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© 2002 American Society for Clinical Oncology Lack of Expression of Galectin-3 Is Associated With a Poor Outcome in Node-Negative Patients With Laryngeal Squamous-Cell CarcinomaByFrom the Department of Oncology and Neurosciences, "G. D’Annunzio" University, Chieti, and Departments of Otolaryngology, Pathology, and Histology, Catholic University, Rome, Italy. Address reprint requests to Mauro Piantelli, MD, Department of Oncology and Neurosciences, c/o SeBi Building, "G. D’Annunzio" University, via dei Vestini, I-66100 Chieti, Italy; email: mpiantelli{at}unich.it
PURPOSE: Galectin-3 is a pleiotropic carbohydrate-binding protein participating in a variety of normal and pathologic processes, including cancer progression. This study was aimed at evaluating the prognostic value of galectin-3 expression in node-negative laryngeal squamous-cell carcinoma (SCC). PATIENTS AND METHODS: Galectin-3 expression was analyzed by immunohistochemistry using M3/38 monoclonal antibody, in a single-institution series of 73 node-negative laryngeal SCC patients (median follow-up, 52 months; range, 2 to 90 months). RESULTS: Forty-two (57.5%) of 73 patients expressed galectin-3. Galectin-3 expression was positively associated with tumor keratinization and histologic grade. A significant correlation was found between galectin-3 tumor positivity and longer relapse-free and overall survival. In univariate analysis, high-grade (grade 3 or 4) tumors, nonkeratinizing tumors, and galectin-3–negative tumors showed a significantly increased risk of relapse and death. In multivariate analysis, only galectin-3 expression retained an independent prognostic significance for both relapse-free and overall survival. CONCLUSION: We conclude that the absence of galectin-3 expression is an independent negative prognostic marker in laryngeal SCC patients. Thus, histochemical detection of galectin-3 in these tumors could be useful for the selection of node-negative patients with potentially unfavorable outcomes, to establish adjuvant therapy protocols.
HEAD AND NECK cancer is a major public health problem. Worldwide, over 500,000 new cases of this cancer occur yearly, and the incidence is rising. Laryngeal squamous-cell carcinoma (SCC) is the most common head and neck cancer, representing about one third of all cases.1 Despite intensive efforts in primary prevention, screening, and therapy, the long-term survival rate of patients with laryngeal SCC has not improved substantially for the past two decades. Standard local treatment consists of disabling surgical procedures associated with radiotherapy.2 Commonly recognized prognostic factors in laryngeal cancer include exact site of the primary lesion, stage, and histologic grade of the tumor. Although useful, these factors do not always allow a definitive discrimination between high- and low-risk patients, especially in node-negative cases.3 Similar to other tumor entities, the quest to define markers with a demonstrable impact on clinical courses is a potentially promising and therapeutically relevant research topic. Galectins are a family of proteins defined by their affinity for beta-galactosides and by conserved-sequence elements.4,5 At least 14 different members of this family have been identified so far, the most extensively investigated being galectin-3 and galectin-1. The function of these molecules is largely unknown, but a body of evidence suggests a role in cell growth and differentiation, adhesion, and migration, and they have been associated with the transformation and progression of human tumors.4-9 In particular, galectin-3 has been described as being involved in the progression of various types of cancer, but contradictory observations have been reported and the clinical relevance of its expression is still controversial.8,9 With regard to head and neck SCCs, previous work has shown that they contain both galectin-1 and galectin-3 and that galectin-3 expression is positively correlated with squamous-cell differentiation.10,11 A decrease in galectin-3 expression in laryngeal SCC correlates with an increasing level of clinically detectable aggressiveness of these tumors.12 However, no association with clinical outcome was evaluated in this latter study. In the present study, we analyzed galectin-3 expression by immunohistochemistry in a single-institution series of 73 node-negative laryngeal SCC patients in order to assess its prognostic importance. We found that about 60% of these tumors expressed the lectin and that the lack of galectin-3 expression was associated with a significantly worse outcome for patients with laryngeal SCC.
Our study included 73 consecutive node-negative laryngeal SCC patients admitted to the Department of Otolaryngology of the "A. Gemelli" Catholic University Hospital, Rome. Patients were treated by surgery alone except for patients with locally advanced tumors (T4), who received postsurgical radiation therapy, as outlined before,13 and received a median of 52 months of follow-up (range, 2 to 90 months). Follow-up was conducted at 3-month intervals for the first year and 6-month intervals thereafter. Recurrences were detected by imaging techniques and confirmed, when required, by histologic examination of bioptic material. At completion of follow-up, patients were categorized as alive with evidence of disease, alive without disease, or dead as a result of laryngeal SCC. No patients in this series had died of a cancer-unrelated cause. Histologic grading and tumor, node, metastasis classification were performed on conventional paraffin sections following the recommendations of the International Union Against Cancer.14 Accordingly, 36 tumors (49%) were graded as well/moderately differentiated (grade 1 or 2) and 37 (51%) were graded as poorly differentiated (grade 3 or 4). Tumors were also classified as keratinizing and nonkeratinizing, according to the presence or absence of keratin, respectively.3 Six tumors (8.2%) were glottic, 26 (35.6%) were supraglottic, and 41 (56.2%) were transglottic. The pathologic analyses were performed in a blinded manner with respect to the clinical information of the patients.
Immunohistochemistry
Statistical Analysis
Galectin-3 and Clinicopathologic Features Of the 73 cases assayed for galectin-3 expression, 42 (57.5%) demonstrated galectin-3 expression in more than 5% of tumor cells and were considered as galectin-3–positive, whereas 31 (42.5%) had no expression or expression was in  5% of tumor cells and were considered as galectin-3–negative. Galectin-3–positive tumors contained malignant cells exhibiting diffuse cytoplasmic specific staining with the antigalectin-3 antibody, although intratumor and intertumor variability was noted (Fig 1). The antibody stained the tumor cell nearest the keratin material (pearl) darkly, and stained progressively less toward the outer edge of the tumor islands. In general, galectin-3 staining was higher in those cells that were more differentiated. In addition to the cytoplasmic staining, eight of 43 galectin-3–positive tumors showed specific staining of a distinct proportion (from 25% to 70%) of cell nuclei. Tumor cases negative for cytoplasmic galectin-3 immunostaining were also negative for the nuclear expression of the lectin. In all cases, the stroma stained weakly, presenting a sharp contrast to the darkly stained tumor cell islands of galectin-3–positive tumor cells.
The relationship between galectin-3 expression and clinicopathologic features is summarized in Table 1. There was no significant difference between the galectin-3–negative and the galectin-3–positive group with respect to age, tumor size, or tumor localization. On the contrary, galectin-3 expression was positively associated with histopathologic grading (P = .012) and tumor keratinization (P = .01).
Galectin-3 and Survival During the follow-up period, locoregional recurrences were observed in 28 of 73 (38.4%) cases. At the end of the study, 22 (30.1%) of 73 patients died of cancer. A significant relationship was found between tumor galectin-3 staining and patient survival. At the end of the follow-up period, the estimated relapse-free survival was 75.6% (95% confidence interval [CI], 62.4% to 88.8%) for patients with galectin-3–positive tumors compared with 33.5% (95% CI, 15.4% to 51.6%) for those with negative tumors (P = .013) (Fig 2A). Similarly, the overall survival (Fig 2B) was 72.2% (95% CI, 44.8% to 99.6%) for patients with galectin-3–positive tumors compared with 37.2% (95% CI, 15.1% to 59.3%) for patients with galectin-3–negative tumors (P = .0001).
Table 2 lists the results of univariate analysis of prognostic variables for relapse-free and overall survival. Patients with high-grade (grade 3 or 4) tumors, nonkeratinizing tumors, and galectin-3–negative tumors showed a significantly increased risk of relapse and death.
In the multivariate analysis, only galectin-3 expression retained an independent prognostic significance for both relapse-free and overall survival. In particular, the relative risks of relapse and death were 2.73 (95% CI, 1.22% to 6.13%) and of 4.73 (95% CI, 1.69% to 13.3%), respectively, for patients with galectin-3–negative tumors (Table 3).
Our data, obtained by evaluating 73 patients with node-negative laryngeal SCC, show that galectin-3 expression allows discrimination between high-risk and low-risk patients, in terms of both relapse-free and overall survival. Hence, galectin-3 staining seems to be a reliable and easy-to-assess marker for identifying laryngeal tumors at increased risk of progression. The mechanisms underlying the detrimental effects of the lack of galectin-3 expression in laryngeal SCC are unclear. Although galectin-3 has been described as being involved in the progression of various types of cancer, contradictory observations have been reported and the clinical relevance of its expression is still controversial.8 The level of expression of galectin-3 depends on the tumor type analyzed. Although downregulation of this lectin is observed in breast,20,21 prostate,22,23 and uterine adenocarcinoma,24 upregulation occurs in the malignant transformation of thyroid tissue.25,26 However, in thyroid cancer, the expression of galectin-3 is significantly decreased in metastatic lesions of the lymph nodes compared with their primary lesions.27 Recent studies indicate that some discrepancies could be explained by a more detailed analysis of galectin-3 expression and localization, as particularly described in the case of colorectal cancer. Data have been presented indicating that decreased28,29 or increased30,31 expression of galectin-3 in colon cancer cells correlates with disease progression. A recent study demonstrated initial downregulation, with increased cytoplasmic expression of galectin-3 at more advanced stages.32 It is likely that galectin-3, because of its pleiotropic and sometimes opposite effects, could be differently expressed during the malignant transformation and tumor progression in different tissues. With regard to head and neck cancer, previous studies have shown that laryngeal SCCs contain both galectin-1 and galectin-3. In addition, the pattern of expression and the sensitivity to treatment with differentiating agents suggests an association between differentiation and galectin-3 expression in these tumors.10,11 A decrease of cytoplasmic galectin-3 and galectin-3 binding sites expression is an event that occurs early during the loss of differentiation in laryngeal SCC and correlates with an increased level of clinically detectable aggressiveness of these tumors.12 However, no association with clinical outcome was evaluated in this study. Our results extend these observations, showing that a reduced expression of cytoplasmic galectin-3 in laryngeal SCC is a predictor of negative prognosis. In contrast to laryngeal SCC, in tongue carcinoma, enhanced expression of cytoplasmic galectin-3 was reported by Honjo et al33 to be associated with a shorter disease-free survival. Interestingly, both these authors and we have used the same monoclonal antibody (TIB-166 M3/38). We believe that the opposite prognostic significance of galectin-3 in tongue and larynx carcinomas may reflect organ-related differences. In fact, as suggested by Choufani et al,12 the heterogeneous expression of galectins in normal and neoplastic head and neck tissues may be related to the embryologic origin of the tissue. In our series, only eight of 73 laryngeal SCCs exhibited galectin-3 staining of cell nuclei, and all of them also expressed galectin-3 in the cytoplasm. Tumor cases negative for cytoplasmic galectin-3 immunostaining were also negative for nuclear expression of the lectin. Because of the limited number of cases, it was not possible to evaluate the clinical significance of nuclear galectin-3. In our opinion, the pattern of cytoplasmic/nuclear expression of galectin-3 in laryngeal SCC is worthy of further investigation, because nuclear galectin-3 may play a role against tumor progression, as shown in prostate cancer.23 The significant association between galectin-3 positivity and tumor keratinizing status suggests that, in laryngeal SCC, the prognostic effectiveness of galectin-3 expression is due, at least in part, to the fact that this lectin is a marker of cell commitment to differentiation. In histologically normal mucosa and dysplasia, galectin-3 can be detected in the intermediate and superficial layers, with little if any staining in the basal cellular layer (data not shown).10 Accordingly, in areas of well-differentiated laryngeal SCC, the expression patterns of galectin-3 and keratins were parallel, although not overlapping; and in areas of poorly differentiated invasive carcinoma, simultaneous disruption of the expression pattern of galectin-3 and keratins occurs.10 In addition, in normal skin, galectin-3 immunostaining was found predominantly in the middle epidermis (spine layer), and a significantly decreased staining was found in basal cell carcinoma of the skin.34 There are many reports that galectin-3 behaves as an adhesion molecule in various stages of embryogenesis and tumor progression.4,8,9,35,36 Therefore, decreased galectin-3 may loosen the connection among tumor cells and between these and matrix proteins, especially laminin, fibronectin, and vitronectin. This looseness may facilitate extracapsular penetration, vessel invasion, and distant metastasis. This hypothesis is also supported by the finding that galectin-3 expression is inversely correlated with the invasive phenotype of trophoblastic tissue.37 However, other studies have challenged this notion (reviewed in8). Taken together, the available data indicate that galectin-3 expression is related to cell-cell or cell-matrix adhesion processes as already reported, but may affect invasion/metastasis in different ways in different organs/tissue. In human laryngeal SCC, as in thyroid papillary carcinoma,27 downregulation of galectin-3 could facilitate the release of tumor cells from the primary lesions, resulting in the lymph node metastases typical of these tumors. Among the biologic parameters investigated to date, galectin-3, S100A2 (a Ca2+-binding protein related to cell commitment to keratinization),13 and methyl-p-hydroxyphenylactate-esterase expression38 represent prognostic indicators that allow discrimination of high- and low-risk patients. In sum, the evaluation of the expression of galectin-3 may allow identification of a subset of node-negative, galectin-3–negative laryngeal SCC patients who are more susceptible to locoregional relapse and thus require that therapy be adapted accordingly. A more aggressive initial management of these galectin-3–negative tumors may be considered, to avoid undertreatment.
Supported in part by grants from Ministero della Università e della Ricerca Scientifica e Tecnologica (MURST Cofin e Progetti di Eccellenza) and Associazione Italiana Ricerca sul Cancro.
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35. Kasai K, Hirabayashi J: Galectins: A family of animal lectins that decipher glycocodes. J Biochem (Tokyo) 119: 1-8, 1996 36. Matarrese P, Fusco O, Tinari N, et al: Galectin-3 overexpression protects from apoptosis by improving cell adhesion properties. Int J Cancer 85: 545-554, 2000[CrossRef][Medline] 37. van den Brûle FA, Price J, Sobel ME, et al: Inverse expression of two laminin binding proteins, 67LR and galectin-3, correlates with the invasive phenotype of trophoblastic tissue. Biochem Biophys Res Commun 201: 388-393, 1994[CrossRef][Medline] 38. Maurizi M, Ferrandina G, Almadori G, et al: Prognostic significance of methyl-p -hydroxyphenylactate-esterase activity in laryngeal squamous cell carcinoma. Br J Cancer 77: 1253-1259, 1998[Medline] Submitted January 17, 2001; accepted June 10, 2002.
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Copyright © 2002 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
