Journal of Clinical Neuroscience 22 (2015) 1282–1287
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Clinical Study
Matrix metalloproteinase-9 and tissue inhibitor of matrix metalloproteinase-2: Prognostic biological markers in invasive prolactinomas Güliz Demirelli Gültekin a,⇑, Burak Çabuk a, Çig˘dem Vural b, Savasß Ceylan a a b
_ Kocaeli, Turkey Department of Neurosurgery, Kocaeli University School of Medicine, Umuttepe, Izmit, Department of Pathology, Kocaeli University School of Medicine, Umuttepe, Izmit, Kocaeli, Turkey
a r t i c l e
i n f o
Article history: Received 29 October 2014 Accepted 4 February 2015
Keywords: Invasion Matrix metalloproteinase-9 Pituitary adenoma Prolactinoma Tissue inhibitor of matrix metalloproteinase-2
a b s t r a c t In this study, the predictive roles of matrix metalloproteinase (MMP)-9 and tissue inhibitor of matrix metalloproteinase (TIMP)-1 and 2 in invasive and noninvasive prolactinomas were examined. Prognostic biomarkers to distinguish between invasive and noninvasive pituitary adenomas are required for the effective treatment of pituitary adenoma patients. We analyzed 57 prolactinoma patients classified as having invasive and noninvasive adenomas for MMP-9, TIMP-1 and TIMP-2 expression using immunohistochemical methods. Significantly higher MMP-9 expression was detected in invasive prolactinomas (p = 0.004). There was also a significant relationship between TIMP-2 expression and invasive behavior (p = 0.004) and TIMP-2 expression and recurrence (p = 0.005). Because MMP-9 expression is significantly increased in invasive prolactinomas, MMP-9 has potential as a marker for invasion. TIMP-2 may be a marker for both invasion and recurrence. These findings require further examination in large scale prospective studies. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Pituitary adenomas are common neoplasms. Although they are generally benign in nature, they may invade adjacent tissues. Recurrence and resistance to treatment are serious problems in invasive pituitary adenoma patients, therefore, when treating pituitary adenomas, biological markers are needed to indicate the invasiveness of the tumor to effectively guide surgical, medical, and radiotherapy treatment modalities and to prevent recurrence. Pituitary adenomas are epithelial tumors that arise from the adenohypophysis. The most frequently detected pituitary adenomas are prolactinomas [1]. Prolactinoma treatment is primarily with dopamine agonist drugs (DA; cabegoline, bromocriptine) [2,3]. In this study, prolactinomas were investigated with a focus on the roles of matrix metalloproteinase (MMP)-9 and tissue inhibitor of matrix metalloproteinase (TIMP)-2 in pituitary adenoma invasion. Only prolactinomas were investigated in order to concentrate the research on the roles of MMP-9 and TIMP-2 in pituitary adenoma invasion. ⇑ Corresponding author. Tel.: +90 050 57891621; fax: +90 380 5497060. E-mail address: [email protected] (G.D. Gültekin). http://dx.doi.org/10.1016/j.jocn.2015.02.021 0967-5868/Ó 2015 Elsevier Ltd. All rights reserved.
The MMP family consists of zinc and calcium-dependent endopeptidases. These enzymes are associated with proteolytic degradation of the extracellular matrix (ECM) [4]. The role of MMP in tumor invasion and metastatic processes has been confirmed. ECM degradation by MMP is a critical process in the progression of malignant tumors, angiogenesis, and tumor invasion and metastasis. Gelatinases (MMP-9 and MMP-2) are thought to be key enzymes in ECM degradation as they degrade some of the main components of the ECM [5,6]. TIMP are low molecular weight proteins that are secreted and bind to active MMP, inhibiting their enzymatic activity [7]. Moreover, MMP-2 and MMP-9 are unique among the MMP in that the latent forms of these proteinases can form complexes with TIMP-2 and TIMP-1, respectively. In addition to their inhibitory role, TIMP can also take part in the activation of MMP [8]. TIMP-2 is a powerful inhibitor of MMP activity and high levels of TIMP-2 are positively correlated with an unfavorable prognosis in cancer patients [9,10]. Previous studies have investigated MMP-9, TIMP-1 and TIMP-2 as prognostic biological markers in breast, lung, colon and many other cancer types, and the correlations of these enzymes with invasion and metastasis have been determined [11–14]. However, MMP-9 studies in pituitary adenomas have yielded
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conflicting results and TIMP-1 and TIMP-2 have not been studied as potential prognostic biological markers in pituitary adenomas. In this study, we evaluated the presence of MMP-9, TIMP-1 and TIMP-2 in invasive and noninvasive prolactinomas and investigated their potential as prognostic predictors using immunohistochemical methods. 2. Methods and materials In the present study, we retrospectively reviewed the records of patients with prolactinoma who had undergone endoscopic transsphenoidal surgery in the Department of Neurosurgery, Kocaeli University School of Medicine between 1999 and 2011. Fifty-seven patients who were diagnosed with prolactinoma based on clinical, radiological and pathological data were included in this study. The mean ages of the patients in the invasive prolactinoma and noninvasive prolactinoma groups were 43.2 years and 35 years, respectively (range: 16–69). There were 11 females and 24 males in the invasive group (n = 35), and 18 females and four males in the noninvasive group (n = 22). The indications for surgery in the invasive group were as follows: resistance to DA treatment (n = 5), intolerance to DA (n = 6), and patient preference (n = 24). The indications for surgery in the noninvasive group were as follows: resistance to DA (n = 9), intolerance to DA (n = 6), and patient preference (n = 7). In the noninvasive group, all patients (n = 22) were first time surgeries, and 31/35 patients in the invasive group were first time surgeries. Four patients in the invasive group were second surgeries. Radiotherapy was not performed in any patient. None of the prolactinoma patients had multiple endocrine neoplasia syndrome. Three patients with atypical meningioma and two with chordoma were included as a positive control group. Grade 3 and 4 tumors, according to the Knosp classification [15], and grade 4D and 4E tumors, according to the modified Hardy classification [16], were considered to be invasive prolactinomas and radiological invasion was intraoperatively confirmed. Thirty-five patients were classified as having invasive prolactinomas and 22 with noninvasive prolactinomas. Treatment outcomes were evaluated according to prolactin (PRL) level in the blood and sella MRI on the first day after operation. Normal PRL values were considered to indicate remission and were monitored for recurrence or cure. A PRL level higher than 21 ng/dl was considered to indicate persistent recurrence. In the noninvasive group, 14/22 patients (64%) were cured and in the invasive group none were cured (0/35; 0%). Persistent recurrence was detected in 31/35 patients (88%) in the invasive group and 7/22 (32%) in the noninvasive group. In the invasive group, late recurrence was detected in 4/35 patients (12%) and the average time to late recurrence was 10 months after surgery. In the noninvasive group, late recurrence was detected in 1/22 patients (4%) and the time to recurrence for that patient was 4 years. Patients were observed for cure and recurrence from 3 to 14 years after surgery. 2.1. Histopathological examination MMP-9 (Thermo Fisher Scientific, Waltham, MA, USA), TIMP-1 (Clone: 102D1; Thermo Fisher Scientific), and TIMP-2 (Clone: 3A4; Thermo Fisher Scientific) antibodies were applied to sections obtained from formalin fixed and paraffin embedded blocks. Sections (4 lm) were mounted on poly-L-lysine coated glass and de-paraffinized after being kept in a 56°C incubator overnight. They were placed in xylol for 30 minutes, absolute alcohol for 15 minutes, 96% alcohol for 15 minutes and then washed with water. Antigens were retrieved by boiling the sections in citrate buffer (pH 6) in a microwave oven for 15 minutes. Endogen peroxidase activity was blocked by quenching in a 3% hydrogen peroxide
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solution for 15 minutes. Tissue sections were incubated at room temperature for 30 minutes with primary antibodies against MMP-9, TIMP-1 and TIMP-2. A secondary antibody was applied and the sections were treated with streptavidin-biotin-peroxidase complex for 30 minutes. 3,3-diaminobenzidine was used as a chromogen and Mayer’s hematoxylin was used for AEC ground staining. The sections were covered with a water-based cover material. Meningioma and chordoma tissues were used as the positive controls for MMP-9, TIMP-1 and TIMP-2 expression. The stained tissues were examined by a single pathologist who was blinded to the clinical data. The extent and intensity of MMP-9 staining in the adenomas were evaluated. The extent of MMP-9 staining was scored as negative (0; from 0–30%), 1 (minimal; from 31–60%), 2 (focal; from 61–100%) or 3 (diffuse). The intensity of MMP-9 staining was scored as negative (0), weak (1), moderate (2) or strong staining (3). TIMP-1 and TIMP-2 were used to distinguish the adjacent pituitary tissue from the adenoma and were evaluated as positive or negative depending on whether staining was detected. 2.2. Statistical analyses Research data were analyzed using the Statistical Package for the Social Sciences (SPSS statistics; version 13.0; IBM Corporation, Armonk, NY, USA). Descriptive statistics and the chi-squared test were used and the significance level was set at p < 0.05. 3. Results 3.1. MMP-9 and invasion MMP-9 expression was detected in all patients (57; 100%). The relationship between MMP-9 expression and invasive behavior was evaluated by the extent of MMP-9 staining measured as the number of adenoma cells showing MMP-9 immunoreactivity. Figure 1 shows MMP-9 staining in one invasive and one noninvasive patient. In invasive prolactinomas, diffuse staining was detected in 85.7% of patients (30/35). A significant relationship was determined between MMP-9 expression and invasive behavior (p = 0.004; Table 1). There was no correlation between MMP-9 staining intensity and invasive prolactinomas. This result conflicted with previous studies, therefore, the possible causative factors of this result were examined. In the invasive group, significant differences in MMP-9 staining intensity were found between patients who took and did not take DA drugs in the preoperative period. Figure 2 shows MMP-9 expression in two patients with invasive giant adenomas, one with DA and one without DA during the preoperative period. In the invasive group, the MMP-9 staining intensities for the DA treated group were strong staining in one (8%), moderate staining in nine (75%) and weak staining in two patients (16%). The MMP-9 staining intensities for the no DA treatment group were strong staining in 10 (75%), moderate staining in 10 (43%) and weak staining in three patients (12%). 3.2. TIMP-1 and invasion TIMP-1 staining was detected in 31/57 patients (54%) and staining in the adenoma was observed in 16. There was no correlation between TIMP-1 expression and invasion. 3.3. TIMP-2 and invasion TIMP-2 staining was detected in the adenoma and adjacent tissue sample in 32/57 patients (56%), while staining in the adenoma
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Fig. 1. Matrix metalloproteinase (MMP)-9 antibody staining of prolactinoma samples (Thermo Fisher Scientific, Waltham, MA, USA). Brown areas show MMP-9 expression, blue areas are where no MMP-9 expression was detected. (A) Patient 44, invasive prolactinoma: MMP-9 staining classified as diffuse (magnification 20). (B) Patient 37, noninvasive prolactinoma: MMP-9 staining classified as focal (magnification 20).
Table 1 The relationship between matrix metalloproteinase-9 expression and invasive behavior of prolactinomas MMP-9 staining
Minimal (60%)
p value
Invasive, n (%) Noninvasive, n (%)
1 (2.8) 7 (31.8)
4 (11.5) 4 (18.2)
30 (85.7) 11 (50)
0.004
MMP = matrix metalloproteinase.
alone was observed in 17. In the present study, TIMP-2 staining in the adenoma was evaluated. Figure 3A shows TIMP-2 staining in an invasive prolactinoma patient. TIMP-2 staining was detected in 15 patients (42.4%) in the invasive group and two (9%) in the noninvasive group. Although TIMP-2 staining was low in both groups, 88.2% of the adenomas showing TIMP-2 staining were in the invasive group. A significant relationship was found between TIMP-2 expression and invasive behavior (p = 0.004; Table 2). 3.4. TIMP-2 and treatment outcome TIMP-2 staining in the adenoma was observed in 17 patients. The invasive to noninvasive patient ratio was 15:2. Patients with TIMP-2 staining consistently resulted in recurrence; a significant relationship was found between TIMP-2 staining and recurrence (p = 0.005; Table 3).
3.5. Correlation of TIMP-2 expression in adjacent tissues and good prognosis, noninvasive behavior Adjacent pituitary tissue was examined in specimens from 14/22 patients in the noninvasive group, but not in specimens from invasive prolactinoma patients. TIMP-2 staining was consistently detected in adjacent pituitary tissue and not in noninvasive adenomas (13/14). Figure 4 shows TIMP-2 staining in adjacent pituitary tissues and a lack of staining in adenomas from noninvasive patients. TIMP-2 staining of adenoma tissues was detected in invasive adenomas. TIMP-2 staining in adjacent tissue was consistently detected in noninvasive adenomas. 3.6. Comparison of MMP-9, TIMP-1 and TIMP-2 expression in pituitary adenoma patients and controls The positive control group included three atypical meningiomas and two clivus chordomas. All of these showed diffuse MMP-9 staining (5/5) and none showed TIMP-1 staining (0/5). TIMP-2 staining was observed in 4/5 controls. When compared with the prolactinoma patients showing TIMP-2 staining, the meningioma and chordoma controls exhibited less intense staining (Fig. 3). Finally, prolactinomas were shown to have greater MMP-9 expression than meningiomas and chordomas (Fig. 3). This pattern conforms with the more aggressive clinical course of invasive prolactinomas compared to atypical meningiomas and chordomas.
Fig. 2. Matrix metalloproteinase (MMP)-9 antibody staining in two invasive giant adenoma patient samples (Thermo Fisher Scientific, Waltham, MA, USA). Brown areas show MMP-9 expression, blue areas are where no MMP-9 expression was detected. (A) Patient 32 treated preoperatively with dopamine agonist drugs (DA; magnification 40). (B) Patient 33, no DA treatment (magnification 40). Whereas MMP-9 staining was diffuse in both patients, staining intensity was different. The (A) DA treated patient sample was classified as moderate MMP-9 staining and the (B) no DA patient as strong.
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Fig. 3. Tissue inhibitor of matrix metalloproteinase (TIMP)-2 antibody staining (Clone 3A4; Thermo Fisher Scientific, Waltham, MA, USA). (A) Patient 26, invasive pituitary adenoma (magnification 200). (B) Control Patient 60, clivus chordoma (magnification 200). The chordoma control showed less intense staining than the invasive prolactinoma.
Table 2 The relationship between tissue inhibitor of matrix metalloproteinase-2 expression and invasive behavior of prolactinomas TIMP-2 expression
Stained
Not stained
Total
p value
Invasive, n (%) Noninvasive, n (%)
15 (42.4) 2 (9)
20 (57.6) 20 (91)
35 22
0.004
TIMP = tissue inhibitor of matrix metalloproteinase.
Table 3 The relationship between tissue inhibitor of matrix metalloproteinase-2 expression and treatment outcome in of prolactinoma patients TIMP-2 expression
Stained
Not stained
Total
p value
Recurrence, n (%) Cure, n (%)
17 (40) 0 (0)
26 (60) 14 (100)
43 14
0.005
TIMP = tissue inhibitor of matrix metalloproteinase.
Fig. 4. Tissue inhibitor of matrix metalloproteinase (TIMP)-2 antibody (Clone 3A4; Thermo Fisher Scientific, Waltham, MA, USA) staining in adjacent pituitary tissue (brown; red arrow) and lack of staining in adenoma tissue (blue; red arrow) in a noninvasive prolactinoma (Patient 31; magnification 100). TIMP-2 staining in adjacent tissue was consistently detected in noninvasive adenomas.
4. Discussion Pituitary adenomas account for 20% of all intracranial tumors and 40–60% of pituitary adenomas are prolactinomas. Invasion is the most important prognostic factor in pituitary adenoma patients. Approximately 30–35% of functional pituitary adenomas
and >50% of nonfunctional pituitary adenomas are associated with dural invasion. Total resection is more difficult for invasive than noninvasive tumors and post-surgery recurrence is common [17,18]. Surgical complications, the inability to achieve remission and inadequate therapy are all related to invasion [19]. However, the molecular mechanism of local invasive behavior is not yet clearly understood. Predicting invasive behavior and recurrence may help in the formulation of treatment decisions in pituitary adenoma patients. Local invasion and metastasis of tumor cells primarily require degradation of the basement membrane. For that purpose, tumor cells express enzymes for proteolysis such as MMP, which are zinc containing endopeptidases that play a role in the degradation of the extracellular matrix in angiogenesis and invasion. MMP-9 is an endopeptidase that degrades type IV collagen and is controlled at the transcriptional level [20]. The endogenous inhibitors of MMP are TIMP-1, 2, 3, and 4 [11]. In recent years, various solid tumor cells have been demonstrated to produce MMP-2, MMP-9, TIMP-1 and TIMP-2 in vitro and these enzymes have been associated with tumor invasion, poor prognosis and differentiation [11]. Many studies on the role of MMP-9 in the invasive behavior of pituitary adenomas have been performed. Kawamoto et al. [21] demonstrated that type IV collagenase (MMP-9) activity was noticeably higher in invasive pituitary adenomas compared to noninvasive pituitary adenomas using immunofluorescence. They concluded that this may be related to cavernous sinus invasion. Furthermore, Liu et al. [22] found that MMP-2 and MMP-9 expression levels were noticeably high in pituitary adenomas with cavernous sinus invasion using both immunohistochemical and real time polymerase chain reaction (RT-PCR) methods. Turner et al. [23] showed remarkably high expression of MMP-9 in invasive macroprolactinomas in comparison with noninvasive adenomas using immunohistochemical methods and suggested that MMP-9 plays an important role in angiogenesis and invasion. Hussaini et al. [19] demonstrated that MMP-9 expression was high in invasive adenomas using gelatin zymography, western blot, RT-PCR and immunohistochemical methods. Gong et al. [24] demonstrated expression levels of MMP-9 3–5 times greater in invasive adenomas compared to noninvasive adenomas. Furthermore, they demonstrated MMP-9 expression at 3 times greater levels in invasive prolactinomas than in all invasive pituitary tumors using gelatin zymography and western blot methods and validated their results using RT-PCR and immunohistochemical methods. Ceylan et al. [25] recently performed the first comprehensive microanatomic and histological study of the normal pituitary gland using immunohistochemical and RT-PCR methods. They suggested
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that the ability of tumor cells to express MMP-9 indicates that the tumor has aggressive and invasive behaviors. Pereda et al., Wang et al. and Pan et al. reached similar conclusions regarding MMP-9 and invasion [26–28]. Conversely, Beaulieu et al., Yokoyama et al., Knappe et al. and Yamada et al. found no correlation between MMP-9 expression and invasion [29–32]. In the present study, significantly higher MMP-9 expression was found in invasive prolactinomas compared to noninvasive prolactinomas using immunohistochemical methods. MMP-9 staining was consistently correlated with invasion (p = 0.004). Diffused MMP-9 staining was observed in 85.7% of the invasive prolactinomas. Moreover, it was consistently detected with diffused MMP-9 staining in the invasive prolactinomas and focal MMP-9 staining in the noninvasive prolactinomas. Our results support those of recent studies on this subject and show that MMP-9 could be used as a prognostic biological marker for prolactinomas and other pituitary adenomas. From our cohort, it was seen that MMP-9 staining intensity was altered from strong to moderate by the administration of DA drugs during the preoperative period and, therefore, we concluded that MMP-9 expression may be slowed by DA. The consistent detection of MMP-9 staining in correlation with invasion was hampered by DA treatment. However, further research is needed to determine if this effect on MMP-9 expression is caused by DA. Prolactinomas have much greater MMP-9 expression than meningiomas and chordomas. This fits the typically more aggressive clinical course of invasive prolactinomas than of atypical meningiomas and chordomas and supports the results of Gong et al. [24] TIMP are low molecular weight proteins that inhibit MMP. This activity has been verified in physiological processes (wound healing, menstrual cycle et cetera) and experimental in vitro research. Therefore, when TIMP-2 increases, MMP-9 will decrease. However, the findings in surgical tumor specimens are different. Recent studies have indicated that TIMP and MMP are multifunctional molecules that have contradictory effects in tumor progression [33]. The present study investigated MMP-9, TIMP-1 and TIMP-2 as they all have complex functions in invasion and metastasis. A few previous studies on TIMP-1 and TIMP-2 in pituitary adenomas have shown that their levels are decreased in invasive adenomas [29,34,31,26], contradicting the results of the present study. We found most of the tumors with TIMP-2 staining (88.2%) to be in the invasive prolactinoma group. Significant relationships were detected between TIMP-2 expression and invasion (p = 0.004) and between TIMP-2 expression and recurrence (p = 0.005). TIMP-2 studies in oral squamous cell carcinoma (SCC) are relevant here because the oral cavity epithelium and the pituitary gland have a common embryological origin through Rathke’s pouch [35]. Both TIMP-2 and MMP have been suggested as prognostic biomarkers in studies of lingual [36], oral [37], laryngeal [38] and head and neck [10] SCC, and have reported relationships with poor prognosis and metastasis. The association of TIMP-2 with poor prognosis has also been demonstrated in studies on bladder [9], breast [39], and colorectal [40] SCC as well as in experimental studies [41]. Strongin et al. [33] explained these results which contradict commonly understood information on a molecular level and recently revealed the dual paradoxical effects of TIMP-2. When TIMP-2 forms a complex with membrane type 1-MMP (MT1-MMP) it induces cell mitosis and migration and promotes invasion. In contrast, it normally limits invasion and metastasis by inhibiting MT1-MMP. Furthermore, Sounni et al. [42] found that TIMP-2 is synthesized by cancer cells, explaining the TIMP-2 paradox. Fujimura et al. [43] found that MMP-9, MMP-2 and TIMP-2 were expressed in significantly higher amounts in the bleeding
sites of cerebral cavernous malformations and showed, with electron microscopy, that the blood-brain barrier, including the endothelial basement membrane, was structurally impaired. The expression of TIMP-2 by adenoma cells may be an indicator of endothelial basal membrane degradation, blood-brain barrier impairment, invasion and/or recurrence and our study lends support to this. TIMP-2 could be used as a prognostic biological marker for pituitary adenomas, particularly prolactinomas, although we detected only low level TIMP-2 staining in prolactinomas raising a potential limitation of TIMP-2 as a prognostic marker. We suggest that TIMP needs to be examined in a larger scale prospective study. The use of these biomarkers and the introduction of further biomarkers will shed light on the invasive behavior of pituitary adenomas and aid in the development of novel therapeutic approaches.
5. Conclusion We conclude that MMP-9 and TIMP-2 can act as prognostic biomarkers of invasive behavior in pituitary adenomas, especially prolactinomas. TIMP-2 can also indicate recurrence risk but this should be confirmed by further large scale studies. MMP-9 expression was shown to be reduced by DA and the correlation between MMP-9 staining intensity and invasion was hampered by DA treatment during the preoperative period. Further research is also needed to determine if MMP-9 expression is reduced by DA.
Conflicts of Interest/Disclosures The authors declare that they have no financial or other conflicts of interest in relation to this research and its publication.
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Clinical Study
Matrix metalloproteinase-9 and tissue inhibitor of matrix metalloproteinase-2: Prognostic biological markers in invasive prolactinomas Güliz Demirelli Gültekin a,⇑, Burak Çabuk a, Çig˘dem Vural b, Savasß Ceylan a a b
_ Kocaeli, Turkey Department of Neurosurgery, Kocaeli University School of Medicine, Umuttepe, Izmit, Department of Pathology, Kocaeli University School of Medicine, Umuttepe, Izmit, Kocaeli, Turkey
a r t i c l e
i n f o
Article history: Received 29 October 2014 Accepted 4 February 2015
Keywords: Invasion Matrix metalloproteinase-9 Pituitary adenoma Prolactinoma Tissue inhibitor of matrix metalloproteinase-2
a b s t r a c t In this study, the predictive roles of matrix metalloproteinase (MMP)-9 and tissue inhibitor of matrix metalloproteinase (TIMP)-1 and 2 in invasive and noninvasive prolactinomas were examined. Prognostic biomarkers to distinguish between invasive and noninvasive pituitary adenomas are required for the effective treatment of pituitary adenoma patients. We analyzed 57 prolactinoma patients classified as having invasive and noninvasive adenomas for MMP-9, TIMP-1 and TIMP-2 expression using immunohistochemical methods. Significantly higher MMP-9 expression was detected in invasive prolactinomas (p = 0.004). There was also a significant relationship between TIMP-2 expression and invasive behavior (p = 0.004) and TIMP-2 expression and recurrence (p = 0.005). Because MMP-9 expression is significantly increased in invasive prolactinomas, MMP-9 has potential as a marker for invasion. TIMP-2 may be a marker for both invasion and recurrence. These findings require further examination in large scale prospective studies. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Pituitary adenomas are common neoplasms. Although they are generally benign in nature, they may invade adjacent tissues. Recurrence and resistance to treatment are serious problems in invasive pituitary adenoma patients, therefore, when treating pituitary adenomas, biological markers are needed to indicate the invasiveness of the tumor to effectively guide surgical, medical, and radiotherapy treatment modalities and to prevent recurrence. Pituitary adenomas are epithelial tumors that arise from the adenohypophysis. The most frequently detected pituitary adenomas are prolactinomas [1]. Prolactinoma treatment is primarily with dopamine agonist drugs (DA; cabegoline, bromocriptine) [2,3]. In this study, prolactinomas were investigated with a focus on the roles of matrix metalloproteinase (MMP)-9 and tissue inhibitor of matrix metalloproteinase (TIMP)-2 in pituitary adenoma invasion. Only prolactinomas were investigated in order to concentrate the research on the roles of MMP-9 and TIMP-2 in pituitary adenoma invasion. ⇑ Corresponding author. Tel.: +90 050 57891621; fax: +90 380 5497060. E-mail address: [email protected] (G.D. Gültekin). http://dx.doi.org/10.1016/j.jocn.2015.02.021 0967-5868/Ó 2015 Elsevier Ltd. All rights reserved.
The MMP family consists of zinc and calcium-dependent endopeptidases. These enzymes are associated with proteolytic degradation of the extracellular matrix (ECM) [4]. The role of MMP in tumor invasion and metastatic processes has been confirmed. ECM degradation by MMP is a critical process in the progression of malignant tumors, angiogenesis, and tumor invasion and metastasis. Gelatinases (MMP-9 and MMP-2) are thought to be key enzymes in ECM degradation as they degrade some of the main components of the ECM [5,6]. TIMP are low molecular weight proteins that are secreted and bind to active MMP, inhibiting their enzymatic activity [7]. Moreover, MMP-2 and MMP-9 are unique among the MMP in that the latent forms of these proteinases can form complexes with TIMP-2 and TIMP-1, respectively. In addition to their inhibitory role, TIMP can also take part in the activation of MMP [8]. TIMP-2 is a powerful inhibitor of MMP activity and high levels of TIMP-2 are positively correlated with an unfavorable prognosis in cancer patients [9,10]. Previous studies have investigated MMP-9, TIMP-1 and TIMP-2 as prognostic biological markers in breast, lung, colon and many other cancer types, and the correlations of these enzymes with invasion and metastasis have been determined [11–14]. However, MMP-9 studies in pituitary adenomas have yielded
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conflicting results and TIMP-1 and TIMP-2 have not been studied as potential prognostic biological markers in pituitary adenomas. In this study, we evaluated the presence of MMP-9, TIMP-1 and TIMP-2 in invasive and noninvasive prolactinomas and investigated their potential as prognostic predictors using immunohistochemical methods. 2. Methods and materials In the present study, we retrospectively reviewed the records of patients with prolactinoma who had undergone endoscopic transsphenoidal surgery in the Department of Neurosurgery, Kocaeli University School of Medicine between 1999 and 2011. Fifty-seven patients who were diagnosed with prolactinoma based on clinical, radiological and pathological data were included in this study. The mean ages of the patients in the invasive prolactinoma and noninvasive prolactinoma groups were 43.2 years and 35 years, respectively (range: 16–69). There were 11 females and 24 males in the invasive group (n = 35), and 18 females and four males in the noninvasive group (n = 22). The indications for surgery in the invasive group were as follows: resistance to DA treatment (n = 5), intolerance to DA (n = 6), and patient preference (n = 24). The indications for surgery in the noninvasive group were as follows: resistance to DA (n = 9), intolerance to DA (n = 6), and patient preference (n = 7). In the noninvasive group, all patients (n = 22) were first time surgeries, and 31/35 patients in the invasive group were first time surgeries. Four patients in the invasive group were second surgeries. Radiotherapy was not performed in any patient. None of the prolactinoma patients had multiple endocrine neoplasia syndrome. Three patients with atypical meningioma and two with chordoma were included as a positive control group. Grade 3 and 4 tumors, according to the Knosp classification [15], and grade 4D and 4E tumors, according to the modified Hardy classification [16], were considered to be invasive prolactinomas and radiological invasion was intraoperatively confirmed. Thirty-five patients were classified as having invasive prolactinomas and 22 with noninvasive prolactinomas. Treatment outcomes were evaluated according to prolactin (PRL) level in the blood and sella MRI on the first day after operation. Normal PRL values were considered to indicate remission and were monitored for recurrence or cure. A PRL level higher than 21 ng/dl was considered to indicate persistent recurrence. In the noninvasive group, 14/22 patients (64%) were cured and in the invasive group none were cured (0/35; 0%). Persistent recurrence was detected in 31/35 patients (88%) in the invasive group and 7/22 (32%) in the noninvasive group. In the invasive group, late recurrence was detected in 4/35 patients (12%) and the average time to late recurrence was 10 months after surgery. In the noninvasive group, late recurrence was detected in 1/22 patients (4%) and the time to recurrence for that patient was 4 years. Patients were observed for cure and recurrence from 3 to 14 years after surgery. 2.1. Histopathological examination MMP-9 (Thermo Fisher Scientific, Waltham, MA, USA), TIMP-1 (Clone: 102D1; Thermo Fisher Scientific), and TIMP-2 (Clone: 3A4; Thermo Fisher Scientific) antibodies were applied to sections obtained from formalin fixed and paraffin embedded blocks. Sections (4 lm) were mounted on poly-L-lysine coated glass and de-paraffinized after being kept in a 56°C incubator overnight. They were placed in xylol for 30 minutes, absolute alcohol for 15 minutes, 96% alcohol for 15 minutes and then washed with water. Antigens were retrieved by boiling the sections in citrate buffer (pH 6) in a microwave oven for 15 minutes. Endogen peroxidase activity was blocked by quenching in a 3% hydrogen peroxide
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solution for 15 minutes. Tissue sections were incubated at room temperature for 30 minutes with primary antibodies against MMP-9, TIMP-1 and TIMP-2. A secondary antibody was applied and the sections were treated with streptavidin-biotin-peroxidase complex for 30 minutes. 3,3-diaminobenzidine was used as a chromogen and Mayer’s hematoxylin was used for AEC ground staining. The sections were covered with a water-based cover material. Meningioma and chordoma tissues were used as the positive controls for MMP-9, TIMP-1 and TIMP-2 expression. The stained tissues were examined by a single pathologist who was blinded to the clinical data. The extent and intensity of MMP-9 staining in the adenomas were evaluated. The extent of MMP-9 staining was scored as negative (0; from 0–30%), 1 (minimal; from 31–60%), 2 (focal; from 61–100%) or 3 (diffuse). The intensity of MMP-9 staining was scored as negative (0), weak (1), moderate (2) or strong staining (3). TIMP-1 and TIMP-2 were used to distinguish the adjacent pituitary tissue from the adenoma and were evaluated as positive or negative depending on whether staining was detected. 2.2. Statistical analyses Research data were analyzed using the Statistical Package for the Social Sciences (SPSS statistics; version 13.0; IBM Corporation, Armonk, NY, USA). Descriptive statistics and the chi-squared test were used and the significance level was set at p < 0.05. 3. Results 3.1. MMP-9 and invasion MMP-9 expression was detected in all patients (57; 100%). The relationship between MMP-9 expression and invasive behavior was evaluated by the extent of MMP-9 staining measured as the number of adenoma cells showing MMP-9 immunoreactivity. Figure 1 shows MMP-9 staining in one invasive and one noninvasive patient. In invasive prolactinomas, diffuse staining was detected in 85.7% of patients (30/35). A significant relationship was determined between MMP-9 expression and invasive behavior (p = 0.004; Table 1). There was no correlation between MMP-9 staining intensity and invasive prolactinomas. This result conflicted with previous studies, therefore, the possible causative factors of this result were examined. In the invasive group, significant differences in MMP-9 staining intensity were found between patients who took and did not take DA drugs in the preoperative period. Figure 2 shows MMP-9 expression in two patients with invasive giant adenomas, one with DA and one without DA during the preoperative period. In the invasive group, the MMP-9 staining intensities for the DA treated group were strong staining in one (8%), moderate staining in nine (75%) and weak staining in two patients (16%). The MMP-9 staining intensities for the no DA treatment group were strong staining in 10 (75%), moderate staining in 10 (43%) and weak staining in three patients (12%). 3.2. TIMP-1 and invasion TIMP-1 staining was detected in 31/57 patients (54%) and staining in the adenoma was observed in 16. There was no correlation between TIMP-1 expression and invasion. 3.3. TIMP-2 and invasion TIMP-2 staining was detected in the adenoma and adjacent tissue sample in 32/57 patients (56%), while staining in the adenoma
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Fig. 1. Matrix metalloproteinase (MMP)-9 antibody staining of prolactinoma samples (Thermo Fisher Scientific, Waltham, MA, USA). Brown areas show MMP-9 expression, blue areas are where no MMP-9 expression was detected. (A) Patient 44, invasive prolactinoma: MMP-9 staining classified as diffuse (magnification 20). (B) Patient 37, noninvasive prolactinoma: MMP-9 staining classified as focal (magnification 20).
Table 1 The relationship between matrix metalloproteinase-9 expression and invasive behavior of prolactinomas MMP-9 staining
Minimal (60%)
p value
Invasive, n (%) Noninvasive, n (%)
1 (2.8) 7 (31.8)
4 (11.5) 4 (18.2)
30 (85.7) 11 (50)
0.004
MMP = matrix metalloproteinase.
alone was observed in 17. In the present study, TIMP-2 staining in the adenoma was evaluated. Figure 3A shows TIMP-2 staining in an invasive prolactinoma patient. TIMP-2 staining was detected in 15 patients (42.4%) in the invasive group and two (9%) in the noninvasive group. Although TIMP-2 staining was low in both groups, 88.2% of the adenomas showing TIMP-2 staining were in the invasive group. A significant relationship was found between TIMP-2 expression and invasive behavior (p = 0.004; Table 2). 3.4. TIMP-2 and treatment outcome TIMP-2 staining in the adenoma was observed in 17 patients. The invasive to noninvasive patient ratio was 15:2. Patients with TIMP-2 staining consistently resulted in recurrence; a significant relationship was found between TIMP-2 staining and recurrence (p = 0.005; Table 3).
3.5. Correlation of TIMP-2 expression in adjacent tissues and good prognosis, noninvasive behavior Adjacent pituitary tissue was examined in specimens from 14/22 patients in the noninvasive group, but not in specimens from invasive prolactinoma patients. TIMP-2 staining was consistently detected in adjacent pituitary tissue and not in noninvasive adenomas (13/14). Figure 4 shows TIMP-2 staining in adjacent pituitary tissues and a lack of staining in adenomas from noninvasive patients. TIMP-2 staining of adenoma tissues was detected in invasive adenomas. TIMP-2 staining in adjacent tissue was consistently detected in noninvasive adenomas. 3.6. Comparison of MMP-9, TIMP-1 and TIMP-2 expression in pituitary adenoma patients and controls The positive control group included three atypical meningiomas and two clivus chordomas. All of these showed diffuse MMP-9 staining (5/5) and none showed TIMP-1 staining (0/5). TIMP-2 staining was observed in 4/5 controls. When compared with the prolactinoma patients showing TIMP-2 staining, the meningioma and chordoma controls exhibited less intense staining (Fig. 3). Finally, prolactinomas were shown to have greater MMP-9 expression than meningiomas and chordomas (Fig. 3). This pattern conforms with the more aggressive clinical course of invasive prolactinomas compared to atypical meningiomas and chordomas.
Fig. 2. Matrix metalloproteinase (MMP)-9 antibody staining in two invasive giant adenoma patient samples (Thermo Fisher Scientific, Waltham, MA, USA). Brown areas show MMP-9 expression, blue areas are where no MMP-9 expression was detected. (A) Patient 32 treated preoperatively with dopamine agonist drugs (DA; magnification 40). (B) Patient 33, no DA treatment (magnification 40). Whereas MMP-9 staining was diffuse in both patients, staining intensity was different. The (A) DA treated patient sample was classified as moderate MMP-9 staining and the (B) no DA patient as strong.
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Fig. 3. Tissue inhibitor of matrix metalloproteinase (TIMP)-2 antibody staining (Clone 3A4; Thermo Fisher Scientific, Waltham, MA, USA). (A) Patient 26, invasive pituitary adenoma (magnification 200). (B) Control Patient 60, clivus chordoma (magnification 200). The chordoma control showed less intense staining than the invasive prolactinoma.
Table 2 The relationship between tissue inhibitor of matrix metalloproteinase-2 expression and invasive behavior of prolactinomas TIMP-2 expression
Stained
Not stained
Total
p value
Invasive, n (%) Noninvasive, n (%)
15 (42.4) 2 (9)
20 (57.6) 20 (91)
35 22
0.004
TIMP = tissue inhibitor of matrix metalloproteinase.
Table 3 The relationship between tissue inhibitor of matrix metalloproteinase-2 expression and treatment outcome in of prolactinoma patients TIMP-2 expression
Stained
Not stained
Total
p value
Recurrence, n (%) Cure, n (%)
17 (40) 0 (0)
26 (60) 14 (100)
43 14
0.005
TIMP = tissue inhibitor of matrix metalloproteinase.
Fig. 4. Tissue inhibitor of matrix metalloproteinase (TIMP)-2 antibody (Clone 3A4; Thermo Fisher Scientific, Waltham, MA, USA) staining in adjacent pituitary tissue (brown; red arrow) and lack of staining in adenoma tissue (blue; red arrow) in a noninvasive prolactinoma (Patient 31; magnification 100). TIMP-2 staining in adjacent tissue was consistently detected in noninvasive adenomas.
4. Discussion Pituitary adenomas account for 20% of all intracranial tumors and 40–60% of pituitary adenomas are prolactinomas. Invasion is the most important prognostic factor in pituitary adenoma patients. Approximately 30–35% of functional pituitary adenomas
and >50% of nonfunctional pituitary adenomas are associated with dural invasion. Total resection is more difficult for invasive than noninvasive tumors and post-surgery recurrence is common [17,18]. Surgical complications, the inability to achieve remission and inadequate therapy are all related to invasion [19]. However, the molecular mechanism of local invasive behavior is not yet clearly understood. Predicting invasive behavior and recurrence may help in the formulation of treatment decisions in pituitary adenoma patients. Local invasion and metastasis of tumor cells primarily require degradation of the basement membrane. For that purpose, tumor cells express enzymes for proteolysis such as MMP, which are zinc containing endopeptidases that play a role in the degradation of the extracellular matrix in angiogenesis and invasion. MMP-9 is an endopeptidase that degrades type IV collagen and is controlled at the transcriptional level [20]. The endogenous inhibitors of MMP are TIMP-1, 2, 3, and 4 [11]. In recent years, various solid tumor cells have been demonstrated to produce MMP-2, MMP-9, TIMP-1 and TIMP-2 in vitro and these enzymes have been associated with tumor invasion, poor prognosis and differentiation [11]. Many studies on the role of MMP-9 in the invasive behavior of pituitary adenomas have been performed. Kawamoto et al. [21] demonstrated that type IV collagenase (MMP-9) activity was noticeably higher in invasive pituitary adenomas compared to noninvasive pituitary adenomas using immunofluorescence. They concluded that this may be related to cavernous sinus invasion. Furthermore, Liu et al. [22] found that MMP-2 and MMP-9 expression levels were noticeably high in pituitary adenomas with cavernous sinus invasion using both immunohistochemical and real time polymerase chain reaction (RT-PCR) methods. Turner et al. [23] showed remarkably high expression of MMP-9 in invasive macroprolactinomas in comparison with noninvasive adenomas using immunohistochemical methods and suggested that MMP-9 plays an important role in angiogenesis and invasion. Hussaini et al. [19] demonstrated that MMP-9 expression was high in invasive adenomas using gelatin zymography, western blot, RT-PCR and immunohistochemical methods. Gong et al. [24] demonstrated expression levels of MMP-9 3–5 times greater in invasive adenomas compared to noninvasive adenomas. Furthermore, they demonstrated MMP-9 expression at 3 times greater levels in invasive prolactinomas than in all invasive pituitary tumors using gelatin zymography and western blot methods and validated their results using RT-PCR and immunohistochemical methods. Ceylan et al. [25] recently performed the first comprehensive microanatomic and histological study of the normal pituitary gland using immunohistochemical and RT-PCR methods. They suggested
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that the ability of tumor cells to express MMP-9 indicates that the tumor has aggressive and invasive behaviors. Pereda et al., Wang et al. and Pan et al. reached similar conclusions regarding MMP-9 and invasion [26–28]. Conversely, Beaulieu et al., Yokoyama et al., Knappe et al. and Yamada et al. found no correlation between MMP-9 expression and invasion [29–32]. In the present study, significantly higher MMP-9 expression was found in invasive prolactinomas compared to noninvasive prolactinomas using immunohistochemical methods. MMP-9 staining was consistently correlated with invasion (p = 0.004). Diffused MMP-9 staining was observed in 85.7% of the invasive prolactinomas. Moreover, it was consistently detected with diffused MMP-9 staining in the invasive prolactinomas and focal MMP-9 staining in the noninvasive prolactinomas. Our results support those of recent studies on this subject and show that MMP-9 could be used as a prognostic biological marker for prolactinomas and other pituitary adenomas. From our cohort, it was seen that MMP-9 staining intensity was altered from strong to moderate by the administration of DA drugs during the preoperative period and, therefore, we concluded that MMP-9 expression may be slowed by DA. The consistent detection of MMP-9 staining in correlation with invasion was hampered by DA treatment. However, further research is needed to determine if this effect on MMP-9 expression is caused by DA. Prolactinomas have much greater MMP-9 expression than meningiomas and chordomas. This fits the typically more aggressive clinical course of invasive prolactinomas than of atypical meningiomas and chordomas and supports the results of Gong et al. [24] TIMP are low molecular weight proteins that inhibit MMP. This activity has been verified in physiological processes (wound healing, menstrual cycle et cetera) and experimental in vitro research. Therefore, when TIMP-2 increases, MMP-9 will decrease. However, the findings in surgical tumor specimens are different. Recent studies have indicated that TIMP and MMP are multifunctional molecules that have contradictory effects in tumor progression [33]. The present study investigated MMP-9, TIMP-1 and TIMP-2 as they all have complex functions in invasion and metastasis. A few previous studies on TIMP-1 and TIMP-2 in pituitary adenomas have shown that their levels are decreased in invasive adenomas [29,34,31,26], contradicting the results of the present study. We found most of the tumors with TIMP-2 staining (88.2%) to be in the invasive prolactinoma group. Significant relationships were detected between TIMP-2 expression and invasion (p = 0.004) and between TIMP-2 expression and recurrence (p = 0.005). TIMP-2 studies in oral squamous cell carcinoma (SCC) are relevant here because the oral cavity epithelium and the pituitary gland have a common embryological origin through Rathke’s pouch [35]. Both TIMP-2 and MMP have been suggested as prognostic biomarkers in studies of lingual [36], oral [37], laryngeal [38] and head and neck [10] SCC, and have reported relationships with poor prognosis and metastasis. The association of TIMP-2 with poor prognosis has also been demonstrated in studies on bladder [9], breast [39], and colorectal [40] SCC as well as in experimental studies [41]. Strongin et al. [33] explained these results which contradict commonly understood information on a molecular level and recently revealed the dual paradoxical effects of TIMP-2. When TIMP-2 forms a complex with membrane type 1-MMP (MT1-MMP) it induces cell mitosis and migration and promotes invasion. In contrast, it normally limits invasion and metastasis by inhibiting MT1-MMP. Furthermore, Sounni et al. [42] found that TIMP-2 is synthesized by cancer cells, explaining the TIMP-2 paradox. Fujimura et al. [43] found that MMP-9, MMP-2 and TIMP-2 were expressed in significantly higher amounts in the bleeding
sites of cerebral cavernous malformations and showed, with electron microscopy, that the blood-brain barrier, including the endothelial basement membrane, was structurally impaired. The expression of TIMP-2 by adenoma cells may be an indicator of endothelial basal membrane degradation, blood-brain barrier impairment, invasion and/or recurrence and our study lends support to this. TIMP-2 could be used as a prognostic biological marker for pituitary adenomas, particularly prolactinomas, although we detected only low level TIMP-2 staining in prolactinomas raising a potential limitation of TIMP-2 as a prognostic marker. We suggest that TIMP needs to be examined in a larger scale prospective study. The use of these biomarkers and the introduction of further biomarkers will shed light on the invasive behavior of pituitary adenomas and aid in the development of novel therapeutic approaches.
5. Conclusion We conclude that MMP-9 and TIMP-2 can act as prognostic biomarkers of invasive behavior in pituitary adenomas, especially prolactinomas. TIMP-2 can also indicate recurrence risk but this should be confirmed by further large scale studies. MMP-9 expression was shown to be reduced by DA and the correlation between MMP-9 staining intensity and invasion was hampered by DA treatment during the preoperative period. Further research is also needed to determine if MMP-9 expression is reduced by DA.
Conflicts of Interest/Disclosures The authors declare that they have no financial or other conflicts of interest in relation to this research and its publication.
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