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Table of Contents   
ORIGINAL RESEARCH  
Year : 2014  |  Volume : 25  |  Issue : 3  |  Page : 346-351
Expression of maspin in benign and malignant salivary gland tumors: An immunohistochemical study


1 Department of Oral and Maxillofacial Pathology, Krishnadevaraya College of Dental Sciences and Hospital, Bengaluru, Karnataka, India
2 Department of Oral and Maxillofacial Pathology, V.S Dental College and Hospital, Bengaluru, Karnataka, India

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Date of Submission27-May-2013
Date of Decision27-Sep-2013
Date of Acceptance28-Apr-2014
Date of Web Publication7-Aug-2014
 

   Abstract 

Context: Maspin is a novel serine protease inhibitor (serpin) with multifaceted tumor-suppressive activities. It was originally identified in normal human breast myoepithelial cells and shows variable expression in different types of cancer cells. Maspin displays anti-metastatic properties in mammary and prostate cancer. Its expression is maintained during ovarian, lung and pancreatic carcinogenesis, indicating that Maspin regulated metastatic potential is tissue specific. Thus, it is possible that Maspin participates in salivary gland tumor biology as well. In this study, expression pattern of maspin in benign and malignant salivary gland tumors is analyzed, to understand the biological behavior of salivary gland tumors with respect to maspin expression.
Aims and Objectives: The aim of this study was to demonstrate, record, and correlate the expression pattern of maspin in benign and malignant salivary gland tumors.
Settings and Design: A retrospective study of maspin expression in 30 diagnosed cases of benign and malignant salivary gland tumors retrieved from archives of our department.
Materials and Methods: Anti-maspin antibody and horseradish peroxidase detection system.
Statistical Analysis: Descriptive statistical analysis and Chi-square/Fisher Exact test.
Results: Intense expression with P < 0.001 is associated with benign tumors, nuclear staining with P < 0.001 is significantly associated with benign tumors and cytoplasmic staining with P = 0.020 is associated with malignant tumors.
Conclusion: Intensity of expression is more in benign tumors when compared with malignant tumors. The benign tumors showed both nuclear and cytoplasmic expression. Some malignant tumors did express maspin, but mainly in the cytoplasm.

Keywords: Mammary epithelial cell, maspin, salivary gland tumors, serpin

How to cite this article:
Reshma V, Rao K, Priya N S, Umadevi H S, Smitha T, Sheethal H S. Expression of maspin in benign and malignant salivary gland tumors: An immunohistochemical study. Indian J Dent Res 2014;25:346-51

How to cite this URL:
Reshma V, Rao K, Priya N S, Umadevi H S, Smitha T, Sheethal H S. Expression of maspin in benign and malignant salivary gland tumors: An immunohistochemical study. Indian J Dent Res [serial online] 2014 [cited 2020 Sep 26];25:346-51. Available from: http://www.ijdr.in/text.asp?2014/25/3/346/138334
Salivary gland tumors are clinically and morphologically diverse group of neoplasms that may pose considerable diagnostic challenges. [1] These tumors show great variation in clinical and histological presentation with a wide range of biologic behavior. [2] Carcinogenesis and progression of malignancies are a complicated multistage process that requires the coordination of multiple genes, including oncogenes and tumor suppressor genes. [3]

Salivary gland tumors can progress from benign to malignant tumors, carcinoma-in-situ can show stromal invasion, a low-grade tumor can convert to high-grade tumor, or a malignant tumor may show dedifferentiation. [4] Inactivation of metastasis suppressor gene is an important factor for metastasis and malignant change of normal cells. [3]

Maspin, a 42-KDa molecule is a recently identified protein belonging to serpin family of protease inhibitors, which inhibits cell motility, invasion, apoptosis, and angiogenesis. [5] The consensus that maspin expression predicts a better prognosis still largely holds for breast, prostate, colon and oral squamous cancers. [6] In contrast, some studies have shown that maspin over expression is correlated with poor prognosis in pancreatic, ovarian cancers and in lung adenocarcinoma. [7] Maspin was originally identified in normal human breast epithelial and myoepithelial cells. Now it has been localized in a broad number of human tissues including placenta, small intestine, uterus, and kidney. [8]

Since maspin is present in the epithelium of other glands, it is possible that it participates in salivary gland biology as well. [8] Thus, the aim of this study was to demonstrate, record, and correlate the differences in maspin expression in benign and malignant salivary gland tumors.


   Materials and methods Top


A total of 30 diagnosed cases of benign and malignant salivary gland tumors were retrieved from archives of the Department of Oral Pathology. The study group comprised of pleomorphic adenoma (PA) (n = 8), Warthin's tumor (n = 7), adenoid cystic carcinoma (ACC) (n = 8) and mucoepidermoid carcinoma (MEC) (n = 7), which were primary neoplasms from major and minor salivary glands. Normal salivary gland tissue (n = 2) was used as a reference to determine the type of cell stained. They were mainly taken from the normal submandibular salivary gland (confirmed by histopathology).

Inclusion criteria

  1. Primary neoplasms arising from major and minor salivary glands.


Exclusion criteria

  1. Secondary or metastatic tumors in the salivary glands.
  2. Other diseases affecting the salivary glands.


Anti-Maspin antibody obtained from Novacastra and Super Sensitive polymer-horseradish peroxidase (HRP) immunohistochemistry detection system HRP/DAB (3,3'- Diaminobenzidine) obtained from Biogenics was used for the study. The anti-maspin antibody was obtained as 1 mg lyophilized vials, which was then mixed with 1 ml of distilled water to attain 1 ml of concentrated anti-maspin antibody. The antigen was retrieved using ethylenediaminetetraacetic acid buffer in a microwave oven, which was run for three cycles of 15 min each with an interval of 5 min between the cycles.

Prostate gland was used as a control slide according to the manufacturer's instructions. A freshly diluted anti-maspin antibody with a dilution ratio of 1:25 (anti-mapsin:distilled water) was used during each staining procedure. Prominent brown staining of the myoepithelial cells or the basal cells lining the prostate gland acini confirmed the positive reactivity. The study sections were evaluated for intensity by comparing with the intensity expressed by the prostate gland acini. Intensity similar to the prostate gland was considered intense; intensity slightly less was considered moderate and weak if there was light brown stain. The sections were thus graded as intense, moderate, weak and negative and a numerical value of 3, 2, 1 and 0 (no expression) were assigned, respectively. The type of cells stained and localization of the stain either as cytoplasmic, nuclear or both within the cells was evaluated.


   Results Top


The normal salivary gland tissue expressed intense staining for myoepithelial cells around the acini with a strong nuclear reactivity. Few striated ducts showed maspin positivity [Figure 1]. The staining was seen mainly in basal cells of the ducts, but a few luminal cells were also positive. No staining was seen in salivary secretory cells or any cells in the supporting stroma.
Figure 1: Normal salivary gland showing myoepithelial and ductal cell positivity for maspin (IHC, ×100)

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Among cases of PAs (n = 8); 6 cases were from major gland and 2 were from minor salivary gland; 7 of them showed intense staining for maspin and expressed maspin in both nucleus and cytoplasm [Figure 2]. One case, which was from major salivary gland showed only cytoplasmic positivity with moderate intensity.

Among cases of Warthin's tumor (n = 7), all of which was from major salivary gland; 5 cases showed both nuclear and cytoplasmic positivity. Among them, 2 cases showed intense positivity, 2 cases showed moderate and 1 case showed weak intensity. The rest of 2 cases showed only cytoplasm positivity, with one being moderate and the other of weak intensity. The overall intensity of staining was less than PA [Figure 3].
Figure 2: Pleomorphic adenoma tumor cells showing strong maspin positivity in both nucleus and cytoplasm (IHC, ×100)

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Figure 3: Warthin's tumor showing ductal cell positivity for maspin (IHC, ×100)

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The ACC cases taken were 8 in number and were graded according to the proportion of tumor with solid pattern as:

  • Grade I: cribriform and tubular patterns, no solid component.
  • Grade II: Mixed tumors with cribriform and tubular architecture with < 30% of solid areas.
  • Grade III: Tumors with 30% or more of solid areas. [9]


The origin of each tumors, their grades and results are tabulated in [Table 1]. The cells showed maspin positivity mainly in cytoplasm [Figure 4].

The cases of MEC were graded in relation to the amount of cyst formation, percentage of epidermoid cells and mucus cells. They were graded as low, intermediate and high-grade according to Seifert et al. [10] In MEC only the epidermoid component showed maspin positivity and was seen mainly in the cytoplasm [Figure 5]. Of the 7 cases of MEC studied, 5 were from minor salivary gland and 2 were from major gland. The intensity of staining and localization of staining is tabulated in [Table 1].
Figure 4: Adenoid cystic carcinoma tumor cells showing faint cytoplasmic positivity for maspin (IHC, ×100)

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Figure 5: Mucoepidermoid carcinoma with epidermoid cells showing cytoplasmic positivity for maspin (IHC, ×100)

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Statistical analysis

Descriptive statistical analysis has been carried out in this study. Results on continuous measurements are presented on mean ± standard deviation (Min-Max) and results on categorical measurements are presented in number (%). Significance is assessed at 5% level of significance.

The following assumptions on data were made.

  1. Dependent variables should be normally distributed.
  2. Samples drawn from the population should be random.
  3. Cases of the samples should be independent.


Chi-square/Fisher Exact test has been used to find the significance of study parameters on categorical scale between two or more groups. Nearly 95% Confidence interval has been computed to find the significant features. Confidence Interval with lower limit > 50% is associated with statistical significance.

The Statistical software namely SAS 9.2, SPSS 15.0, Stata 10.1, MedCalc 9.0.1, Systat 12.0 and R environment version 2.11.1 were used for the analysis of the data and Microsoft Word and Excel have been used to generate tables.

Based on these statistical criteria, the results obtained were as follows:

  1. In a total of 15 cases of benign tumors studied (PA = 8 and Warthin tumor = 7), all the cases showed positive maspin expression. Of 15 cases of malignant tumors studied (ACC = 8 and MEC = 7), 3 cases of ACC and 5 cases of MEC were positive for maspin.
  2. Comparison of intensity of staining in benign and malignant tumors showed that intense expression is significantly associated with benign salivary gland tumors with a P < 0.001** [Table 2].
  3. Comparison of nuclear staining of maspin in benign and malignant salivary gland tumors showed that presence of nuclear staining is significantly more associated with benign tumors in comparision to malignant tumors (80.0% vs. 13.3%) with a P < 0.001** [Table 3].
  4. Comparison of cytoplasmic staining of maspin in benign and malignant salivary gland tumors showed that presence of cytoplasmic staining is significantly more associated with malignant tumors when compared with benign tumors (13.3% vs. 53.3%) with a P = 0.020* (*Moderately significant [P value: 0.01 < P ≤ 0.05]) [Table 4].
  5. Comparison of maspin expression in nucleus and cytoplasm of different benign and malignant salivary gland tumors showed that PA is significantly more associated with both nucleus and cytoplasm staining and MEC is more associated with cytoplasm staining with a P < 0.001** (**strongly significant [P value: P ≤ 0.01]) [Table 5].
  6. Comparison of intensity of maspin staining in individual benign and malignant tumors showed that intense maspin expression was significantly associated with PA P = 0.002** [Table 6].
Table 1: Grading, origin and staining pattern of malignant salivary gland tumors


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Table 2: Comparison of intensity in benign and malignant salivary gland tumors


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Table 3: Comparison of maspin expression in nuclear staining in benign and malignant salivary gland tumors


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Table 4: Comparison of maspin expression in cytoplasm staining in benign and malignant salivary gland tumors


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Table 5: Expression of maspin in nucleus and cytoplasm of different benign and malignant salivary gland tumors (n=30)


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Table 6: Intensity of maspin in nucleus and cytoplasm of different benign and malignant salivary gland tumors (n=30)


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   Discussion Top


Tumor metastasis is a complex process and requires multiple cellular functions over time. From cellular invasion, extravasation from the primary tumor, intravasation to the secondary organs, to successful colonization, tumor cells utilize many cellular and biochemical mechanisms to complete the metastatic spread. [5] Cell-matrix interaction is essential for neoplastic invasion and metastasis. In this context, protease and protease inhibitors play important roles by degrading extracellular matrix and preventing this process respectively. [8]

Maspin protein is suggested to have inhibitory effects on tumor-induced angiogenesis, tumor cell motility, invasion and metastasis. It was first isolated from normal mammary epithelial cells, is highly expressed in various other normal cells and in different types of cancerous cells of breast, ovarian, lungs, prostate, stromal cells of cornea, and oral squamous cells. [11],[12] In mammary epithelial cells, maspin localizes primarily in the cytoplasm, but can also localize in the nucleus and the cell surface. [12] This study was undertaken to identify the expression pattern of maspin in benign and malignant salivary gland tumors and correlate with its biological behavior.

The benign tumors (PA and Warthin's tumor) and the malignant tumors (ACC and MEC) were considered for the study.

The normal salivary gland reference samples expressed intense staining for myoepithelial cells lining the acini with a strong nuclear immunoreactivity for maspin. Few striated ducts also showed maspin positivity mainly in the nucleus. These results were consistent with the results obtained by Navarro R de et al. [8]

All benign salivary gland tumors considered in the study expressed maspin that correspond to benign biological behavior of these tumors, which states that high rates of maspin expression correlated with better survival rates. [8] In contrast, only 8 cases of malignant tumors expressed maspin.

The localization of maspin in cytoplasm or nucleus or both and its associated significance has been debated by various authors. Depending on the localization of maspin, different functions can be attributed. Nuclear maspin may regulate gene transcription in response to cellular state induced by inflammation, tissue injury and remodeling. Cytoplasmic maspin may regulate the glutathionine redox system of cell and therefore play an important role in maintenance of cellular homeostasis and response to cellular stress. [11] Sood et al. demonstrated that mixed nuclear and cytoplasmic maspin localization in ovarian cancer is indicative of a more benign lesion than neoplasms with cytoplasmic expression only, suggesting an important tumor-suppressive role for nuclear maspin. [13]

Earlier studies described maspin exclusively in cytoplasm and close to cytoplasmic membrane. Recent studies have shown maspin to be expressed in both cytoplasm and nucleus of salivary gland neoplasms. [8] Studies carried out by Martins et al. have shown both nuclear and cytoplasmic expression of maspin in PA. They opined that positivity in the cytoplasm disappeared first as this benign tumor progressed toward carcinoma-ex PA. Areas with faint immunostaining showed nuclear staining only. [14]

In contrast, Schwarz et al. studied maspin in various salivary gland malignancies and correlated its prognostic implications. They found that nuclear positivity was more frequent than cytoplasmic positivity. Loss of nuclear maspin was associated with poor survival, whereas loss of cytoplasmic staining was without statistically significant prognostic impact. Negative nuclear maspin expression showed significant lower survival rates than positive staining. The survival was poorest if both nuclear and cytoplasmic staining was negative. [11]

In our study, PA showed positivity for maspin in most of the abluminal cells and in sheets of cells; but few cases also showed luminal and abluminal cell staining. There was no significant variation in staining pattern in tumors arising from major or minor salivary glands. These results are consistent with the studies done by Navarro R de et al. [8] and Martin et al. [14] who concluded that cells with myoepithelial differentiation and occasional epithelial (luminal) cells were positive for maspin. Most of the cases showed prominent nuclear and cytoplasmic positivity which were consistent with the studies done by Schwarz et al. [11]

Cases of Warthin's tumor considered in our study expressed maspin in ductal cells with both nuclear and cytoplasmic positivity. The intensity of expression was generally lesser than that of PA. Luminal cells lining the lymphoid stroma in Warthin's tumor showed similar expression for maspin as that of striated ducts of the normal salivary glands. [15] We performed a PubMed, Google scholar and science direct search to find out if any studies were done on maspin expression in Warthin's tumor, to the best of our knowledge no such studies were done. Though Warthin's tumor is a benign tumor, the intensity of expression of maspin was less as compared to PA, only 2 cases of Warthin's tumor showed intense staining. More studies on Warthin's tumor needs to be done to arrive at an appropriate conclusion.

In this study, ACC showed negative staining in most of the cases with only few showing cytoplasmic positivity. The cases that showed cytoplasmic positivity were mainly from minor salivary glands and correlated with the grades of the tumors. One case, which was a grade I tumor arising from minor salivary gland showed both nuclear and cytoplasmic positivity [Table 1]. These results were consistent with studies done by Navarro R de et al. where areas of anaplasia were totally negative for Maspin. [8]

Cases of MEC showed predominantly negative nuclear staining in almost all cases. Two low-grade tumors arising from major salivary gland showed weak cytoplasmic positivity. One minor salivary gland tumor of intermediate grade expressed moderate cytoplasmic positivity and one low-grade minor salivary gland tumor expressed intense staining in both nucleus and cytoplasm. This outcome differs from that of Schwarz et al. [11] who concluded that both cytoplasm and nucleus stained in high proportions in MEC.

In our study, when the malignant tumors expressed maspin most of it was in the cytoplasm with loss of nuclear maspin expression, which would correspond to aggressive behavior of these tumors, poor survival rates and correlates with the view of Schwarz et al. [11] who stated that loss of nuclear maspin presents with poor survival rates. Furthermore, as the tumor grade increased the localization and intensity of expression also varied. Benign and low-grade tumors predominantly showed intense nuclear and cytoplasmic staining, whereas high-grade tumors showed weak cytoplasmic staining.


   Conclusion Top


These results point to the fact that maspin can be used as a tool for assessing the biological behavior of benign and malignant salivary gland tumors. Intense expression and mainly nuclear maspin expression is associated with benign biological behavior of the salivary gland tumor in question.

 
   References Top

1.Al-Khateeb TH, Ababneh KT. Salivary tumors in north Jordanians: A descriptive study. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007;103:e53-9.  Back to cited text no. 1
    
2.Nagarkar NM, Bansal S, Dass A, Singhal SK, Mohan H. Salivary gland tumors-Our experience. Indian J Otolaryngol Head Neck Surg 2004;56:31-4.  Back to cited text no. 2
    
3.Wang MC, Yang YM, Li XH, Dong F, Li Y. Maspin expression and its clinicopathological significance in tumorigenesis and progression of gastric cancer. World J Gastroenterol 2004;10:634-7.  Back to cited text no. 3
    
4.Cheuk W, Chan JK. Advances in salivary gland pathology. Histopathology 2007;51:1-20.  Back to cited text no. 4
    
5.Chen EI, Yates JR. Maspin and tumor metastasis. IUBMB Life 2006;58:25-9.  Back to cited text no. 5
    
6.Pemberton PA, Wong DT, Gibson HL, Kiefer MC, Fitzpatrick PA, Sager R, et al. The tumor suppressor maspin does not undergo the stressed to relaxed transition or inhibit trypsin-like serine proteases. Evidence that maspin is not a protease inhibitory serpin. J Biol Chem 1995;270:15832-7.  Back to cited text no. 6
    
7.Hirai K, Koizumi K, Haraguchi S, Hirata T, Mikami I, Fukushima M, et al. Prognostic significance of the tumor suppressor gene maspin in non-small cell lung cancer. Ann Thorac Surg 2005;79:248-53.  Back to cited text no. 7
    
8.Navarro Rde L, Martins MT, de Araújo VC. Maspin expression in normal and neoplastic salivary gland. J Oral Pathol Med 2004;33:435-40.  Back to cited text no. 8
    
9.Perez-Ordonez B. Selected topics in salivary gland tumour pathology. Curr Diagn Pathol 2003;9:355-65.  Back to cited text no. 9
    
10.Seifert G, Miehlke A, Hanbrich J, Chilla R. Diseases of the Salivary Glands. Stuttgart: George Thieme Veilag Thieme Inc.; 1986.  Back to cited text no. 10
    
11.Schwarz S, Ettl T, Kleinsasser N, Hartmann A, Reichert TE, Driemel O. Loss of Maspin expression is a negative prognostic factor in common salivary gland tumors. Oral Oncol 2008;44:563-70.  Back to cited text no. 11
    
12.Ghazy SE, Helmy IM, Baghdadi HM. Maspin and MCM2 immunoprofiling in salivary gland carcinomas. Diagn Pathol 2011;6:89.  Back to cited text no. 12
    
13.Sood AK, Fletcher MS, Gruman LM, Coffin JE, Jabbari S, Khalkhali-Ellis Z, et al. The paradoxical expression of maspin in ovarian carcinoma. Clin Cancer Res 2002;8:2924-32.  Back to cited text no. 13
    
14.Martins MT, Altemani A, Freitas L, Araújo VC. Maspin expression in carcinoma ex pleomorphic adenoma. J Clin Pathol 2005;58:1311-4.  Back to cited text no. 14
    
15.Faur A, Laza¢r E, Cornianu M, Dema A, Vidita CG, Ga¢luºcan A. Warthin tumor: A curious entity - Case reports and review of literature. Rom J Morphol Embryol 2009;50:269-73.  Back to cited text no. 15
    

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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0970-9290.138334

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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]

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