immunoreaction products. Twenty-three specimens (17.7%) were immunoreactive for caveolin-1 in less than 25% of cells; 49 specimens (37.7%) were immunoreactive in 25 to 50% of cells; 19 specimens (14.6%) were immunoreactive in 50 to 75% of cells; and 39 specimens (30.0%) were immunoreactive in 75 to 100% of cells. According to the criteria of the current study, 58 specimens (44.6%) were positive for caveolin-1 (Fig. 1).

thumbnail image

Figure 1. Immunohistochemic staining for caveolin-1. A) Smooth muscle cells are stained as an internal control, but tumor cells are not stained. B) Almost all the cancer cell cytoplasm stained for caveolin-1. (×200)

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Prognostic Significance of Caveolin-1 Expression

Caveolin-1 immunostaining correlated with the P stage, N classification, and M classification (Table 1). The overall five year survival rate was 51.2%. The survival curve of patients with caveolin-1-immunopositive tumors was worse than that for patients with caveolin-1-immunonegative tumors (Fig. 2). Univariate analyses identified caveolin-1 positivity, T classification, N classification, M classification, lymphatic invasion, and a positive surgical margin as negative predictors; multivariate analyses indicated that T classification, N classification, and a positive surgical margin were independent prognostic factors. Caveolin-1 positivity was not an independent factor (Table 2).

Table 1. Relationship between Clinicopathologic Features and Caveolin-1 Expression in Surgical Specimens of Esophageal Squamous Cell Carcinomaa
VariableCaveolin-1 positive (n = 58)Caveolin-1 negative (n = 72)P value
  • a

    TNM classification system of the International Union Against Cancer.

  • b

    Significant.

Gender
 Male52610.446
 Female611
Age
 ≥ 603848> 0.999
 < 602024
Double cancer
 Yes11150.829
 No4757
p-Stage
 I, II30510.029b
 III, IV2821
p-Grade
 G113190.684
 Others4553
pT classification
 T123330.593
 Others3539
pN classification
 N022420.023b
 N13630
pM classification
 M043650.018b
 M1157
Tumor size
 < 4.5cm21390.052
 ≥ 4.5cm3733
Lymphatic invasion
 Positive34370.476
 Negative2435
Vascular invasion
 Positive1924> 0.999
 Negative3948
Surgical margin
 Positive720.077
 Negative5170
Adjuvant therapy
 Yes24280.858
 No3444
thumbnail image

Figure 2. Comparison of overall survival curves for patients with caveolin-1-positive and negative tumors in 130 patients who underwent curative resection of esophageal squamous cell carcinoma.

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Table 2. Univariate and Multivariate Analysis of Caveolin-1 and Pathologic Parameters in Patients Undergoing Curative Resection of Esophageal Squamous Cell CarcinomaUnivariate
FactorHazard ratio (95% C I)P value
Caveolin-11.907 (1.090–3.338)0.0238a
Gender2.820 (0.877–9.074)0.082
Age1.046 (0.581–1.883)0.8815
Double cancer0.700 (0.339–1.445)0.3347
p-Grade1.749 (0.786–3.891)0.1704
pT classification3.424 (1.786–6.569)0.0002a
pN classification5.623 (2.843–11.120)<0.0001a
pM classification3.269 (1.761–6.068)0.0002a
Tumor size1.729 (0.965–3.099)0.0658
Lymphatic invasion2.565 (1.406–4.681)0.0021a
Vascular invasion1.536 (0.847–2.784)0.1577
Surgical margin5.181 (2.288–11.732)<0.0001a
Adjuvant therapy1.018 (0.579–1.789)0.8193
Multivariate
  • CI: confidence interval

  • a

    Significant.

Caveolin-11.325 (0.725–2.422)0.361
pT classification2.170 (1.080–4.362)0.0296a
pN classification4.443 (1.992–9.911)0.0003a
pM classification1.082 (0.511–2.289)0.8368
Lymphatic invasion0.873 (0.423–1.802)0.7124
Surgical margin2.600 (1.020–6.626)0.0452a

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. STATISTICAL ANALYSIS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Tumors often utilize multifunctional molecules to grow or to survive in the harsh environment of the host. Activation of Ras is a well-established example of a method used by tumors to promote tumor progression.13, 14 However, the role of caveolin-1 in tumors is less clear. In vitro data seem to show that caveolin-1 suppresses tumor growth7, 15 while paradoxically being associated with a poor prognosis in prostate carcinoma.10 To confuse the matter further, caveolin-1 expression is increased in prostate carcinoma and breast carcinoma,11 but is reduced in lung carcinoma, colon carcinoma, and sarcoma.16–18 Thus, caveolin-1 seems to behave in a tissue-dependent manner.

The current data suggest that the significance of caveolin-1 expression in ESCC is comparable to that in prostate carcinoma, in that caveolin-1 over-expression correlates with the Gleason score, a positive surgical margin, and lymph node involvement. Additionally, caveolin-1 immunoreactivity is an independent predictor for time to disease progression in lymph node negative prostate carcinomas.10 Caveolin-1 immunoreactivity in ESCC similarly correlated with N classification, a positive surgical margin, and poor prognosis. Thus, caveolin-1 immunoreactivity in ESCC seems to share common features with immunoreactivity in prostate carcinoma.

It is well-documented that the incidence of esophageal carcinoma is much higher in men than in women, and at least one study of dependency on sex hormones in this disease has been reported.19 Prostate carcinoma is also androgen-dependent, and in prostate carcinoma, caveolin-1 mediates testosterone-stimulated survival/clonal growth and promotes metastatic activity.20 Moreover, over-expression of caveoin-1 potentiates ligand-dependent androgen receptor activation.21 Similar mechanisms may be at work in ESCC.

The current results suggest that over-expression of caveolin-1 may be a marker for lymph node metastasis and poor prognosis after surgical resection. Caveolin-1 positivity has a 54.5% sensitivity, a 65.6% specificity, a 62.1% positive predictive value, and a 41.7% negative predict value for nodal metastasis. Still, this marker alone cannot be relied upon to predict lymph node metastases. However, preoperative studies of biopsy specimens obtained by endoscopy might allow clinicians to make better informed therapeutic decisions in conjunction with tumor markers.

Most patients in the current study had early stage disease, and that population has an inherently better prognosis. Although based only on this retrospective study of patients with ESCC whose disease was sufficiently early to permit surgical resection, we concluded that 1) caveolin-1 may play a role in tumor spreading; 2) caveolin-1 may be a marker for lymph node metastasis; and 3) caveolin-1 inversely correlates as a predictor for long-term survival.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. STATISTICAL ANALYSIS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

The authors thank Mr. Hiraku Shida for technical support in performing the immunohistochemic analyses.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. STATISTICAL ANALYSIS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
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