Ann Thorac Surg 2003;76:213-218
© 2003 The Society of Thoracic Surgeons
Original article: general thoracic
Prognostic significance of CD44v5 expression in human thymic epithelial neoplasms
Shih-Chun Lee, MDa,
Horng-Jyh Harn, PhDc,
Torng-Sen Lin, MDd,
Kun-Tu Yeh, MDe,
Yao-Chi Liu, MDb,
Chien-Sung Tsai, MDb,
Yeung-Leung Cheng, MDa,f*
a Division of Thoracic Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan
b Department of Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan
c Division of Molecular Medicine, Department of Pathology, Buddhist Tzu-Chi General Hospital, Hualien, Taiwan
d Division of Cardiothoracic Surgery, Department of Surgery, Changhua Christian Hospital, Changhua, Taiwan
e Department of Pathology, Changhua Christian Hospital, Changhua, Taiwan
f Graduate Institution of Medical Science, National Defense Medical Center, Taipei, Taiwan
Accepted for publication February 5, 2003.
* Address reprint requests to Dr Cheng, Division of Thoracic Surgery, Department of Surgery, Tri-Service General Hospital, 325 Cheng-Gong Rd, Section 2, Taipei 114, Taiwan.
e-mail: ndmc0928{at}yahoo.com.tw
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Abstract
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BACKGROUND: Cell surface glycoproteins of the CD44 family play roles in cell-cell and cell-matrix interactions. Their aberrant expression has been implicated in tumor invasion and metastasis of a variety of neoplasms, but not, to date, of thymic epithelial tumors.
METHODS: To investigate the expression of CD44 molecules, immunohistochemical staining using monoclonal antibodies against human CD44 standard form (CD44 s) and two common splicing variant (CD44v) isoforms, CD44v5 and CD44v6, was performed on 64 resected thymomas and 20 normal thymuses. These tumors were categorized histologically according to the World Health Organization (WHO) histologic classification, and the pathologic staging was classified according to the definitions of Masaoka.
RESULTS: The positive expression rates in these patients were as follows: CD44 s (normal thymuses, 10%; thymomas, 22%), CD44v5 (normal thymuses, 0%; thymomas, 67%), and CD44v6 (normal thymuses, 0%; thymomas, 26%). CD44 s and CD44v5 immunoreactivity showed a positive correlation with tumor stages (p = 0.034 and 0.027, respectively). The CD44v5 expression of neoplastic cells in tumor capsules has significant correlation with tumor stages (II, 5%; III, 70%; IVA, 100%; p < 0.001). On the basis of univariate survival analysis, the Masaoka staging system, WHO histologic classification, and CD44v5 expression showed a statistically significant positive relation to survival (p < 0.001, 0.002, 0.011, respectively). Using Cox’s regression model, increasing CD44v5 expression, the Masaoka staging, and the WHO classification system were found to be significant independent prognostic factors.
CONCLUSIONS: CD44v5 expression is independently positively correlated with the aggressiveness of thymic epithelial tumors. The expression of CD44v5 may be a potential trigger of tumor invasion in thymomas.
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Introduction
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CD44, a polymorphic integral membrane glycoprotein and a cell surface hyaluronate receptor [1], is one of the molecules involved in cell adhesion and widely expressed on both hematopoietic cells (eg, lymphocytes) and nonhematopoietic cells (eg, epithelial cells, fibroblasts, and keratinocytes) [2, 3]. The CD44 molecule participates in various physiologic functions, including lymphocyte activation [4], circulation and homing to specific lymph node tissue [1, 3], and cell-cell and cell-matrix adhesion and interaction [3].
CD44 is a family of 80- to 250-kDa membrane glycoproteins derived from a single gene located on the short arm of chromosome 11 [5]. The human CD44 gene consists of 20 exons distributed over a length of about 60 kb. Alternative splicing of the 10 exons located between exons 5 and 16 (exon V1 to V10) may generate variant isoforms (CD44v) [6]. The CD44 standard (CD44 s) or hematopoietic form is the most abundant CD44 protein in tissues.
Furthermore, CD44 or its splice variants have been demonstrated to be involved with the metastasis potential of certain tumors. The correlation between tumor progression and metastases appears to be different in different tumor types. Enhanced expression of CD44 isoforms have been reported in human breast carcinoma [7], colorectal carcinoma [8], gastric carcinoma [8–10], and lung cancer [11]. Conversely, downregulation of CD44 expression in cancer cells has been reported in Barrett’s esophagus carcinoma [12] and extrahepatic bile duct/ampullary carcinoma [13]. Our previous immunohistochemical studies of CD44 expression in human gastric cancers found CD44v5 and CD44v6 overexpression in poorly differentiated, advanced, and metastatic gastric carcinomas [9, 10, 14].
Thymoma, the most common anterior mediastinal tumor in the adult, occurs mostly in patients older than 40 years. Although their microscopic appearance is benign, thymomas invade nearby tissue either grossly or microscopically and have therefore been considered to be malignant lesions and to have the least favorable prognosis. To our knowledge, CD44 expression in thymomas has not been reported. Therefore, we measured CD44, CD44v5, and CD44v6 expression in thymomas, and determined the relationship between the expression of these molecules and the clinicopathologic features and prognosis of patients.
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Material and methods
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Patients and tumor specimens
Formalin-fixed and paraffin-embedded tissues from 64 surgically resected thymomas were obtained from the archives of the Department of Pathology, Tri-Service General Hospital, Taipei, and Changhua Christian Hospital, Changhua, Taiwan. These patients were chosen only because of the availability of their surgically resected tumors, the availability of sufficient follow-up data, and interpretability of the immunostaining results. Patients who died of myasthenia gravis (MG), other diseases, or accidents were excluded. Hematoxylin and eosin–stained slides in each case were reviewed to confirm the primary diagnosis. There were 35 men and 29 women, and their mean age at surgery was 45 years (range, 21 to 71 years). All patients underwent complete resection of their thymomas. Twenty normal thymuses as control were obtained from open-heart surgery by our cardiovascular surgeon (C.S. Tsai). These tumors were categorized according to the World Health Organization (WHO) histologic classification as types A, AB, B1, B2, and B3 [15]. The surgicopathologic staging was based on the definitions of Masaoka and associates [16]. Patients were followed up every 3 months in the first postoperative year, at 6-month intervals during the second year, and once a year thereafter. Data on recurrences and causes of death were obtained from all patients. Thirteen died, and 51 were alive and thymoma-free. The mean follow-up time for these patients was 8 years (range, 21 months to 15 years) after surgical resection.
Immunohistochemical staining
Surgically resected specimens containing tumor tissues or residual thymus were fixed in 10% buffered formalin at room temperature and embedded in paraffin for histologic diagnosis. Consecutive 5 µm-thick sections were cut from each trimmed paraffin block, and mounted on gelatin-dichromate-coated glass slides (Dako Corp, Santa Barbara, CA). The expression of various CD44 molecules was determined by immunohistochemical staining of histologic sections using specific monoclonal antibodies as previous described [9]. In brief, after dewaxing with xylene and rehydration through a graded series of ethanol, the slides were microwaved in 10 mmol/L phosphate citrate buffer at 90°C for 15 minutes. Endogenous peroxidase was deactivated with 0.3% H2O2 in methanol, followed by incubation with primary antibody (in phosphate-buffered saline [PBS] with 1% bovine serum albumin) for 1 hour at room temperature. The monoclonal antibodies (MAbs) BMS113 (anti-CD44 s, 1:100 dilution), BMS115 (anti-CD44), 1:50 dilution, and BMS125 (anti-CD44), 1:50 dilution were purchased from Bender MedSystems (Vienna, Austria). After washing in PBS, the sections were incubated with secondary biotinylated antibody (Dako) for 30 minutes, followed by rinsing in PBS. After incubation with streptavidin-biotin-peroxidase complex for 30 minutes, sections were incubated with 3% 3,3-amino-9-ethylcarbazole (Sigma Chemical Co, St. Louis, MO) in N',N-dimethylformamide for 5 to 10 minutes. After stopping the reaction with tap water, the sections were counterstained with hematoxylin, mounted with glycerol-gelatin, and then viewed by microscopy.
Staining interpretation
All stained slides were interpreted by two experienced pathologists. Positive cells were defined as those showing a strong plasma membrane staining pattern. Cells that lacked membranous staining or showed only cytoplasmic staining were considered to be negative. The CD44 s, CD44v5, and CS44v6 expressed by epithelial cells in the tumors and capsules were observed. According to the estimated percentage of positively stained cells, tumors were classified as "diffusely stained" (more than 50% positive neoplastic cells), "focally stained" (less than 50% positive neoplastic cells), or "unstained" (no positive tumor cells).
Statistical analysis
The correlation of CD44 s, CD44v5, and CD44v6 expression with clinicopathologic characteristics, such as age, gender, status of MG, pathologic staging, and histologic subtypes of thymoma, was analyzed by 
2 test or Fisher’s exact test. The survival curves were analyzed by means of the Kaplan-Meier method, and their difference by the log-rank test. Multivariate analysis for survival was performed using Cox’s proportional hazards models. The SPSS 10.0 statistical software system (SPSS Inc, Chicago, IL) was employed for all analysis. The criterion for significance level was considered at p less than 0.05.
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Results
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According to the WHO classification, there were 11 (17%) type A, 14 (22%) type AB, 19 (30%) type B1, 15 (23%) type B2, and 5 (8%) type B3 tumors. Twenty-seven patients were in stage I (42%), 20 (31%) in stage II, 13 (20%) in stage III, and 4 (6%) stage IVA. Eighteen patients (28%) presented with symptoms related to MG. The clinicopathologic features of these patients (patient age, gender, histologic subtypes, pathologic staging, and status of MG) and the correlations with the expression of CD44-related molecules are summarized in Table 1.
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Table 1. Correlation Between the Expression of CD44s, CD44v5, and CD44v6, and Clinicopathologic Characteristics of 64 Patients With Thymoma
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In nonneoplastic thymus tissues, weak immunoreactivity for CD44 seconds in 10% (2/20) and negative expression for CD44v5 and CD44v6 were observed in epithelial cells. In contrast, the expression of CD44 s, CD44v5, and CD44v6 in neoplastic cells of thymomas [CD44 s (T), CD44v5(T), and CD44v6(T)] showed focal or diffuse immunostaining in 22% (14/64), 67% (43/64), and 26% (16/64) of the specimens, respectively. Furthermore, the expression of CD44 molecules was evaluated in the neoplastic cells of the tumor capsules [CD44 s (C), CD44v5(C), and CD44v6(C)], and categorized as mentioned above, and 0%, 34% (22/64), and 6% (4/64) of the specimens showed positive immunohistochemical staining, respectively. Correlations of patients’ age, gender, or MG status with the expression of CD44 seconds, CD44v5, and CD44v6 were not found. However, the immunoreactivity of CD44 s (T), CD44v5(T), and CD44v5(C) had significant correlation with the pathologic staging of thymomas (p = 0.034, 0.027, < 0.001, respectively; Fisher’s exact test), but CD44 s (C), CD44v6(T), and CD44v6(C) did not. There was no correlation of WHO classification with the expression of CD44.
The pathologic stages and their association with patients’ gender, age, WHO classification, and status of MG are shown in Table 2,
and only the histologic subtypes had significant correlation with pathologic staging (p = 0.016; Fisher’s exact test). Most patients with early stages were WHO subtype A or type AB (stage I, 63%; II, 30%; III, 15%; IVA, 0%), whereas most patients with late stages were WHO subtype B (B1, B2, or B3) (stage I, 37%; II, 70%; III, 85%; IVA, 100%). The respective proportions of invasive thymomas with WHO classification types A, AB, B1, B2, and B3 were 18.3%, 42.7%, 57.6%, 86.7%, and 100%, respectively.
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Table 2. Correlation Between the Pathologic Staging and Other Clinicopathologic Characteristics of 64 Patients With Thymoma
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According to the univariate survival analysis (the Kaplan-Meier survival curves), the Masaoka staging system, WHO histologic classification, and CD44v5 expression exhibited statistically positive relationship with
survival (Figs 1–3).
After using Cox’s proportional hazards modeling,
the Masaoka staging system (p < 0.001; relative risk, 3.581; 95% confidence interval [95% CI], 2.106 to 6.089), WHO classification (p = 0.017; relative risk, 2.709; 95% CI, 1.616 to 4.541), and CD44v5 expression (p = 0.026; relative risk, 2.352; 95% CI, 1.164 to 4.761) were found to be independent prognostic factors, whereas gender, age, association with MG, expression of CD44 s, or CD44v6 were not.
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Fig 1. Kaplan-Meier survival curves according to the Masaoka stage of thymoma. There were significant differences between patients with stage I versus III, stage I versus IVa, stage II versus III, stage II versus IVa, and stage III versus IVa (p < 0.001, < 0.001, 0.031, < 0.001, 0.044, respectively), but not in stage I versus II.
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Fig 2. Survival curves according to the WHO histologic classification of thymoma. There were significant differences between patients with type A versus B2, A versus B3, AB versus B2, AB versus B3, B1 versus B2, and B1 versus B3 (p = 0.022, 0.002, 0.022, 0.004, 0.033, 0.023, respectively). But there were no significant differences between patients with type A, AB, or B1.
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Fig 3. Survival curves according to the expression of CD44v5 in thymomas. Patients with increasing expression of CD44v5 showed a significantly better survival compared with patients who had no expression of CD44v5 (p = 0.011).
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Comment
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Thymoma is a common anterior mediastinal tumor. Histologically, thymoma is a benign neoplasm, but its invasiveness has been associated with its staging prognosis. According to Rosai’s classification [17], thymoma can be divided into benign (noninvasive or encapsulated) and malignant (invasive or metastatic thymoma without overt cytologic atypia). Malignant thymomas possess invasiveness or metastatic behavior, and patients with these tumors have poorer outcome than those with benign thymomas. To date, the Masaoka staging system and the WHO histologic classification of thymic epithelial neoplasms proposed in 1999 were considered as the most significant clinicopathologic prognostic indicators [18, 19]. Many biomolecular studies of tumorigenesis and biological characteristics of thymomas have been reported, and several factors were considered to be associated with the biological actions of thymomas [20, 21], but few were clearly linked to their different behaviors.
CD44, one of the well-known cell adhesion molecules, is broadly expressed on cells of hematopoietic, epithelia, and mesothelial origin. CD44 is considered to mediate functions of lymphocyte homing, cell activation, and cell-cell and cell-matrix interactions [2–4, 22]. The relation between tumor progression and metastases appears to differ in different types of tumors [7, 8, 11–13]. In our previous studies, we showed that expression of CD44 and certain of its isoforms possibly play a role in the differentiation and metastatic potential of human brain tumor [23], hepatoma [24], and gastric carcinoma [9, 10, 14]. The present study demonstrated that the expression of CD44 s and CD44v5 were positively correlated with pathologic stages of thymomas, and CD44v5 expression in the tumor capsules was a significant predictor for the invasive behavior of thymomas. Furthermore, the WHO histologic subtypes had significant correlation with pathologic staging. The results were similar to previous reports [18, 19]. Nevertheless, the expression of CD44v5 did not correlate with WHO histologic classification (Table 1).
The results of survival analysis showed that Masaoka staging system and WHO histologic classification were significant prognostic factors, a conclusion compatible with that in previous reports [18, 19]. Furthermore, our results showed that the increasing expression of CD44v5 was positively correlated with survival of thymoma patients. The multivariate analysis showed that Masaoka staging system, WHO histologic classification, and CD44v5 expression were significant independent indicators for predicting the aggressiveness of thymomas.
The molecular mechanism of CD44v5 expression in tumor metastasis is not clear yet. The results of our previous study of gastric cancer suggested that CD44v5 might be related to hyaluronate binding in the extracellular matrix. Direct hyaluronate binding studies of CD44 v4-v7 isoforms transfected into the human gastric carcinoma cell line, SC-M1, have indicated that the v4-v7 isoforms themselves, in addition to glycosylation, can alter hyaluronate binding [14]. Further investigation is needed to determine whether this model applies to malignant thymoma, or whether there is an alternative explanation (eg, a new ligand present in the variant domain).
In summary, our results support the conclusion that increasing CD44v5 expression is correlated with invasiveness of thymoma. Invasiveness is the most important feature determining the therapeutic and prognostic outcome in patients with thymoma. CD44v5 expression in thymic neoplastic cells may play an important role in invasiveness, and as a useful molecular marker to predict the biological behaviors of thymoma. More aggressive therapy may be considered for treating patients with early-stage thymoma who manifest increased CD44v5 expression.
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Abstract
Introduction
