Acta Neuropathol (2004) 107 : 91–96 DOI 10.1007/s00401-003-0770-6
R E G U L A R PA P E R
Hidehisa Horiguchi · Toshiaki Sano · Zhi Rong Qian · Mitsuyoshi Hirokawa · Noriko Kagawa · Takehiko Yamaguchi · Takanori Hirose · Shinji Nagahiro
Expression of cell adhesion molecules in chordomas: an immunohistochemical study of 16 cases Received: 7 March 2003 / Revised: 7 August 2003 / Accepted: 7 August 2003 / Published online: 8 November 2003 © Springer-Verlag 2003
Abstract Chordomas are thought to be tumors originating from notochord remnants characterized histologically by cohesive cells with epithelial features and by immunohistochemical expression of epithelial markers. To investigate the expression and distribution of cell adhesion molecules in chordomas, we immunohistochemically studied the expression of representative cell adhesion molecules, E-cadherin, P-cadherin, N-cadherin, β-catenin, CD44, ICAM-1 (CD54), NCAM (CD56), and VCAM-1 (CD106) in 16 tumors from 16 patients (skull base, n=5; cervical, n=2; sacral, n=9) and 3 cases of fetal notochord. Of 16 tumors, 12 (75.0%) expressed membranous immunoreactivity for NCAM, 10 (62.5%) for VCAM-1, 9 (56.3%) for CD44, 8 (50.0%) for N-cadherin, 6 (37.5%) for β-catenin, 4 (25%) for ICAM-1, and 1 (6.3%) for P-cadherin. Nuclear staining for E-cadherin was recognized in 11 (68.8%) tumors, and membranous staining for E-cadherin in 3 (18.8%); none of the tumors showed both nuclear and membranous staining. Intranuclear accumulation of β-catenin was not found in any chordoma. One fetal notochord case showed immunoreactivity for N-cadherin, E-cadherin (some cells showed staining in both cytoplasm and nuclei), CD44 and β-catenin. These results indicate that chordomas frequently
H. Horiguchi (✉) · T. Sano · Z. R. Qian · M. Hirokawa · N. Kagawa Department of Pathology, University of Tokushima School of Medicine, 3-18-15 Kuramoto, 770-8503 Tokushima, Japan Tel.: +81-88-6337064, Fax: +81-88-6339423, e-mail:
[email protected] S. Nagahiro Department of Neurological Surgery, University of Tokushima School of Medicine, Tokushima, Japan T. Yamaguchi Department of Pathology, Koshigaya Hospital, Dokkyo University School of Medicine, Koshigaya 343-8555, Japan T. Hirose Department of Pathology, Saitama Medical School, Moroyama, Saitama 350-0495, Japan
express immunoreactivity for multiple adhesion molecules including VCAM, CD44 and N-cadherin, as well as for NCAM and E-cadherin, as previously reported. These molecules may participate in producing the cellular cohesion evident in tumor morphological structure. Although the precise underlying mechanisms remain to be elucidated, the high frequency of nuclear expression of E-cadherin (11 of 16 cases) may be diagnostically useful. Keywords Chordoma · Cell adhesion molecules · E-cadherin · Immunohistochemstry
Introduction Chordomas are relatively rare bone tumors. They occur exclusively in the axial skeleton such as the clivus and a midline lesion of the vertebrae and sacrum and are thought to originate from notochord remnants [5]. Histologically, chordomas often exhibit a myxoid extracellular matrix and cartilaginous differentiation as osteochondroid features. However, they have some epithelial features such as cell cohesiveness, a cord-like structure, and lobulation. The tumor cells are usually immunoreactive for cytokeratin (CK) and epithelial membrane antigen as epithelial markers. Their S-100 protein positivity is also a useful diagnostic indicator. Ultrastructurally, intracytoplasmic intermediate filaments and desmosome-like junctions are recognized in the tumor cells [16, 23]. These findings suggest the existence of cell adhesion molecules in chordomas. Cell adhesion molecules that have a function in cellcell and cell-matrix adherence junctions have been found in human epithelial cells [4, 14], and epithelial tumors express various adhesion molecules. Some carcinomas show a reduction in these molecules [3, 31], while in others there is overexpression of specific adhesion molecules [10, 27]. Moreover, the expression of cell adhesion molecules in several mesenchymal tumors has been reported [11, 12]. We used immunohistochemical methods to study the expression of representative cell adhesion molecules, E-cadherin, N-cadherin, P-cadherin, β-catenin, CD44, ICAM-1
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(CD54), NCAM (CD56), and VCAM-1 (CD106) in tumor samples from 16 chordoma patients, and discuss the implications of the expression of these cell adhesion molecules.
activity was scored from negative to 3+ according to the percentage of positive cells (1+: 50%). Tissues of small cell carcinoma of lung for N-cadheirn, E-cadherin and NCAM; uterine cervix for CD44, ICAM-1 and VCAM-1; lung with adenocarcinoma for P-cadherin; colon for β-catenin and pheochromocytoma for pan-neurofilament were used as positive control specimens.
Materials and methods We obtained samples from 16 chordoma patients and 3 fetal autopsy cases (8, 21 and 35 weeks) at University of Tokushima School of Medicine, Dokkyo University School of Medicine and their associated hospitals (Table 1). Resected tumors were fixed in 10% formalin and embedded in paraffin. Tissues (4 µm in thickness) were used for routine hematoxylin and eosin (HE) staining and immunohistochemical study. All cases were histologically diagnosed as chordoma by HE staining. The antibodies shown in Table 2 were used for immunohistochemistry. Antigens were retrieved by microwave in citrate buffer (pH 7.0 for ICAM-1, pH 8.0 for VCAM-1 and pH 6.0 for the other antibodies). Immunohistochemistry was performed using the ENVISION kit and autostainer (DAKO, Carpenteria, CA), and diaminobenzidine for visualization. Immunore-
Results
Table 1 Results of cases and immunohistochemistry (N-cad N-cadherin, P-cad P-cadherin, E-cad E-cadherin, β-cat β-catenin, ICAM-1 CD54, NCAM CD56, VCAM-1 CD106, NF neurofilament, – nega-
tive, 1+ 50%, MS membranous staining, NS nuclear staining, Recur./Meta. recurrence/metastasis, ND not detected, NC notochord)
Clinicopathology The tumor patients, 14 men and 2 women, were aged between 34 and 74 years. Of the 16 tumors, 9 were sacral, 5 were of cranial origin, and 2 were cervical. Follow-up data were available for 6 patients; 5 experienced local recurrence at 9 months to 8 years from the first surgery, 1 sacral tumor had metastasized to the thigh at 2 years after the initial surgery (Table 1).
Case
Age/sex
Site
N-cad
P-cad
E-cad
β-cat
CD44
ICAM-1 NCAM
VCAM-1
NF
Rec./Meta.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 NC1 NC2 NC3
63/M 64/M 68/M 64/M 51/M 70/M 47/M 70/F 55/M 55/M 58/M 42/M 34/M 55/M 63/M 74/F 8 weeks 21 weeks 35 weeks
Sacral Skull Sacral Skull Sacral Sacral Skull Cervical Sacral Skull Sacral Sacral Sacral Sacral Cervical Skull
1+ – 2+ 2+ – 1+ 1+ 1+ – – – – 2+ 1+ – – 3+ – –
– 1+ – – – – – – – – – – – – – – – – –
– 3+ (MS) 3+ (NS) 3+ (NS) 2+ (NS) 1+ (NS) 1+ (MS) 1+ (NS) 2+ (NS) 1+ (MS) 2+ (NS) 1+ (NS) 2+ (NS) 2+ (NS) 1+ (NS) – 3+a – –
– 2+ 1+ 2+ – 1+ 1+ 1+ – 1+ – – – 1+ – – 3+ – –
1+ – 2+ – 1+ 1+ – 1+ – – 1+ 1+ – 1+ 1+ – 1+ – –
– 1+ – 1+ – – 1+ – – – – – – 1+ – – – – –
– 1+ 2+ 2+ 2+ 1+ – – 1+ – 2+ 1+ – 2+ 1+ – – – –
– – – – – – – – – – – – – – – – – – –
ND Rec., 9 months Recur., 7 years ND ND ND ND Recur., 6 years Recur., 3 years ND ND Meta., 2 years ND ND ND Recur., 8 years
aSome
3+ 3+ 2+ 2+ 2+ 1+ 1+ 1+ 1+ 1+ 1+ 1+ – – – – 1+ – –
cells showed only cytoplasmic staining and the others demonstrated both nuclear and cytoplasmic staining
Table 2 Antibodies used for immunohistochemistry
Antibody (clone names and source)
Dilution
Antigen retrieval by microwave
E-cadherin (36, Transduction Laboratories, Lexington, USA) N-cadherin (3B9, ZYMED, South San Francisco, USA) P-cadherin (56C1, Novocastra, Newcastle, UK) CD44 (F10–44–2, Novocastra) ICAM-1 (CD54, 23G12, ICAM-1, Novocastra) NCAM (CD56, 123C3, Santa Cruz Biochemistry, Santa Cruz, USA) VCAM-1 (CD106, 1.4C3, VCAM-1) Pan-neurofilament (ZYMED) β-catenin (14, Transduction Laboratories)
1:500 1:100 1:100 1:50 1:50 1:100 1:100 1:50 1:500
done done done done done done done done done
93 Fig. 1 Immunohistochemistry of cell adhesion molecules in chordoma showing membranous staining. A NCAM, B VCAM-1, C CD44, D N-cadherin, E ICAM-1, F β-catenin. Chordoma frequently show immunoreactivity for NCAM (75.0%), VCAM-1 (62.5%), CD44 (56.3%) and N-cadherin (50.0%). Immunoreactivity for β-catenin is seen in membrane in 6 (37.5%) of the 16 tumors. Nuclear staining for β-catenin is not seen in any of the present study cases
Histological findings All but three tumors had a well-lobulated structure; in the three exceptions there was comparatively scant tissue. The nuclei were oval to spindle-shaped, and there was remarkable cell cohesiveness in distinct lobules. Most cases revealed cytoplasmic vacuolation and a mucous background, and several cases manifested physaliphorous cells as a typical feature of chordoma. Chondroid differentiation was observed in none of the cases. In seven cases there were prominent cord-like structures in the myxoid matrix. Mitoses and necrosis were rare. Immunohistochemical findings As summarized in Table 1, the expression of cell adhesion molecules varied among the 16 cases; 12 (75.0%) were
immunoreactive for NCAM, 10 (62.5%) for VCAM-1, 9 (56.3%) for CD44, 8 (50%) for N-cadherin, 4 (25.0%) for ICAM-1 and 6 (37.5%) for β-catenin; all of which demonstrated membranous immunoreactivity (Fig. 1A–F). Just 1 case was immunoreactive for P-cadherin. Membranous reactivity for E-cadherin was present in 3 cases (18.8%) and 11 cases (68.8%) manifested nuclear staining without cytoplasmic or membranous positivity (Fig. 2). None of the 16 cases was immunoreactive for pan-neurofilaments. In 3 notochord tissues, 1 (8 weeks) showed immunoreactivity for N-cadherin, E-cadherin, β–catenin, CD44 and NCAM. E-cadherin immunoreactivity was seen not only in cytoplasm but also in nuclei in some cells. The other 2 tissues (21 and 35 weeks) did not show immunoreactivity for any antibodies.
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Fig. 2 Immunohistochemistry for E-cadherin in chordoma. Eleven (68.8%) of the 16 tumors show characteristic nuclear staining in this study. These 11 cases had no cytoplasmic or membranous staining
Discussion Human notochord tissue and its hamartomatous remnant, ecchordosis physaliphora [9], are incidentally found at spinal surgery and autopsy. They have been exclusively recognized in axial skeletons, such as the skull, vertebrae, and sacrum. Histologically, notochordal tissue consists of eosinophilic or vacuolated cells with mucous fluid and myxoid stroma. Ultrastructurally, intracytoplasmic vacuoles, distended endoplasmic reticulum, and desmosomal junctions have been found in notochordal tissue [9, 30]. Immunohistochemically, notochord cells strongly express S-100 protein, CKs and epithelial membrane antigen as characteristic features [5, 24]. As several features of chordoma histologically and immunohistochemically resemble notochord, these tumors are thought to originate from notochord remnants and morphological similarities, e.g., shared cell adhesion characteristics, would not be surprising. Immunohistochemically, chordomas have epithelial markers such as CKs, epithelial membrane antigen, and carcinoembryonic antigen [5, 17]. Naka et al. [19], who found that both chordomas and notochords immunohistochemically express CK 8 and 19, suggested that this indicates the notochord origin of chordoma and that the presence of CK 8 and 19 facilitates a differential diagnosis of chordoma from myxoid chondrosarcoma. Dorfman and Czerniak [5] described that some chordomas are positive for neurofilament; however, the immunoreactive implications remain unclear. None of our samples were immunoreactive for pan-neurofilament peptides. The presence of microscopic cellular cohesiveness, ultrastructural cell-cell junctional apparati, and CK immunoreactivity suggests that cell-cell adhesion molecules function in chordomas as they do in other epithelial tumors. Some kinds of adhesion molecules play an important role
in embryogenesis and tissue morphogenesis. NCAM is transiently expressed by the notochord in the early stage of chick embryogenesis [28]. NCAM expression has also been reported in non-neuronal tumors [11, 21]. Of the classical cadherins, N- and P-cadherins have been observed in the notochord of mouse and chick embryos [22, 26]. These observations indicate that NCAM and N-cadherin have an important role not only in neuronal differentiation but also in notochord embryogenesis and in chordoma tumorigenesis. Naka et al. [20] reported that E-cadherin, α, β, γ-catenins and NCAM are frequently expressed immunohistochemically in chordomas. All of their immunoreactive cases manifested cytoplasmic positivity. Our study confirms the expression of NCAM in chordomas, and we additionally found that these many tumors express other adhesion molecules, especially N-cadherin. Our findings indicate that chordomas retain characteristics of the fetal notochord. We posit that NCAM and N-cadherin can exist as essential elements for tumoral morphogenesis, while their presence does not confirm a neurogenic origin for chordomas. Laskin and Miettinen [12] found that none of 11 chondroid tumors, including extraskeletal myxoid chondrosarcomas and chondrosarcomas of the bone, were immunoreactive for N-cadherin. On the other hand, 8 of our 16 chordomas manifested N-cadherin immunoreativity. This observation may be helpful in distinguishing chordomas from other tumors. CD44, ICAM-1 and VCAM-1 are representative cell adhesion molecules, whose expression has been recognized in various tumoral and non-tumoral cells. The overexpression of isoforms of CD44 plays a role in the metastatic potential of some malignant tumors and in the disease-free survival of patients [7, 10, 31]. However, the relationship between the expression of cell adhesion molecules and the prognosis of cancer patients remains controversial. In patients with colorectal cancer, Maeda et al. [15] reported a close association between the immunohistochemical reduction of ICAM-1 and metastasis and prognosis. On the other hand, despite the absence of immunoreactivity in normal pancreatic tissue, in pancreas cancers there is enhanced expression of ICAM-1 and VCAM-1 [27]. More than half of our chordoma samples exhibited immunoreactivity for CD44 and VCAM-1, suggesting that these cell adhesion molecules may enhance the invasive and metastatic potential of chordomas. In the present study, we could not get enough follow-up cases to assess the relationship between expression of these molecules and biological behavior of tumors. Further clinicopathological studies are needed to test this hypothesis. E-cadherin normally localizes adjacent to the cell membrane. In the human notochord, E-cadherin expression is recognized in the early fetal stages and is lost by weeks 12–13 of gestation [6]. In the present study, notochord at 8 weeks of gestation showed E-cadherin immunoreactivity in both cytoplasm and nuclei of some cells, while two cases (21 and 35 weeks) had no immunoreactivity. This supports the above-mentioned finding of E-cadherin expression in fetal notochord [6]. In some malignant tumors, the reduction of E-cadherin expression results in a poor
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prognosis [3, 13]. On the other hand, β-catenin, a proteinbinding E-cadherin, has been found to participate in cell adhesion [1]. The nuclear accumulation of β-catenin is thought to represent a dysfunction that induces tumorigenesis [2, 29]. Mori et al. [18] reported the immunohistochemical expression of E-cadherin at the intercellular adhesion site of several chordomas, while Laskin and Miettinen [12] found no evidence of E-cadherin immunoreactivity in any of their 11 chordomas. We unexpectedly detected immunoreactivity for E-cadherin in the nuclei of tumor cells in 68.8% of our cases and found normal membrane localization of β-catenin. Repeat immunostaining for E-cadherin confirmed its intranuclear immunoreactivity. There are only few tumors with nuclear accumulations of E-cadherin. Han et al. [8] reported the nuclear localization of E-cadherin expression in most of the 35 Merkel cell carcinomas they studied; approximately half of their cases manifested membranous staining by E-cadherin. Sauer et al. [25], who examined fine needle aspirates, noted that the nuclei were positive in 12 of 56 (21.4%) breast carcinomas. In some instances of theirs, both nuclei and membranes were stained. In the present study, one notocohrd case showed immunoreactivity in both nuclei and cytoplasm. However, none of our tumor cases with nuclear immunoreactivity for E-cadherin manifested cytoplasmic and/or membranous staining. According to these results two possibilities come to mind to explain these contrary observations. One is a cross-reaction with some other epitopes in the nucleus, possibly attributable to differences in the antibodies used in the respective studies. Alternatively, the E-cadherin protein may have migrated into the nucleus by unknown mechanisms. Although comparison with other chondroid tumors is necessary, a high frequency of nuclear immunoreactivity for E-cadherin may be a useful diagnostic finding in chordomas. Our study revealed that chordomas frequently show immunohistochemical expression of various cell adhesion molecules such as NCAM, VCAM-1, CD44, N-cadherin and E-cadherin (intranuclear). N-cadherin can be a useful diagnostic marker for distinction chordoma from other chondroid tumors. We posit that some of them contribute to the maintenance of the morphological nature of the notochord, while others play a role only in cell cohesiveness. Moreover, the biological behavior, e.g., local aggressiveness and metastasis, should be compared to expression of various adhesion molecules. Although the underlying mechanism(s) remain to be clarified, we conclude that the expression of E-cadherin in the tumor cell nuclei is a characteristic feature of chordomas. Acknowledgements We thank Dr. Yoshiyuki Fujii (Tokushima Red Cross Hospital), Dr. Nobuya Sano (Hyogo Prefectural Awaji Hospital), Dr. Sadao Shinomiya (Health Insurance Naruto Hospital), Dr. Yoshikazu Yamazaki (Kochi Red Cross Hospital), and Ms. Noriko Amo for kindly providing of tissue materials and technical assistance.
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