10.1245/ASO.2005.07.002
Annals of Surgical Oncology 12:925-934 (2005)
© 2005 Society of Surgical Oncology
Increased Expression of Valosin-Containing Protein (p97) Is Correlated With Disease Recurrence in Follicular Thyroid Cancer
Shinji Yamamoto, MD1,
Yasuhiko Tomita, MD2,
Takashi Uruno, MD3,
Yoshihiko Hoshida, MD2,
Ying Qiu, MD2,
Norishige Iizuka, MD2,
Itsuko Nakamichi, MD2,
Akira Miyauchi, MD3 and
Katsuyuki Aozasa, MD2
1 Department of Surgery and Clinical Oncology, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka, 565-0871, Japan
2 Department of Pathology (C3), Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka, 565-0871, Japan
3 Division of Surgery, Kuma Hospital, 8-2-35 Shimoyamate, Chuo, Kobe, 650-0011, Japan
Correspondence: Address correspondence and reprint requests to: Yasuhiko Tomita, MD; E-mail: yt{at}molpath.med.osaka-u.ac.jp.
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ABSTRACT
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Background: Valosin-containing protein (VCP) is involved in the ubiquitin/proteasome-degradation pathway, which works in proliferation and antiapoptosis in human cancer cells. Our previous study showed that VCP expression levels correlated with the recurrence and prognosis of several human cancers, such as hepatocellular carcinoma, gastric carcinoma, and colorectal carcinoma. In this study, the correlation of VCP expression with the prognosis of differentiated thyroid carcinoma was examined.
Methods: VCP expression in 332 patients who underwent operation for differentiated thyroid carcinoma—257 with papillary thyroid carcinoma and 75 with follicular thyroid carcinoma (FTC)—was analyzed by immunohistochemistry. The staining intensity in tumor cells was categorized as weaker than (low expression), equal to (intermediate expression), or stronger than (high expression) that in endothelial cells in noncancerous tissue.
Results: One hundred ten (33.5%) cases showed low VCP expression, 117 (28.0%) showed intermediate expression, and 101 (30.8%) showed high expression. VCP expression significantly correlated with histological subtype (P < .05) and lymph node metastasis (P < .01). However, it correlated with neither any clinicopathologic factor nor prognosis in papillary thyroid carcinoma. VCP expression correlated with extrathyroidal extension (P < .05), pT classification (P < .05), and lymph node metastasis (P < .01) in FTC. Patients with low VCP expressing FTC showed better disease-free and overall survival rates than those with intermediate or high expression (P < .01 and P < .05, respectively). Multivariate analysis revealed VCP expression and extracapsular extension to be independent prognostic factors for disease-free survival in cases of FTC.
Conclusions: The prognostic utility of VCP expression in FTC was demonstrated.
Key Words: Valosin-containing protein • Prognosis • Follicular thyroid carcinoma • Metastasis
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INTRODUCTION
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Thyroid carcinomas are relatively uncommon, with an incidence representing approximately 1% of all kinds of cancers in Western countries.1,2 Histologically, they are roughly divided into (1) differentiated and undifferentiated carcinoma and (2) other types.2 Surgical resection is the main modality of treatment for differentiated thyroid carcinoma, with generally favorable results: the disease-specific overall survival rate at 10 years is approximately 90%.2,3 However, recurrence of the tumor is not infrequent, and in such cases, patients usually show an unfavorable course.3 Therefore, it is important to know the tendency for relapse or metastasis of thyroid cancer.
Several clinicopathologic factors, such as age at diagnosis, size of the primary tumor, extracapsular invasion, histological differentiation, and the occurrence of distant metastasis, have been reported to be prognosticators for differentiated thyroid carcinoma.3–5 However, clinical outcome is not predictable in some cases, even when these findings are taken into account.4,5 Therefore, it is important to identify biological factors that affect disease recurrence and the survival of patients with differentiated thyroid carcinoma.
Histologically, differentiated thyroid carcinoma is divided into two categories: papillary thyroid carcinoma (PTC), which comprises most differentiated thyroid carcinomas, and follicular thyroid carcinoma (FTC).2,3 Metastasis to the lymph nodes is relatively frequent in PTC but rather infrequent in FTC.2,3 However, hematogenous metastasis—mainly to lung, bone, and brain—is frequent in FTC and results in a poorer prognosis than for PTC.2,3
Recently, by using the messenger RNA subtraction technique, we identified the gene encoding valosin-containing protein (VCP; also known as p97) to be associated with the metastasis of a murine osteosarcoma cell line.6 VCP, a member of the adenosine tri-phosphatases associated with various cellular activities superfamily, is known to be involved in the ubiquitin/proteasome-degradation pathway, which works in both the upregulation of cell proliferation and the downregulation of cell death in human cancer cells.7 The cell line transfected with VCP showed a decreased rate of apoptosis after stimulation of tumor necrosis factor 
and showed increased metastatic potential.6 Therefore, it is postulated that the expression level of VCP could be used as a prognosticator for cancers. Indeed, our previous study showed that the VCP expression level in cancer cells was correlated with the rate of disease recurrence and the prognosis of patients with cancer in the liver, stomach, prostate, colorectum, pancreas, and lung.8–13
In this study, the expression level of VCP in differentiated thyroid carcinoma was examined by immunohistochemical analysis, and its correlation with clinicopathologic factors and prognosis was evaluated.
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PATIENTS AND METHODS
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Patients
Surgical specimens were obtained from 332 patients who had undergone a resection of primary thyroid cancer at the Division of Surgery, Kuma Hospital, Kobe, Japan, during the period from February 1987 to December 1995. The patients included 36 males and 296 females with ages ranging from 14 to 86 years (median, 47.0 years). The operation used was enucleation of the lesions in 1 patient, isthmectomy in 3, near-lobectomy in 14, lobectomy in 46, lobectomy and isthmectomy in 86, subtotal thyroidectomy in 38, near-total thyroidectomy in 2, and total thyroidectomy in 142. Cervical lymphadenectomy was performed in 245 patients. The resected thyroid was macroscopically examined to determine the location and size of the tumor. The location of tumors was as follows: right lobe in 162 cases, isthmus in 22, left lobe in 147, and total thyroid involvement in 1. The size of the tumor ranged from .4 to 10.5 cm (median, 2.8 cm). Samples obtained from the thyroid lesions and dissected lymph nodes were fixed in 10% formalin and routinely processed for paraffin embedding. Histological sections, cut at 4 µm, were stained with hematoxylin and eosin and immunoperoxidase procedures (avidin-biotin complex method). Histological sections were reviewed by one of the authors (Y.H.) to examine the extent and mode of invasion in the thyroid, lymph node metastasis, and histological subtype. FTCs were diagnosed according to the following two criteria: observation of capsular or vascular invasion and no evidence of a papillary structure or intranuclear cytoplasmic inclusion throughout the tumor. Follicular variants of PTC were diagnosed on the basis of the presence of intra-nuclear cytoplasmic inclusion in some of the tumor cells and the absence of papillary structures throughout the tumors. Tumor stage was defined according to the pathologic tumor-node-metastasis classification.14
After surgery, patients were followed up with periodic evaluations of blood tests, ultrasonography, and computed tomography at 3- to 6-month intervals. Adjuvant chemotherapy was performed in 199 patients at high risk for tumor recurrence, i.e., presence of lymph node metastasis, huge tumor, and extension of tumor to adjacent tissue. Protocols for adjuvant therapy were thyroid stress hormone–suppression therapy with (2 cases) or without (197 cases) radiotherapy. The follow-up period for survivors ranged from 1 to 189.7 months (median, 133.4 months) after surgery.
Immunohistochemical Analysis
Immunohistochemistry was performed on the paraffin-embedded tissue sections with immunoperoxidase procedures. Briefly, antigen retrieval was performed by heating the deparaffinized and rehydrated sections in 10 mM of citrate buffer for 5 minutes. Mouse monoclonal anti-VCP (p97) antibody (Progen Biotechnik, Heidelberg, Germany) was used as the primary antibody at a dilution of 1/3000. Sections were lightly counterstained with methyl green. For negative controls, nonimmunized mouse immunoglobulin G (Vector Laboratories, Burlingame, CA) was used as the primary antibody. Stained sections were evaluated in a blinded manner by two authors (S.Y. and Y.T.) without prior knowledge of the clinicopathologic features of patients. VCP expression was mainly observed in the cytoplasm. Staining intensity in the cytoplasm of tumor cells was categorized as follows: weaker than (low expression), equal to (intermediate expression), or stronger than (high expression) that in endothelial cells, which showed a constant positive immunoreactivity. When the staining intensity of tumor cells varied in different areas of the tumor, the predominant pattern was chosen as the expression level.
Statistical Analysis
Statistical analyses were performed with JMP software (SAS Institute Inc., Cary, NC). The 
2 test and Fisher’s exact probability test were used to analyze the correlation between VCP expression and clinicopathologic features. Kaplan-Meier methods with the log-rank test were used to calculate the overall survival rate and differences in survival curves.15 Cox’s proportional hazards regression model with a stepwise analysis was used to analyze the independent prognostic factors.16 P values of <.05 were considered to be statistically significant.
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RESULTS
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Histological Findings
Histologically, 257 tumors were PTCs, of which 20 were of the encapsulated type, 12 were of the follicular type, and 3 were of the diffuse-sclerosing type. Seventy-five tumors were FTCs, among which five were of the oxyphilic cell type and three were of the clear cell type. There were no cases of Hürthle cell cancers in this series. The tumor was 
2 cm and localized to the thyroid (pT1) in 33 patients, 2 to 4 cm and localized to the thyroid (pT2) in 195 patients, either >4 cm or with minimal extrathyroidal extension (pT3) in 75 patients, and with massive extension (pT4) in 29 patients. Sixty patients were node negative (pN0), 68 showed lymph node metastasis at level VI (pN1a), and 117 had metastasis in other nodes (pN1b). Eighty-seven patients did not receive a lymph node dissection.
Patient Outcomes
The 10-year disease-free and overall survival rates were 90.7% and 98.0%, respectively. Forty-one patients showed tumor recurrence: local recurrence in 34, lymph node in 2, lung in 10, and bone in 2. Five patients died from the tumor.
Immunohistochemical Detection of VCP Expression
Four (1.2%) of 332 patients who did not have endothelial staining were regarded as having poor antigen preservation and were excluded from further analysis. The remaining 328 cases showing endothelial staining were evaluated for VCP expression. The 2 observers made the same judgment in 304 of 328 cases examined. A split judgment was made in the remaining 24 cases, and the observers discussed each case until they reached a consensus. Ninety-six cases showed a low level of VCP expression throughout the tumor, whereas the expression level was high at the periphery but low in the larger central area of the tumor in 14 cases. In total, 110 (33.5%), 117 (35.7%), and 101 (30.8%) cases were classified as having low, intermediate, and high expression, respectively (Fig. 1
).
Received for publication July 1, 2004.
Accepted for publication May 11, 2005.
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ABSTRACT
INTRODUCTION
). Multivariate analysis with factors proven to be significant in the univariate analysis revealed VCP expression and extracapsular invasion to be independent prognostic factors for disease-free survival (Table 5
B, a transcription factor whose activity is correlated with antiapoptosis, cell proliferation, and invasion.7 Therefore, it is postulated that VCP plays a crucial role in tumor invasion and metastasis. 
