Characteristic Formation of Hyaluronan-Cartilage Link Protein-Proteoglycan Complex in Salivary Gland Tumors.

RESEARCH ARTICLE

Characteristic Formation of Hyaluronan-Cartilage Link Protein-Proteoglycan Complex in Salivary Gland Tumors Hiroko Kuwabara, MD,* Akira Nishikado, MD,w Hana Hayasaki, PhD,z Zenzo Isogai, MD,y Masahiko Yoneda, PhD,8 Ryo Kawata, MD,w and Yoshinobu Hirose, MD*

Abstract: Hyaluronan (HA) and its binding molecules, cartilage link protein (LP) and proteoglycan (PG), are structural components of the hydrated extracellular matrix. Because these molecules play important roles in the tumor microenvironment, we examined the distribution of HA, LP, versican, and aggrecan in salivary gland tumors using histochemical and immunohistochemical methods, including double staining. LP was present in pleomorphic adenoma (PA) and adenoid cystic carcinoma (ACC) tissues, and aggrecan was absent in the malignant tumors that we investigated. LP colocalized with both HA and aggrecan in the chondromyxoid matrix of PA, suggesting the presence of a HA-LP-aggrecan complex. Furthermore, the HA-LP-versican complex could be observed in the pseudocystic space of the cribriform structures in ACC. The characteristic HA-LP-PG complex in PA and ACC might play a role in the behavior of tumors, and immunohistochemical analysis of these molecules could represent a diagnostic adjunct for salivary gland tumors. Key Words: cartilage link protein, versican, aggrecan, hyaluronan, salivary gland tumors, immunohistochemistry (Appl Immunohistochem Mol Morphol 2015;00:000–000)

H

yaluronan (HA) and proteoglycan (PG) are major components of the hydrated extracellular matrix (ECM), and these molecules form large complexes that provide structural integrity and mechanical functions in tissues.1,2 Cartilage link protein (LP), also known as “HA and PG binding protein-1” or “HAPLN-1,” plays a role in the formation and stabilization of HA and large chondroitin sulfate PGs, such as aggrecan and versican/ PG-M.3 Therefore, HA, chondroitin sulfate PG and LP cooperatively maintain the structural integrity of the exReceived for publication December 6, 2014; accepted February 21, 2015. From the Departments of *Pathology; wOtorhinolaryngology, Osaka Medical College, Takatsuki; zDepartment of Anatomy, Kansai University of Welfare Science, Kashiwara; yDepartment of Advance Medicine, National Center for Geriatrics and Gerontology, Obu; and 8Biochemistry and Molecular Biology Laboratory, Department of Nursing and Health, Aichi Prefectural University, Nagoya, Japan. The authors declare no conflict of interest. Reprints: Hiroko Kuwabara, MD, Department of Pathology, Osaka Medical College, 2-7 Daigaku-machi, Takatsuki, Osaka 569-8686, Japan (e-mail: [email protected]). Copyright r 2015 Wolters Kluwer Health, Inc. All rights reserved.

Appl Immunohistochem Mol Morphol

tracellular and pericellular matrices. LP, which was originally isolated from cartilage, is also expressed in other tissues, such as the brain, sclera, aorta, heart, retina, muscle tendon, skin, and digestive tract.4 HA and PG in the tumor microenvironment play important roles in determining the migratory phenotype of a cell by initiating, directing, and terminating cell movement.5 Each salivary gland tumor has a characteristic ECM, and, notably, pleomorphic adenoma (PA) and adenoid cystic carcinoma (ACC) contain abundant glycosaminoglycans, including HA and chondroitin sulfate.6 PA, the most common type of salivary gland neoplasm, consists of epithelial and modified myoepithelial elements that intermingle with mesenchyme-like tissues, yielding varying admixtures of myxoid, chondroid, hyalinized, and fibrous structures.7 The expression of HA and a cartilage-specific PG, aggrecan, and cartilage-associated proteins, such as cartilage-derived morphogenic protein-1 and chondromodulin-I, are present in PA.8–11 In ACC, which comprises approximately 10% of all epithelial salivary neoplasms, variable morphologic configurations can be observed, including cribriform, tubular, and solid growth patterns.7 A cribriform pattern, the most common pattern, is characterized by nests of cells with cylindromatous microcystic spaces, known as pseudocysts, which are surrounded by myoepithelial cells. The pseudocystic space contains HA and versican, in addition to type IV collagen-positive and laminin-positive replicating basal lamina adjacent to the myoepithelial cells.6,12–14 On the basis of this background, we speculated that the HA-LP-PG complex plays a key role in salivary gland tumors, particularly in PA and ACC. Herein, we investigate the distribution of HA, LP, versican, and aggrecan in salivary gland tumors, including PA, ACC, mucoepidermoid carcinoma (MEC), salivary duct carcinoma (SDC), and basal cell adenoma (BCA). We show that the peculiar distribution of the HA-LP-PG complex in PA and ACC, and the formation of different matrix complexes among each salivary gland tumor represents a useful diagnostic adjunct.

MATERIALS AND METHODS Patients and Materials Surgical specimens of 15 PA (5 male, 10 female; mean age = 41 y; 15 parotid glands), 15 ACC (8 male,

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7 female; mean age = 48 y; 7 parotid glands, 3 submandibular glands, and 5 oral mucosa), 12 MEC (6 male, 6 female; mean age = 51 y; 11 parotid glands and 1 oral mucosa), 5 SDC (4 male, 1 female; mean age = 61 y; 5 parotid glands), and 5 BCA (3 male, 2 female; mean age = 50 y; 5 parotid glands) were subjected to histochemical and immunohistochemical analyses. Slides were reviewed to confirm diagnoses and the adequacy of tissue, and only tumors with definite patterns were included in this study. All specimens included normal salivary gland tissues. The study was approved by the Osaka Medical College Ethics Committee. Tissue samples were routinely fixed in 10% buffered formalin, dehydrated in a graded ethanol series, and embedded in paraffin wax. Serial sections 3-mmthick were then prepared, and these were stained using hematoxylin-eosin, HA staining, or immunohistochemistry.


without HABP were used as negative controls. The staining intensity was evaluated as none (), weak (+), moderate (++), or intense (+++).

Immunohistochemistry Immunostaining was performed using a BONDMAX autoimmunostainer (Leica Microsystems, Wetzlar, Germany). Deparaffinized and rehydrated sections were subjected to endogenous peroxidase blocking. After heating in antigen unmasking solution, slides were incubated with one of the following antibodies: LP (9/30/8-A-4, 1:400, Developmental Studies Hybridoma Bank, DSHB, Iowa City, IA),16,17 versican (2B1, 1:1000, Seikagaku Corp., Tokyo, Japan), or aggrecan (#179509, 1:100, R&D Systems, Minneapolis, MN). Color development was carried out using DAB, and slides were counterstained with hematoxylin. Sections of human lung tissues, including tracheal cartilage, were used as positive controls, and procedures without primary antibodies were used as negative controls. The staining intensity was evaluated according to the criteria described in the section for HA staining.

Staining for HA The distribution of HA was detected by applying biotinylated hyaluronan binding protein (HABP). A cartilage-derived HABP with high affinity and specificity was extracted from bovine nasal cartilage and biotinylated.15 After blocking endogenous peroxidase activity with 3% hydrogen peroxide, slides were incubated with biotinylated HABP (1:50) and horseradish peroxidase-conjugated avidinbiotin complex (1:200, Vector Laboratories, Burlingame, CA). Then, 3,30 -diaminobenzidine tetrahydrochloride (DAB) and hematoxylin were used as a chromogen and a counterstain, respectively. Sections of human cutaneous dermal tissues were used as positive controls, and procedures

Double Immunostaining Double-immunofluorescence staining of PA and ACC was performed using the following combinations: aggrecan (goat polyclonal, 1:50, R&D Systems) and LP (1:200); HABP (1:25) and LP; HABP (1:25) and aggrecan (goat polyclonal); versican (rabbit monoclonal, EPR 12277, 1:200, Epitomics, Cambridge, UK) and LP; and HABP and versican (rabbit monoclonal). The secondary

TABLE 1. HA, LP, Versican, and Aggrecan Expression in Salivary Gland Tumors PA HABP

++ to Mesenchymal areas (15/15) +++ (myxoid, chondroid, hyalinized, fibrous) ++ Myoepithelial cells (14/15) +++ Capsule(14/14)

LP

++ to Mesenchymal areas (15/15) +++ (myxoid, chondroid)

Versican

+

Mesenchymal areas (10/15) (fibrous)

+

Myoepithelial cells (3/15)

Aggrecan +++ Mesenchymal areas (15/15)

ACC

MEC

++

Stroma (15/15)

+ to ++ +

Pseudocyst (14/15) Myoepithelial cells (3/15)

+++ Pseudocyst (13/15) +++ Basal lamina (8/15) +++ Pseudocyst (15/15) + Myoepithelial cells (5/15) + Stroma (14/15) –

(15/15)

++ Stroma (12/12)

–

(12/12)

+ to Stroma (9/12) ++ + Epithelial cells (2/12)

–

(12/12)

SDC

BCA

++ Stroma ++ Stroma (5/5) (5/5)

–

(5/5)

++ Hyalinized stroma (1/5)

+ to Stroma ++ Stroma (5/5) ++ (5/5) + Basal cells (5/5)

–

(5/5)

(myxoid, chondroid) ++ to Myoepithelial cells (10/15) +++

++ Spindle cells and associated stroma (3/5)

Parentheses indicate number of cases. The intensity of staining: –, none; +, weak; ++, moderate; +++, intense. ACC indicates adenoid cystic carcinoma; BCA, basal cell adenoma; HA, hyaluronan; HABP, hyaluronan binding protein; LP, link protein; MEC, mucoepidermoid carcinoma; PA, pleomorphic adenoma; SDC, salivary duct carcinoma.

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HA-cartilage LP-PG Complex in Salivary Gland Tumors

FIGURE 1. Representative images of pleomorphic adenoma. A, Hematoxylin-eosin. B, Hyaluronan binding protein. C, Aggrecan. D, Link protein (LP). E, Versican. Hyaluronan, aggrecan, and LP expression in the chondromyxoid matrix.

antibodies used were Alexa Fluor 488 chicken anti-goat, Alexa Fluor 546 goat anti-mouse, Alexa Fluor 488 streptavidin, Alexa Fluor 546 chicken anti-goat, Alexa Fluor 488 goat anti-rabbit, and Alexa 546 goat antirabbit (Life Technologies, Carlsbad, CA), diluted in 1:100 in phosphate-buffered saline. These stained slides were Copyright

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examined using a LSM510 laser-scanning confocal microscope (Carl Zeiss, Oberkochen, Germany).

Statistical Analyses The fitness of LP and aggrecan molecules in each salivary gland tumor was analyzed statistically www.appliedimmunohist.com |


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FIGURE 2. Representative images of adenoid cystic carcinoma. A, Hematoxylin-eosin. B, Hyaluronan binding protein. C, Versican. D, Link protein (LP). E, Aggrecan. Hyaluronan, versican and LP expression in pseudocystic spaces.

using a goodness-of-fit test (likelihood ratio test) with JMP (SAS institute Japan, Tokyo, Japan). As a control, a positive/negative ratio of 1 (50%/50%) was used. A P-value < 0.05 was considered to indicate statistical significance.


RESULTS Normal Salivary Glands HA was observed in the stroma, but not in acinar or ductal epithelial cells. Versican and aggrecan were posCopyright

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FIGURE 3. Hematoxylin-eosin and immunostaining. A and B, Mucoepidermoid carcinoma [versican (B)]. C and D, Salivary duct carcinoma [versican (D)]. Versican expression in the stroma adjoining tumor cells. E and F, Basal cell adenoma (F, aggrecan). Aggrecan positivity in spindle cells and their surrounding stroma.

itively stained in the periductal tissues and vessel walls, respectively. LP was completely absent. The histochemistry and immunohistochemistry results for HABP, LP, versican, and aggrecan in salivary gland tumors are summarized in Table 1. Copyright

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PA HA could be observed throughout mesenchymal tissues (myxoid, chondroid, hyalinized, and fibrous), myoepithelial cells, and capsular tissues. Strong immunoreactivity for aggrecan was observed in myxoid and www.appliedimmunohist.com |


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aggrecan

LP

merge

DIC

HABP

LP

merge

DIC

HABP

aggrecan

merge

DIC

FIGURE 4. Double-immunostaining of pleomorphic adenoma. Upper: aggrecan and LP; middle: HABP and LP; lower: HABP and aggrecan. The colocalization of aggrecan, LP, and HA. DIC indicates differential interference contrast microscope; HABP, hyaluronan binding protein; LP, link protein.

chondroid mesenchymal areas and myoepithelial cells, but not in the capsule. LP was detected in myxoid and chondroid areas, where both HA and aggrecan were present. Versican was observed in the fibrous mesenchymal areas and in some myoepithelial cells (Fig. 1).

ACC HA was detected weakly to moderately both in the pseudocystic space and stroma. The mucinous materials in the pseudocystic spaces were positive for versican and LP, but not for aggrecan. LP was also positive in basal lamina (Fig. 2).

MEC and SDC HA was detected diffusely in the stroma, and versican was present in the stromal sections adjoining tumor cells (Figs. 3A–D). LP and aggrecan were completely absent.


BCA HA and versican were present in the stroma. LP staining was negative, except for 1 membranous BCA case, where LP was present in the hyalinized stroma. In contrast, spindle cells, which are interpreted as myoepithelial cells, and their surrounding stroma were positive for aggrecan (Figs. 3E, F).

HA-LP-PG Complexes in PA and ACC LP was mainly observed in the PA and ACC, and the colocalization of LP/PG, HA/LP, and HA/PG were investigated by double-immunofluorescence staining. In the PA, LP colocalized with both aggrecan and HA, and the colocalization of aggrecan and HA was observed (Fig. 4). By contrast, in the pseudocystic space of the ACC, LP colocalized with both versican and HA, and colocalization of versican and HA was observed (Fig. 5). Copyright

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versican

LP

merge

DIC

HABP

LP

merge

DIC

HABP

versican

merge

DIC

FIGURE 5. Double-immunostaining of adenoid cystic carcinoma. Upper: versican and LP; middle: HABP and LP; lower: HABP and versican. The colocalization of versican, LP, and HA. DIC indicates differential interference contrast microscope; HABP, hyaluronan binding protein; LP, link protein.

Controls

DISCUSSION

In the tracheal cartilage, aggrecan and LP were localized in chondrocytes and their territorial matrix. Versican was detected in fibrous tissue surrounding the cartilage. In cutaneous tissue, HABP was localized in the dermis. No staining for HABP or any antibody was observed in the negative controls.

Statistical Analyses The fitness of LP and aggrecan molecules in each salivary gland tumor is shown in Table 2. LP was notably present in the chondromyxoid matrix of PA and pseudocystic space of ACC, and it was significantly absent in MEC and SDC. In contrast, aggrecan was significantly present in PA, and it was absent in malignant tumors, including ACC, MEC, and SDC. Copyright

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In this present study, we report for the first time the expression of LP in PA and ACC, and that LP creates a specific ECM by binding to HA and PG. In PA, the HA-LPaggrecan complex was present in the chondromyxoid area, and this finding is in accordance with previous reports that aggrecan is abundantly located in the chondromyxoid areas of PA.8,13 Aggrecan is quite specific for cartilage, and provides an extremely high-fixed charge density, which is central to creating the high osmotic environment necessary to retain water in tissues, via its chondroitin sulfate chains.18 HA and LP interact with a globular domain G1 of aggrecan to form aggrecan aggregates, generating tensile strength and elasticity,19 and this complex might induce chondroid ECM differentiation in PA, thereby stabilizing aggregated aggrecan. Moreover, we are the first to report the HA-LPversican complex in the pseudocystic space of ACC. www.appliedimmunohist.com |


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TABLE 2. Characteristics of LP and Aggrecan Positivity LP P Aggrecan P

PA (Chondromyxoid)

ACC (Pseudocyst)

MEC

SDC

BCA

15/15 < 0.0001 15/15 < 0.0001

13/15 0.0027 0/15 < 0.0001

0/12 < 0.0001 0/12 < 0.0001

0/5 0.0085 0/5 0.0085

1/5 0.165 3/5 0.6536

P-value calculated by goodness-of-fit test. ACC indicates adenoid cystic carcinoma; BCA, basal cell adenoma; LP, link protein; MEC, mucoepidermoid carcinoma; PA, pleomorphic adenoma; SDC, salivary duct carcinoma.

These 3 components are profoundly associated with organogenesis, such as cardiac cushion development and stem cell differentiation.20 Genetic deficiency in LP results in impairment of growth and the development of several tissues, including the cartilage, heart, and central nervous system.21–23 In addition, versican is essential for ventricular septal formation subsequent to atrioventricular cushion development, which affects LP deposition, and it has been suggested that the HA-LP-versican complex is required for versican aggregate deposition to create spaces for the cardiac jelly.24 Gene expression profiles showed that versican is tumor-specific in ACC,25 and that the HA-LP-versican complex seems to produce a carcinoma-specific rigid cribriform space in ACC. In contrast, LP was absent in MEC, SDC, and BCA, except for a membranous BCA case. Membranous BCA can be histologically characterized by a hyalinized fibrous stroma, and it has a recurrence rate of 25%.7 Membranous BCA has a different stroma from conventional BCA, which may influence the behavior of a tumor cell. Aggrecan was negative in malignant tumors, including ACC, MEC, and SDC, and this finding is useful for distinguishing PA from these malignant tumors. For example, PA with squamous metaplasia and cribriform architecture can mimic MEC and ACC, respectively,26 and the presence of aggrecan seems to favor the diagnosis of PA. Interestingly, aggrecan was positive in the richly cellular stroma of BCA. These stromal cells, which are thought to be modified S-100 positive myoepithelial cells, are diagnostic indicators of BCA.3,27 Although we have no appropriate explanation for this positivity, PA and the richly cellular stroma of BCA may have common ECM features. In addition, aggrecan positivity in malignant salivary gland tumors seems to facilitate the diagnosis of carcinoma ex-PA. SDC is the most common histologic subtype of carcinoma ex-PA,28 and carcinoma ex-PA and its de novo carcinomas exhibit a different genetic background and prognosis,29 therefore, positive aggrecan immunostaining might distinguish carcinoma ex-PA from de novo carcinoma. Versican was present in the stroma adjoining the tumor cells in MEC and SDC. Generally, versican is strongly expressed in the stroma of many malignant tumors, including gastric, colonic, melanoma, breast, uterine, and ovarian carcinomas.30 Versican regulates cell adhesion, proliferation, migration, survival, differentiation, and angiogenesis, and its expression is increased in tumors having a higher grade and a worse


outcome. The expression of versican in salivary gland tumors might facilitate tumor cell growth, as observed for other tumors. The investigated ECM molecules that showed a peculiar localization among each neoplastic tumor, as compared with normal salivary glands. In addition, versican and aggrecan were present in neoplastic myoepithelial or basal cells, but not in normal ones, which suggests that these ECM molecules are produced by neoplastic myoepithelial or basal cells. This is supported by immunoelectron microscopic observation of aggrecan in PA, which showed aggrecan positivity in the vacuoles of neoplastic myoepithelial cells.31 In conclusion, HA-LP-aggrecan and HA-LP-versican complexes were present in PA and ACC, respectively, and aggrecan was absent in the malignant tumors that we studied. Immunohistochemical detection of LP, versican and aggrecan could be a useful tool in the diagnosis of salivary gland tumors, and LP might organize and maintain the peculiar ECM of PA and ACC, interacting with PG and HA. ACKNOWLEDGMENTS The authors gratefully thank Shizuka Akashi and Kaname Shimokawa for providing expert technical assistance, and Yasuichiro Nishimura for helpful discussions regarding the statistical analyses. REFERENCES 1. Watanabe H, Cheung SC, Itano N, et al. Identification of hyaluronan-binding domains of aggrecan. J Biol Chem. 1997;272: 28057–28065. 2. Wight TN. Versican: a versatile extracellular matrix proteoglycan in cell biology. Curr Opin Cell Biol. 2002;14:617–623. 3. Matsumoto K, Shionyu M, Go M, et al. Distinct interaction of versican/PG-M with hyaluronan and link protein. J Biol Chem. 2003;278:41205–41212. 4. Binette F, Cravens J, Kahoussi B, et al. Link protein is ubiquitously expressed in non-cartilaginous tissues where it enhances and stabilizes the interaction of proteoglycan with hyaluronic acid. J Biol Chem. 1994;269:19116–19122. 5. Cattaruzza S, Perris R. Proteoglycan control of cell movement during wound healing and cancer spreading. Matrix Biol. 2005;24: 400–417. 6. Nakanishi K, Kawai T, Suzuki M, et al. Glycosaminoglycans in pleomorphic adenoma and adenoid cystic carcinoma of the salivary gland. Arch Pathol Lab Med. 1990;114:1227–1231. 7. Barnes L, Eveson JW, Reichart P, et al. World Health Organization Classification of Tumours. Pathology and Genetics of Head and Neck Tumours. Lyon: IARC; 2005. 8. Zhao M, Takata T, Ogawa I, et al. Immunohistochemical evaluation of the small and large proteoglycans in pleomorphic adenoma of salivary glands. J Oral Pathol Med. 1999;28:37–42.

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