Extracellular matrix metalloproteinase inducer expression has been focus of research for variety of neoplasms owing to its potential role played in invasion,  angiogenesis and metastasis through interactions with other molecules.  This study was designed to determine the immunohistochemical expression of EMMPRIN in benign and malignant salivary glands tumors in local population.  This descriptive study was conducted at the Department of Morbid Anatomy and Histopathology/ Oral Pathology, University of Health Sciences Lahore,  Pakistan. Biopsies and detailed clinical data of 85 cases of salivary gland neoplasm’s (25 pleomorphic adenoma, 06 Warthin tumour, 25 adenoid cystic  carcinoma, 25 mucoepidermoid carcinoma and 02 each basal cell adenocarcinoma and carcinoma ex pleomorphic adenoma) were obtained from different  local tertiary care hospitals in Lahore from Jan. 2014 to Sep 2015. After confirming the histologic diagnosis on hematoxylin and eosin stained sections,  immunohistochemical expression of EMMPRIN was determined. SPSS version 21.0 was used to determine the association between expression of EMMPRIN in  benign, malignant and individual tumors. Chi-square and Fischer Exact tests were applied and p<0.05 was considered to be statistically significant. Expression of EMMPRIN in malignant tumors was significantly higher than benign tumors (p< 0.0001). The staining pattern of cells was also significantly  associated with type of tumour (p<0.0001). Significant EMMPRIN expression was noted with grades in AdCC (p <0.0001) & MEC (p=0.016). The current study concludes that EMMPRIN expression is significantly higher in malignant salivary gland neoplasm’s and may help the pathologists for  assessing tumor differentiation and malignant potential if added in a panel of other conventional markers.

EMMPRIN;CD147;Benign salivary gland tumors; Malignant salivary gland tumors.

Hussain S, Sahaf R, Siraj MR, Rehman F, Anjum S, et al. (2018) Immunohistochemical Expression of EMMPRIN (CD147) in Salivary Gland Tumors. J Cancer Biol Res 6(1): 1112

EMMPRIN: Extracellular Matrix Metalloproteinase Inducer; PA: Pleomorphic Adenoma; WT: Warthin Tumour; AdCC: Adenoid Cystic Carcinoma; MEC: Mucoepidermoid Carcinoma; BCA: Basal Cell Adenocarcinoma; CEPA: Carcinoma Ex Pleomorphic Adenoma; SPSS: Statistical Package for the Social Sciences; IgG1: Immunoglobulin G1; DAB: 3,3’-Diaminobenzidine; PBS: Phosphate Buffered Saline; DPX: Distyrene, Plasticizer, and Xylene ; USA: United States of America; F:M: Female: Male; PNI: Peri-Neural Invasion; VI: Vascular Invasion; MMP-2: Matrix Metalloproteinase-2; MMP-9:Matrix Metalloproteinase-9; mRNA: Messenger Ribonucleic Acid; OSCC: Oral Squamous Cell Carcinoma; FGFR: Fibroblast Growth Factor Receptor

Extracellular matrix metalloproteinase inducer (EMMPRIN) or CD147 is a member of immunoglobulin super family [1]. It consists of three domains; cytoplasmic, transmembrane and intracellular [1]. Expressed on variety of cells [1], EMMPRIN plays critical roles in the invasion and metastasis by tumour cells through its complex interactions with various matrix metalloproteinases [2]. Over expression of EMMPRIN is noted in a number of human carcinomas including oral and head & neck squamous cell carcinomas [1]. The mechanism underlying the proliferation and metastasis in head & neck squamous cell carcinoma is via fibroblast growth factor (FGFR) while in oral squamous cell carcinoma (OSCC) invasion is mediated via epithelial-mesenchymal transition (EMT) by the activation of matrix metalloproteinases (MMPs) [1] (proteolytic enzymes which degrade the extracellular matrix) [3]. Other mechanisms include angiogenesis through the stimulation of vascular endothelial growth factor (VEGF) production [4].

Salivary gland tumors exhibit tremendous morphological variability in their histologic profile including features like hybrid tumors, anaplasia, lack of proper grading systems and tendency for benign tumors to transform into malignant ones, so necessitating the use of specialized techniques for proper diagnosis and prediction of their biological behavior [5].

Owing to the diagnostic & prognostic roles played by EMMPRIN in various aspects of cancer progression [6] , this study was designed to determine, for the first time in Pakistan, the expression of EMMPRIN in salivary gland tumors.

This study was conducted at the Department of Morbid Anatomy and Histopathology/Oral Pathology, University of Health Sciences, Lahore. A total of 85 biopsies, 25 each of pleomorphic adenoma (PA), adenoid cystic carcinoma (AdCC) & mucoepidermoid carcinoma (MEC), 6 of Warthin tumour (WT) and 2 each of carcinoma ex pleomorphic adenoma (CEPA) and basal cell adenocarcinoma (BCA) of salivary glands reported at Histopathology Departments of University of Health Sciences, King Edward Medical College/Mayo hospital, Sheikh Zaid Hospital and Fatima Jinnah Medical College /Ganga Ram Hospital, Lahore from January, 2015 to September, 2015 were included in the study. Detailed clinical data was retrieved from the respective departmental records.

Hematoxylin and eosin staining

Paraffin embedded tissue sections were made from biopsy specimens. Tissue sections of 4µm were cut using rotary microtome and were stained with hematoxylin and eosin stain. Diagnosis was confirmed by 2 oral pathologists/histopathologists. Subtype determination of PA was done according to the criteria provided by Seifert [7].

Grading of AdCC was done according to the grading criteria provided by Spiro [8] where mostly tubular or cribriform (no stipulations or minor solid components) was given grade I, 50% solid pattern was grade II and mostly solid was named grade III.

Grading of MECs was done on the basis of less than 20% cystic component (+2), presence of neural invasion (+2), necrosis (+3), ≥ 4 mitoses per 10 high power fields (+3) and anaplasia (+4). Sum of the point values was used to determine low (0-4), intermediate (5-6) or high (7-14) grade MEC [9].

Immunohistochemistry

About 4 µm thick tissue sections were cut with the help of rotary microtome and taken on poly-L-lysine coated slides for immunohistochemical staining with anti-EMMPRIN (CD-147) antibody. Two sections were taken from each block, dried at 60°C for 50 minutes followed by de-waxing in xylene and rehydration in alcohol. Next, the slides were placed in Coplin jars containing citrate buffer (pH 6.0) solution and then in hot water bath (95°C) for 40 minutes in order to retrieve antigens (Heat Induced Epitope Retrieval). After removing the slides from hot water bath, they were allowed to cool at room temperature and hydrogen peroxide was added to block endogenous peroxidase activity followed by thorough washing with PBS (phosphate buffered saline). Sections were then incubated with 1-2 drops of protein blocker for 10 minutes to block endogenous enzymatic activity and then again washed with PBS. This was followed by incubation with primary antibody, mouse IgG1 kappa monoclonal CD147 antibody [HIM6] (Bio Legend; USA), diluted to concentration of 1:25 µg/ml (suggested dilution by the manufacturer) for 1 hour. Then, sections were incubated successively with Biotinylated Secondary Antibody for 10 minutes and Streptavidin Peroxidase Reagent for 10 minutes before application of DAB (di-aminobenzidine) (2 minutes) to avoid false positive staining. All incubation steps were separated by thorough washing with PBS. Counter staining with hematoxylin was done followed by dehydration and mounting of sections with coverslips using DPX. Positive (oral mucosa and skin) and negative (omission of primary antibody) controls were run with each batch of 20 histological sections of salivary gland tumors. EMMPRIN staining was evaluated on the basis of extent and intensity immunolabeling of tumor cells [10].

The intensity (qualitative variable) of staining was scored:

0 (absent), 1 (weak), 2 (moderate) and 3 (strong)

The extent/proportion (quantitative variable) of tumor cells staining was semi-quantitatively evaluated as:

0 (no or <10% positive tumor cells);

1 (10% - 24% positive tumor cells);

2 (25% to 49% positive tumor cells);

3 (50% to 74% positive tumor cells); and

4 (75% or more positive tumor cells).

Total/Final Score: The sum of the intensity and extent scores was the final score (0–7).

Negative: 0-1

Weak positive (1+): 2-3

Moderate positive (2+): 4-5

Strong positive (3+): 6-7

The clinical, histological and immunohistochemical data was analyzed statistically using SPSS 21.0. Chi-square and Fischer Exact tests were applied and p-value <0.05 was considered to be statistically significant.

The clinical parameters of the salivary gland tumors studied are summarized in Table 1.

The mean age for benign tumors was found to be 32.52 ± 16.326 years with an age range of 12-70 years. Most patients were seen in 2nd and 3rd (25.8% each) decades of life. Almost equal gender predisposition was noted (F:M , 1.06:1). Parotid gland (61.3%) was the most frequent site affected followed by minor salivary glands (22.6%) with palate being the commonest site (71.4%) (Table 1).

Mean age for the malignant cases was calculated to be 35.65 ± 14.130 years with an age range of 9-70 years. Most patients were seen in 5th decade (37%) of life. A slight male predilection of 1:1.25 was noted in malignant salivary gland tumors. Most of these tumors arose in minor salivary glands (48%) followed by parotid gland (42.6%). Of the minor salivary gland sites, palate (34.6%) was the most frequent site involved (Table 1).

Statistically significant association was noted among tumour type and age of patients (p=0.028), gland involved (p<0.0001) and laterality (p= 0.041) (Table 1).

Cell-rich or cellular subtype of PA (n=12; 48%) was the commonest subtype noted in PA, closely followed by classic (n=11; 44%). Only 2(8%) cases of stroma rich/hypo-cellular subtype were seen in the current study.

Regarding tumour morphology of malignant tumours, 17 (68%) cases of AdCC were of grade I and 8(32%) were grade III. Cribriform pattern (n=15; 60%) was the predominant pattern noted in AdCC followed by tubular (n=6; 24%) and solid (n=4; 16%). As for MEC, 9(36%) were grade I, 7(28%) were grade II and 9(36%) were grade III. Both cases of basal cell adenocarcinoma were of solid subtype characterized by solid nests of cells delineated by basement membrane like material.

Positive nodes were noted in 2(8%) of AdCC, 12(48%) cases of MEC and 2(100%) cases of carcinoma ex PA.

Perineural invasion (PNI) was noted in 9(36%) and 10(40%) cases of MEC and AdCC respectively. One (50%) case of basal cell adenocarcinoma showed PNI.

Vascular invasion (VI) was noted in 18(72%) cases in of AdCC and 1(50%) case of basal cell adenocarcinoma.

The staining reaction for anti-EMMPRIN (CD147) in the normal salivary gland tissue was strong membranous and cytoplasmic in the ductal structures while in acini the intensity was less profound than ducts and was limited to the cell membranes (Figure 1A).

Figure 1 Photomicrograph (A) showing normal salivary gland tissue showing strong cytoplasmic and membranous staining in ducts (arrows) and membranous only in acinar cells (EMMPRIN 100X), (B) moderate positive staining in epithelial component of PA (EMMPRIN 40X), (C) moderate staining in mesenchymal component of PA (EMMPRIN 40X) and (D) showing moderate positive staining cytoplasmic and membranous staining in WT (EMMPRIN 40X).

The total scores for anti-EMMPRIN (CD147) in benign and malignant salivary gland tumours are summarized in Table 2.

The staining pattern for anti-EMMPRIN (CD147) in individual tumours is shown in Table 3.

The anti-EMMPRIN (CD147) positive scores were significantly higher in malignant neoplasms with a p-value <0.0001. Significant association was noted among the total score of EMMPRIN staining in the benign tumours (p=0.004), however, no significant association was noted within the malignant group (p=0.449). In contrast, the type of staining pattern was statistically significant (differed significantly) not only in benign and malignant tumours but also within the groups (p<0.0001).

The anti-EMMPRIN (CD147) positivity was moderate in 17(54.8%) of benign tumours followed by weak in 8(25.8%) and strong in 6(19.4%) (Table 2) (Figure 1B-Figure1D). Predominant staining pattern was cytoplasmic and membranous in 20(64.5%) cases while cytoplasmic in 10(32.3%). Only one case showed nuclear staining in addition to cytoplasmic and membranous. No significant association was noted between the staining pattern and type of tumour (p=0.172). Eighteen (72%) PA showed cytoplasmic and membranous pattern (Figure 1B, Figure 1C) while most WT showed cytoplasmic reaction (n=4; 66.7%) with occasional cells showing both cytoplasmic/membranous pattern (Figure 1D). Stroma showed negative or weak staining in most cases (93.5%) and only 2 (9%) cases of PA showed moderate reaction in the stroma.

No significant association was noted between the subtypes of PA and anti-EMMPRIN (CD147) antibody staining reaction.

All the malignant tumours were strong positive for antiEMMPRIN (CD147) antibody and the staining pattern was mostly cytoplasmic and membranous (n=33; 61.1%) followed by membranous alone in 18(33.3%) cases. Only 1 (1.9%) case showed cytoplasmic localization alone and 2(3.5%) showed nuclear reaction in addition to cytoplasmic and membranous staining (Table 2, Table 3)(Figure 2-Figure 4).

Figure 2 Photomicrograph (A) showing strong positive antiEMMPRIN staining in tubular pattern of adenoid cystic carcinoma, (B) Cytoplasmic and/or membranous staining in most cells of tubular pattern of AdCC (EMMPRIN 100X), (C) Strong positive staining in cribriform pattern of AdCC (EMMPRIN 40X), (D) High power view of strong positive staining in cribriform pattern of AdCC (EMMPRIN 100X), (E) Moderate positive staining in solid pattern (EMMPRIN 40X) and (F) High power view of moderate positive staining in solid pattern of AdCC (EMMPRIN 100X).

Figure 3 Photomicrograph (A) showing strong positive staining reaction in grade I MEC (EMMPRIN 40X), (B) Membranous staining in most cells (EMMPRIN 100X), (C) Strong positive staining in grade II MEC (EMMPRIN 40X), (D) Moderate membranous staining in grade II MEC (EMMPRIN 100X), (E) Strong membranous staining in grade III MEC (EMMPRIN 100X) and (F) Showing strong membranous staining in grade III MEC (EMMPRIN 100X).

Figure 4 Photomicrograph (A) showing strong positive cytoplasmic plus membranous staining reaction in BCA (EMMPRIN 100X), (B) Moderate membranous staining in most cells of BCA (EMMPRIN 100X), (C) Strong positive cytoplasmic plus membranous staining reaction in CEPA (EMMPRIN 40X) and (D) Showing high power view (EMMPRIN 100X). Note the strong positivity in the invaded nodal tissue.

All cases of AdCC showed cytoplasmic and membranous staining (Figure 2A-Figure 2F). On the other hand most MECs (n=17; 68%) showed membranous localization and only 8(32%) cases showed cytoplasmic reaction in addition to membranous (Figure 3A-Figure 3F). Basal cell adenocarcinoma showed membranous and cytoplasmic staining reaction in one case each (50%) (Figure 4A-Figure 4B) while both cases of CEPA showed membranous and cytoplasmic reaction (Figure 4C-Figure 4D).

Anti-EMMPRIN (CD147) staining score and staining pattern were significantly associated with the grades in AdCC and MEC (p<0.0001& 0.016 respectively). No significant association of anti-EMMPRIN (CD147) antibody staining was noted with lymph node involvement, peri-neural invasion or vascular invasion in both AdCC and MEC.

Stroma in AdCC and MEC was moderately reactive in 15(60%) and 10(40%) cases respectively.

Both cases of each BCA and CEPA were strong positive for EMMPRIN. Weak stromal reaction was noted in BCA while moderate to strong reaction was seen in CEPA.

Salivary gland tumors, both benign and malignant, affect a younger age group in our population with a slight female predilection in benign tumors and male predilection in malignant tumours. Statistically significant difference was noted for antiEMMPRIN (CD147) antibody expression and staining pattern between benign and malignant salivary gland tumors (p<0.0001). It can be concluded that EMMPRN (CD147) can be utilized as a marker to characterize benign and malignant salivary gland tumors if added in the panel of conventional markers.

The authors acknowledge the encouragement extended by the Vice Chancellor of University of Health Sciences, Lahore Pakistan. Also, to Mr. Ghulam Rasool, the laboratory staff of Oral Pathology Department of University of Health Sciences, Lahore, Pakistan for their technical and logistic support. We also acknowledge the support of the Heads of Histopathology Departments of King Edward Medical University, Fatima Jinnah Medical College, Postgraduate Medical Institute and Sheikh Zaid Hospital, Lahore for their assistance in providing access to material required for this research project.

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