Abstract
Objective
To evaluate the expression of matrix metalloproteinase-2 (MMP-2), MMP-9 and tissue inhibitor of metalloproteinase-2 (TIMP-2) in pituitary tumors and investigate their correlation with circulating plasma proteins and cavernous sinus invasion. In addition, the Ki-67 index was also assessed.
Methods
Seventy-four patients (37 females) with pituitary adenomas were included, with preoperative peripheral blood collected in 29 cases. Tumor samples were evaluated for MMP-2, MMP-9, TIMP-2 and Ki-67 expression by immunohistochemistry. Protein plasma was semi-quantitatively detected using a commercial membrane antibody array.
Results
Sixteen patients presented tumors invading the cavernous sinus. MMP-2 and TIMP-2 were slightly increased in these tumors compared to the noninvasive group, but the difference was not statistically significant. MMP-9 and TIMP-2 plasma concentrations did not correlate with tumor protein expression and also did not differ between the two groups. MMP-2 was not detected in plasma in any case. No statistically significant difference was observed when different tumor subtypes were considered. A significant difference was observed in tumor size (3.4 cm (2.8–4.9) vs 1.9 cm (1.3–2.6); P < 0.001) and in the Ki-67 index (1.8% (0.3–2.5) vs 0.5% (0.2–1.0); P = 0.01) between the invasive and noninvasive groups.
Conclusions
In this cohort, we found no significant correlation between tissue and plasma levels of MMP-2, MMP-9 and TIMP-2 and cavernous sinus invasion in pituitary tumors. Further investigation is needed to elucidate the potential role of these markers in the invasiveness of pituitary tumors.
Introduction
Pituitary tumors are incidentally discovered in approximately 10% of the general population and constitute around 15% of tumors in the central nervous system (Molitch 2008). Although most of these tumors do not lead to distant metastases, 5–35% exhibit invasiveness, extending into local tissues such as the cavernous sinus, sphenoid sinus, orbit and clivus (Turner et al. 2000, Liu et al. 2005a, Moreno et al. 2005). The degree of invasion is the main determinant for therapeutic efficacy and prognosis in pituitary tumors, influencing postoperative outcomes, rates of remission/recurrence and patient survival (Knosp et al. 1993, Trouillas et al. 2013, Lefevre et al. 2024). However, the underlying differences in tumor behavior remain poorly understood, and as of now, there are no reliable pathophysiological mechanisms to predict tumor invasiveness (Mete et al. 2012, Lefevre et al. 2024).
Matrix metalloproteinases (MMPs) represent a family of zinc-dependent endopeptidases capable of degrading multiple extracellular matrix components, playing crucial roles in tissue remodeling, angiogenesis and tumor invasion processes (Kawamoto et al. 1996b, Ceylan et al. 2011). Over the past two decades, several studies have correlated the expression of MMP-2 and MMP-9 with the invasive behavior of pituitary tumors, particularly those invading the cavernous sinus (Gültekin et al. 2015, Kawamoto et al. 1996a, 1996b, Turner et al. 2000, Liu et al. 2005a, 2005b, Qiu et al. 2011). Moreover, elevated serum levels of MMP-9 have been observed in various human malignancies, including gastrointestinal tract and breast cancers, with notable associations with histological grade and lymph node/vascular metastasis (Zucker et al. 1993, Langenskiöld et al. 2005, Vasaturo et al. 2013). Furthermore, the tissue inhibitor of metalloproteinase-2 (TIMP-2) is being investigated as a potential protective factor, not only inhibiting MMP-2 but also acting as an endogenous inhibitor of angiogenesis and tumor growth (Bourboulia et al. 2013).
However, conflicting results have been reported in studies evaluating the expression of MMP-2, MMP-9 and TIMP-2 in pituitary tumors (Beaulieu et al. 1999, Knappe et al. 2003, Gültekin et al. 2015). Therefore, in this study, we aimed to comprehensively analyze MMP-2, MMP-9 and TIMP-2 expression in tumor tissue between invasive and noninvasive groups and correlate these findings with their plasma levels to evaluate the preliminary utility of these markers as preoperative predictors of tumor behavior.
Material and methods
Patients
This study employed a retrospective design utilizing archived tumor specimens, complemented by a prospective collection of matched tissues and serum samples. A convenience sample of 74 patients was selected, with 29 cases providing serum and tumor tissues. Specifically, prospective samples were consecutively obtained from a subset of patients who underwent transsphenoidal or transcranial surgery at our institution between August 2014 and October 2015 and received a confirmed histopathological diagnosis of pituitary adenoma (Fig. 1). Patients with a history of prior radiotherapy were excluded from the study. The research was approved by the Ethics Committee for Analysis of Research Projects – CAPPesq, HCFMUSP – CAAE 22787414.0.0000.0068, and written informed consent was obtained from all participants.
Flow chart showing sample collection and analysis process.
Citation: Endocrine Oncology 5, 1; 10.1530/EO-24-0037
Clinical and hormonal assessments and imaging
A comprehensive clinical evaluation of all individuals was carried out through detailed anamnesis, physical examination and extensive review of medical records. In addition, hormonal and radiological assessments were performed in the preoperative period. Clinical characteristics included gender, age at diagnosis, tumor subtype, tumor size and cavernous sinus invasion. Magnetic resonance imaging (MRI) was utilized to define invasion, with tumors classified as Knosp 3A, 3B and 4 considered invasive to the cavernous sinus (Knosp et al. 1993).
Tumor protein assessment by immunohistochemistry
Once the tumor sample was collected, it was immediately placed in a fixative solution, usually 10% formalin, to preserve the tissues. Tumor samples were then embedded in paraffin for histopathological evaluation. After the evaluation of hematoxylin and eosin tumor sections, 51 samples were selected for tissue microarray (TMA) construction. The remaining 23 samples, which were insufficient for TMA, were traditionally evaluated by whole-section analysis. Protein markers were assessed in all 74 samples using standard immunohistochemical (IHC) procedures. In summary, slides were deparaffinized, rehydrated and subjected to antigen retrieval by boiling in 10 mM citrate buffer (pH 6.0). Endogenous peroxidase activity was blocked with 6% aqueous hydrogen peroxide solution for 20 min. Subsequently, the sections were incubated overnight at 4°C with the primary antibody: MMP-2 (Sigma-Aldrich, USA) at 1:50 dilution, MMP-9 (Novus Biologicals, USA) at 1:400 dilution, TIMP-2 (Sigma-Aldrich, USA) at 1:50 dilution and Ki-67 (Dako Omnis, USA) at 1:200 dilution. The antibodies were diluted in phosphate-buffered saline (PBS) containing 1% bovine serum albumin and 0.1% sodium azide in a humidity chamber. Ki-67 was assessed using the MIB-1 antibody.
Thereafter, the slides were treated with Post Primary Block (NovoLink Max Polymer, Leica Biosystems, UK) for 30 min at 37°C and incubated with NovoLink Polymer (NovoLink Max Polymer, Leica Biosystems, UK) for 30 min at 37°C. Color development was achieved using a chromogenic substrate solution composed of 100 mg of 3,3′-diaminobenzidine tetrahydrochloride (Sigma, USA), 1 mL dimethyl sulfoxide, 1 mL of 6% hydrogen peroxide solution and 100 mL PBS for 5 min at 37°C. The slides were counterstained with Harris hematoxylin for 30 s. Controls included a positive control (placenta for MMP-2 and MMP-9 and chordoma for TIMP-2) and a negative control with omission of the primary antibody.
Each slide was independently examined by two experienced pathologists (RSSM and FPF), who were blinded to the diagnosis, using an optical microscope at 400× magnification. For the TMA slides, histological fields were evaluated integrally. For whole-section slides, approximately 100 cells from five randomly chosen fields were evaluated. MMP-2, MMP-9 and TIMP-2 expressions were assessed using the H-score method on a continuous scale of 0–300, based on the percentage of positive cells. IHC staining was recorded in four categories: 0 for ‘no staining’, 1 + for ‘light staining’, 2 + for ‘intermediate staining’ and 3 + for ‘dark staining’. The percentage of cells at different staining intensities was determined by visual assessment, with the score calculated using the following formula: 1 × (% of 1 + cells) + 2 × (% of 2 + cells) + 3 × (% of 3 + cells) (Jordan et al. 2012). The proliferative Ki-67 index was measured in areas of high cell density with positive nuclear immunostaining at 400× magnification. Antibody-labeled cells were counted, and their fraction of the total number of cells was determined, with the result reported as percentage.
Plasma protein assessment by immunoassay
Preoperative peripheral venous blood samples, approximately 5 mL each, were collected in heparinized tubes. The blood was promptly centrifuged at 4,000 g for 10 min, and the plasma was then recovered, aliquoted and stored at −80°C. Plasma concentrations of MMP-2, MMP-9 and TIMP-2 were measured in these samples using a multiplex immunoassay with a commercially available Abcam® Human MMP Antibody Array-Membrane kit (Abcam, UK). The assay consisted of arrays of immobilized antibody spots, each specific to a target protein subtype, allowing simultaneous detection of 7 human MMPs (1–3, 8–10 and 13) and 3 TIMPs (−1, −2 and −4). Detection was performed using a complementary antibody cocktail that is biotinylated (similar to a sandwich assay), horseradish peroxidase-conjugated streptavidin detection and chemiluminescence reagents. All procedures were performed according to the manufacturer’s instructions (https://www.abcam.com/en-br/products/antibody-arrays/human-mmp-antibody-array-membrane-10-targets-ab134004#tab=support).
An Alliance 4.7 Uvitec® imaging system (Cleaver Scientific Ltd, UK) was used to image the membranes. The intensity of the generated spots was semi-quantified as density values using the ImageJ software (http://rsb.info.nih.gov/ij). Densitometry data from each array were expressed as relative expression after background subtraction and normalization with control spots, in accordance with the manufacturer’s instructions (https://www.abcam.com/en-br/products/antibody-arrays/human-mmp-antibody-array-membrane-10-targets-ab134004#tab=support and the Supplemental Method (see section on supplementary materials given at the end of the article)). For this study, only data for MMP-2, MMP-9 and TIMP-2 were subjected to statistical analysis.
Statistical analysis
The interobserver variation in H-scores was calculated using the Spearman rank test. Qualitative data were assessed using the chi-square test or Fisher’s exact test, as appropriate, and presented as absolute values. Quantitative data were evaluated using the non-parametric Wilcoxon rank-sum test and expressed as median with the 25th–75th percentiles. Spearman’s rank correlation method was employed to test the associations between variables. All statistical analyses were performed using the STATA software, version 14.0 (StataCorp LLC, USA). A P value <0.05 was considered statistically significant.
Results
Patients’ characteristics
We investigated a cohort of 74 patients (37 males and 37 females) with a mean age at diagnosis of 43.8 ± 14.9 years (range: 16–79). Of these, 37 (50%) were diagnosed with non-functioning pituitary adenoma, 21 (28%) with somatotropinoma, 14 (19%) with corticotropinoma and 2 (3%) with prolactinoma. MRI revealed pituitary invasive tumors involving the cavernous sinus in 16 (22%) patients and noninvasive tumors in 58 (78%) patients. Invasive tumors exhibited a significantly larger size compared to noninvasive ones (3.4 cm (2.8–4.9) vs 1.9 cm (1.3–2.6); P < 0.001; Table 1) and a higher Ki-67 index (1.8% (0.3–2.5) vs 0.5% (0.2–1.0); P = 0.01; Table 1).
Clinical and radiological characteristics of the invasive and noninvasive groups to the cavernous sinus, along with MMP-2, MMP-9 and TIMP-2 immunohistochemical expression and protein plasma levels in both groups.
| Invasive (n = 16) | Noninvasive (n = 58) | Overall (n = 74) | P* | |
|---|---|---|---|---|
| Sex (female:male) | 10(63):6(37) | 27(47):31(53) | 37(50):37(50) | 0.26 |
| Age at diagnosis (years) | 40.4 ± 13.4 | 45.5 ± 14.1 | 44.4 ± 14 | 0.79 |
| Tumor subtype | ||||
| Non-functioning | 9 (30) | 28 (70) | 37 (50) | 0.54 |
| Somatotropinomas | 3 (14) | 18 (86) | 21 (28) | |
| Corticotropinomas | 3 (21) | 11 (79) | 14 (19) | |
| Prolactinomas | 1 (50) | 1 (50) | 2 (3) | |
| Largest tumor diameter (cm) | 3.4 (2.8–4.9) | 1.9 (1.3–2.6) | 2.3 (1.5–3.3) | <0.001 |
| Ki-67 index | 1.8 (0.3–2.5) | 0.5 (0.2–1.0) | 0.5 (0.2–1.5) | 0.01 |
| Tumor protein expression (H-score) | ||||
| MMP-2 | 105 (30–155) | 40 (12–90) | 45 (20–110) | 0.09 |
| MMP-9 | 93 (46–152) | 95 (30–155) | 95 (30–155) | 0.80 |
| TIMP-2 | 191 (62–277) | 145 (65–212) | 154 (65–217) | 0.26 |
| Plasma protein levels (immunoblotting) | ||||
| MMP-9 | 832 (499–1326) | 304 (175–929) | 449 (210–1,040) | 0.11 |
| TIMP-2 | 3,568 (2,474–4,216) | 3,038 (2,100–3,770) | 3,054 (2,474–4,082) | 0.47 |
Statistically significant values are highlighted in bold. IHC: immunohistochemical. Data are described in absolute values (%), mean ± standard deviation or median (25th–75th percentiles).
Abbreviations: MMP-2, matrix metalloproteinase-2; MMP-9, matrix metalloproteinase-9; TIMP-2, tissue inhibitor of metalloproteinase-2.
These differences remained significant when comparing invasive somatotropinomas to noninvasive ones (size = 3.5 cm (2.3–6) vs 1.3 cm (1.2–1.7), P = 0.01; Ki-67 = 1.7% (1.5–2.5) vs 0.2% (0.1–0.5), P < 0.01; Table 2). In corticotropinomas, only tumor size differed between invasive and noninvasive groups (3.2 cm (2.5–5.2) vs 1.2 cm (0.8–1.8), P = 0.01; Table 2). No other statistically significant differences were observed between the invasive and noninvasive groups regarding gender, age at diagnosis or tumor subtype (Tables 1 and 2).
Clinical and radiological characteristics of the invasive and noninvasive groups to the cavernous sinus according to pituitary tumor subtype, along with MMP-2, MMP-9 and TIMP-2 immunohistochemical expression and protein plasma levels.
| Invasive | Noninvasive | Overall | P* | |
|---|---|---|---|---|
| Non-functioning | (n = 9) | (n = 28) | (n = 37) | |
| Sex (female:male) | 4(44):5(56) | 9(32):19(68) | 13(35):24(65) | 0.69 |
| Age at diagnosis (years) | 44.7 ± 12.5 | 52.8 ± 13.8 | 50.8 ± 13.8 | 0.12 |
| Largest diameter (cm) | 3.3 (3.2–4.6) | 2.5 (2.3–3.8) | 3.0 (2.5–4) | 0.09 |
| Ki-67 index | 2.0 (0.5–2.5) | 0.8 (0.25–1.3) | 1.0 (0.3–2.0) | 0.08 |
| MMP-2 (tissue) | 147 (35–160) | 42 (10–132) | 50 (10–147) | 0.15 |
| MMP-9 (tissue) | 100 (45–140) | 50 (3.7–138) | 50 (20–140) | 0.48 |
| TIMP-2 (tissue) | 195 (157–292) | 162 (102–221) | 185 (105–240) | 0.17 |
| MMP-9 (plasmatic) | 536 (448–623) | 276 (159–469) | 355 (206–489) | 0.27 |
| TIMP-2 (plasmatic) | 3,568 (3,054–4,082) | 3,060 (2,364–3,355) | 3,132 (2,627–3,637) | 0.44 |
| Somatotropinomas | (n = 3) | (n = 18) | (n = 21) | |
| Sex (female:male) | 2(67):1(33) | 7(39):11(61) | 9(43):12(57) | 0.55 |
| Age at diagnosis (years) | 35.7 ± 8.3 | 39.6 ± 12.7 | 39.1 ± 12 | 0.50 |
| Largest diameter (cm) | 3.5 (2.3–6) | 1.3 (1.2–1.7) | 1.5 (1.2–1.9) | 0.01 |
| Ki-67 index | 1.7 (1.5–2.5) | 0.2 (0.1–0.5) | 0.2 (0.1–0.7) | <0.01 |
| MMP-2 (tissue) | 105 (65–210) | 35 (25–85) | 60 (25–105) | 0.20 |
| MMP-9 (tissue) | 85 (82–300) | 120 (35–162) | 120 (82–300) | 0.52 |
| TIMP-2 (tissue) | 10 (2.5–212) | 57 (30–130) | 50 (25–130) | 0.40 |
| MMP-9 (plasmatic) | 1,388 (1,388–1,388) | 304 (146–643) | 474 (146–929) | 0.33 |
| TIMP-2 (plasmatic) | 4,745 (4,745–4,745) | 3,272 (2,762–4,213) | 3,742 (2,763–4,598) | 0.33 |
| Corticotropinomas | (n = 3) | (n = 11) | (n = 14) | |
| Sex (female:male) | 3(100):0(0) | 10(91):1(9) | 13(93):1(7) | 0.59 |
| Age at diagnosis (years) | 44.7 ± 15.5 | 37.9 ± 7.4 | 38.5 ± 9 | 0.65 |
| Largest diameter (cm) | 3.2 (2.5–5.2) | 1.2 (0.8–1.8) | 1.6 (0.8–2.0) | 0.01 |
| Ki-67 index | 0.1 (0.1–0.3) | 0.3 (0.2–1.0) | 0.2 (0.1–0.5) | 0.19 |
| MMP-2 (tissue) | 38.7 (25–105) | 45 (12.5–65) | 42.5 (20–65) | 0.70 |
| MMP-9 (tissue) | 105 (47.5–235) | 142 (70–270) | 135 (70–235) | 0.61 |
| TIMP-2 (tissue) | 277 (55–277) | 195 (150–235) | 202 (150–270) | 0.48 |
| MMP-9 (plasmatic) | 1,183 (1,040–1,326) | 1,628 (210–2,749) | 1,254 (625–2,411) | 0.86 |
| TIMP-2 (plasmatic) | 2,433 (2,392–2,474) | 2,939 (1,278–4,696) | 2,657 (1,835–3,867) | 0.64 |
| Prolactinomas | (n = 1) | (n = 1) | (n = 2) | |
| Sex (female:male) | 1(100):0(0) | 1(100):0(0) | 2(100):0(0) | – |
| Age at diagnosis (years) | 16 | 31 | 23.5 ± 10 | – |
| Largest diameter (cm) | 4.5 | 2.8 | 3.6 (2.8–4.5) | – |
| Ki-67 index | 3.2 | 1 | 2.1 (1–3.2) | – |
| MMP-2 (tissue) | 15 | 40 | 27.5 (15–40) | – |
| MMP-9 (tissue) | 0 | 12.5 | 6.2 (30–12.5) | – |
| TIMP-2 (tissue) | 10 | 0 | 5 (0–10) | – |
| MMP-9 (plasmatic) | 498.6 | – | 498.6 | – |
| TIMP-2 (plasmatic) | 4,216 | – | 4,216 | – |
Statistically significant values are highlighted in bold. IHC: immunohistochemical. Data are described in absolute values (%), mean ± standard deviation or median (25th–75th percentiles).
Abbreviations:. MMP-2, matrix metalloproteinase-2; MMP-9, matrix metalloproteinase-9; TIMP-2, tissue inhibitor of metalloproteinase-2.
MMP-2, MMP-9 and TIMP-2 tissue expression versus sinus cavernous invasion
Immunostaining of MMP-2, MMP-9 and TIMP-2 was predominantly observed in the cytoplasm of tumor cells (Fig. 2). Interestingly, the median of MMP-2 H-scores were higher in the invasive group but did not reach statistical significance when compared to the noninvasive group (105 (30–155) vs 40 (12–90), P = 0.09; Table 1). In addition, there was no significant difference in the tissue expression of MMP-2, MMP-9 and TIMP-2 between invasive and noninvasive groups when considering pituitary tumor subtypes (Table 2). Regarding the Knosp grading scale, no statistically significant differences were observed in the immunoexpression of MMP-2 (P = 0.25), MMP-9 (P = 0.74) and TIMP-2 (P = 0.68) between grades 0, 1, 2, 3A, 3B and 4 (Table 3).
Immunohistochemistry staining of matrix metalloproteinases-2 and MMP-9 and tissue inhibitor of metalloproteinase-2 in PitNET. (a) Placenta and chordoma positive control; (b) H-score = 0; (c) H-score = 300. Magnification ×10.
Citation: Endocrine Oncology 5, 1; 10.1530/EO-24-0037
Immunoexpression levels of MMP-2, MMP-9 and TIMP-2 by Knosp grade of pituitary tumors.
| Knosp grade | MMP-2 | MMP-9 | TIMP-2 |
|---|---|---|---|
| 0 | 40 (10–75) | 47.5 (7.5–165) | 120 (45–210) |
| 1 | 45 (5.0–70) | 115 (45–142.5) | 120 (60–225) |
| 2 | 115 (35–160) | 107.5 (40–165) | 160 (130–212.5) |
| 3A | 32.5 (12.5–147.5) | 102.5 (45–165) | 172.5 (55–215.5) |
| 3B | 160 (105–210) | 82.5 (47.5–185) | 277.5 (212.5–292.5) |
| 4 | 72 (35–105) | 55 (0.0–107.5) | 111 (10–277.5) |
| P value | 0.12 | 0.85 | 0.30 |
Data are described in median (25th–75th percentiles).
Abbreviations: MMP-2, matrix metalloproteinase-2; MMP-9, matrix metalloproteinase-9; TIMP-2, tissue inhibitor of metalloproteinase-2.
Alternatively, protein expression was categorized according to the percentage of positive tumor cells as 1 – minimum expression (<30%), 2 – focal (30–60%) and 3 – diffuse (>60%) (Gültekin et al. 2015). However, no differences were observed between invasive and noninvasive groups for all evaluated markers (data not shown).
Supplemental Table 1 provides individual data from all patients analyzed in this study, including IHC and immunoblotting results.
Plasma protein levels of MMP-2, MMP-9 and TIMP-2
Plasmatic levels of MMP-2 were not detected in any case. However, concentrations of MMP-9 and TIMP-2 showed no significant differences between the invasive and noninvasive groups with respect to cavernous sinus invasion (MMP-9 = 832 (499–1,326) vs 304 (175–929), P = 0.11; TIMP-2 = 3,568 (2,474–4,216) vs 3,038 (2,100–3,770), P = 0.47; Table 1). No significant differences were observed in the comparison of plasmatic MMP-9 and TIMP-2 between invasive and noninvasive groups across pituitary tumor subtypes (Table 2). Furthermore, plasmatic levels of MMP-9 and TIMP-2 did not correlate with the IHC H-score of these proteins (MMP-9: rho = 0.29, P = 0.13 and TIMP-2: rho = −0.54, P = 0.26). Figure 3 shows a representative example of plasmatic MMP/TIMP protein detection using a dot-blot membrane array.
Representative examples of protein plasmatic detection. (a) Membranes were hybridized with a 1:1 plasma dilution in a human matrix metalloproteinases (MMP) antibody array. Hybridization signals for MMP-2, MMP-9 and tissue inhibitor of metalloproteinase-2 are indicated. On each membrane, positive (Pos) and negative (Neg) hybridization controls were included. (b) Semi-quantitative protein dots analysis based on pixel intensity. The spot intensity graph was generated using the ImageJ software.
Citation: Endocrine Oncology 5, 1; 10.1530/EO-24-0037
Discussion
This study aimed to explore the correlations between the tissue expression of MMP-2, MMP-9 and TIMP-2 in pituitary tumors and their invasiveness. Uniquely, it also examined the potential of these proteins’ plasma levels as preoperative markers for tumor behavior. However, we did not find significant differences in the median H-score for MMP-2, MMP-9 and TIMP-2 between pituitary tumors invading the cavernous sinus and noninvasive ones. Similarly, no significant difference was observed in protein expression when assessed using the categorical score. Our findings align with those of Knappe and coworkers (Knappe et al. 2003), who also reported no significant differences in MMP-2 and MMP-9 expression via immunohistochemistry between invasive and noninvasive pituitary tumors. Beaulieu and coworkers (Beaulieu et al. 1999), employing western blotting, detected MMP-2 presence in most pituitary tumors without a correlation to tumor size or invasiveness.
Further complexity arises from varied findings present in the literature. Liu and coworkers (Liu et al. 2005b), using an immunohistochemistry categorical score, reported significantly higher MMP-2 and MMP-9 expression in invasive pituitary tumors. In addition, other studies have identified significantly elevated levels of these metalloproteinases, particularly MMP-9, in invasive pituitary tumors (Kawamoto et al. 1996b, Liu et al. 2005b, Gong et al. 2008) and in patients with a lower postoperative survival rate (Guo et al. 2019). Interestingly, some researchers found increased MMP-9 expression exclusively in invasive macroprolactinomas and pituitary carcinomas (Turner et al. 2000, Gültekin et al. 2015). Notably, ACTH-secreting tumors with elevated MMP-9 levels were associated with higher recurrence rates and shorter recurrence-free intervals (Liu et al. 2018). In our study, while tissue expression of MMP-2 was elevated in invasive pituitary tumors, particularly in invasive somatotropinomas and non-functioning tumors, this increase did not achieve statistical significance. These divergent results underscore the intricate relationship between MMPs and pituitary tumor behavior, necessitating further exploration to uncover underlying mechanisms and potential clinical implications.
TIMP-2 is a critical inhibitor of MMP-2 (Johansson et al. 2000), and its potential protective role against pituitary tumor invasiveness has been suggested in certain studies, which noted higher expression levels in noninvasive tumors (Beaulieu et al. 1999, Knappe et al. 2003). Conversely, Gültekin et al. (2015) reported significantly elevated TIMP-2 expression in invasive pituitary tumors, suggesting a possible paradoxical dual effect of TIMP-2. The authors proposed that when TIMP-2 forms a complex with MMP-4, it might induce mitosis and cell migration, thereby promoting invasion through the Ras–Raf–ERK mitogenic signaling cascade (Sounni et al. 2010, Strongin 2010, Gültekin et al. 2015). In our study, TIMP-2 expression did show a significant difference between the invasive and noninvasive groups, indicating that further investigations are needed. These studies should focus on TIMP-2 localization and interaction with other MMPs to fully elucidate its role in pituitary tumor invasion.
A meta-analysis by Liu et al. (2016) evaluated studies on the expression of MMP-9, MMP-2 and TIMP-2 in pituitary tumors. This analysis included 24 studies with a total of 1,320 patients (611 with invasive pituitary tumors and 709 with noninvasive). The findings revealed significantly higher expression of MMP-9 and MMP-2 in invasive pituitary tumors compared to noninvasive ones at both protein and mRNA levels (Liu et al. 2016). However, only five studies provided data on TIMP-2 expression, and these did not show a significant difference between invasive and noninvasive tumors. Despite these results, the authors cautioned against overinterpretation due to the limited number of studies, their retrospective nature, heterogeneity and a lack of strict quality control (Liu et al. 2016).
Another important issue to be considered is the accuracy of the radiologic criteria used to define cavernous sinus invasion. A meta-analysis indicated that Knosp grade 3 had a specificity of 90% for detecting cavernous sinus invasion (Dhandapani et al. 2016). Furthermore, Micko et al. (2015) demonstrated a significantly lower invasion rate for Knosp grade 3 tumors when the superior compartment was involved (grade 3A). Specifically, the invasion rate in grade 3A tumors was 26.5%, compared to a 70.6% surgically observed invasion rate in grade 3B tumors (Micko et al. 2015). This variability may have limited our ability to differentiate between invasive and noninvasive tumors, potentially explaining the conflicting results seen in different studies. Despite this, in our cohort, no relationship was observed between the immunoexpression of MMP-2, MMP-9 and TIMP-2 and Knosp grade.
Several studies have demonstrated a correlation between plasma levels of MMP-2 and MMP-9 and tumor stage in gastrointestinal and breast cancers (Wu et al. 2007, Vasaturo et al. 2013). Our study was the first to evaluate the plasma levels of these markers, specifically in pituitary tumors. Interestingly, we did not detect any MMP-2 plasma expression in our cases, and there was no significant difference in the plasma expression of MMP-9 and TIMP-2 between the invasive and noninvasive groups. These findings suggest that the pituitary gland may contribute minimally, if at all, to the circulating levels of these markers. Furthermore, no correlation was observed between the plasma expression of MMP-9 and TIMP-2 and their expression within the tumor tissue.
In our study, Ki-67, a well-established marker of cell proliferation in pituitary tumors, was significantly more expressed in the invasive group. Consistent with our findings, several other studies also demonstrated a higher Ki-67 index in invasive tumors compared to noninvasive ones (Thapar et al. 1996, Mastronardi et al. 1999, Chacko et al. 2010). Thapar et al. (1996) demonstrated that using a cutoff of 3% could distinguish invasive from noninvasive pituitary tumors with 97% specificity and 73% sensitivity. However, the 2017 World Health Organization classification of tumors of the pituitary gland does not recommend a specific Ki-67 cutoff value (Lopes 2020). In addition, other studies have found an association between the Ki-67 index and cavernous sinus invasion (Mastronardi et al. 1999, Pan et al. 2005, Chacko et al. 2010).
This study has several limitations that could affect its findings. With a sample size of only 74 patients and preoperative blood samples from just 29, the study may lack the statistical power needed to detect subtle differences or associations, especially in subgroup analyses. The observed increases in MMP-2 levels in invasive tumors, which did not reach statistical significance, may indicate either a true lack of association or an insufficient sample size to detect a real effect. Future research should consider using larger sample sizes and more sensitive detection methods to better elucidate the role of MMPs and TIMP-2 in pituitary tumor invasion.
In conclusion, our study concurs with the literature’s mixed results regarding the expression of MMP-2, MMP-9 and TIMP-2 in pituitary tumors and fails to demonstrate the usefulness of their plasma levels as indicators of tumor behavior. Therefore, additional research is necessary to clarify the role of these proteins in tumor invasiveness.
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/EO-24-0037.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the work reported.
Funding
This research was supported by Grant 2014/10462–4 from the São Paulo Research Foundation (FAPESP) and by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) institutional scholarship (158472/2014–4). The authors declare that they have no financial relationship with the organizations that sponsored this research. The resources were used to purchase the materials and reagents necessary for the development of the research. The sponsors had no involvement in the study design, data collection, analysis, interpretation, report writing or the decision to submit the article for publication.
Author contribution statement
ACB was involved in investigation, conceptualization, formal analysis, resources and writing original draft; RSJ was involved in resources an investigation; RLB was involved in resources, investigation and formal analysis; AG, MCM, GO, VAC and MBCCN were involved in resources; FPF was involved in investigation; RSSM was involved in investigation and formal analysis; EBT was involved in conceptualization, formal analysis, investigation, review and editing of the manuscript, preparation of all figures and tables, supervision and project administration. All authors reviewed the manuscript.
Declaration of generative AI and AI-assisted technologies in the writing process
During the preparation of this work, the author(s) used ChatGPT to enhance the writing and review process in the English language. After using this tool/service, the author(s) reviewed and edited the content as needed and take full responsibility for the content of the publication.
Acknowledgments
We would like to express our heartfelt gratitude to Marcello Delano Bronstein (in memorian), whose contributions to this work were invaluable. His dedication, expertise and insights have left a lasting impact on this research. He will be remembered for his significant contributions to the field. We would also like to acknowledge the contributions of the researchers Juliana Guerra and Leonardo T Araujo at the Núcleo de Patologia Quantitativa of Instituto Adolfo Lutz for their technical support in metalloproteinase IHC staining.
References
Beaulieu E, Kachra Z, Mousseau N, et al. 1999 Matrix metalloproteinases and their inhibitors in human pituitary tumors. Neurosurgery 45 1432–1441. (https://doi.org/10.1097/00006123-199912000-00033)
Bourboulia D, Han HY, Jensen-Taubman S, et al. 2013 TIMP-2 modulates cancer cell transcriptional profile and enhances E-cadherin/beta-catenin complex expression in A549 lung cancer cells. Oncotarget 4 163–173. (https://doi.org/10.18632/ONCOTARGET.801)
Ceylan S, Anik I, Koc K, et al. 2011 Microsurgical anatomy of membranous layers of the pituitary gland and the expression of extracellular matrix collagenous proteins. Acta Neurochir 153 2435–2443. (https://doi.org/10.1007/S00701-011-1182-3)
Chacko G, Chacko AG, Kovacs K, et al. 2010 The clinical significance of MIB-1 labeling index in pituitary adenomas. Pituitary 13 337–344. (https://doi.org/10.1007/s11102-010-0242-7)
Dhandapani S, Singh H, Negm HM, et al. 2016 Cavernous sinus invasion in pituitary adenomas: systematic review and pooled data meta-analysis of radiologic criteria and comparison of endoscopic and microscopic surgery. World Neurosurg 96 36–46. (https://doi.org/10.1016/j.wneu.2016.08.088)
Gong J, Zhao Y, Abdel-Fattah R, et al. 2008 Matrix metalloproteinase-9, a potential biological marker in invasive pituitary adenomas. Pituitary 11 37–48. (https://doi.org/10.1007/s11102-007-0066-2)
Gültekin GD, Çabuk B, Vural Ç, et al. 2015 Matrix metalloproteinase-9 and tissue inhibitor of matrix metalloproteinase-2: prognostic biological markers in invasive prolactinomas. J Clin Neurosci 22 1282–1287. (https://doi.org/10.1016/J.JOCN.2015.02.021)
Guo H, Sun Z, Wei J, et al. 2019 Expressions of matrix metalloproteinases-9 and tissue inhibitor of metalloproteinase-1 in pituitary adenomas and their relationships with prognosis. Cancer Biother Radiopharm 34 1–6. (https://doi.org/10.1089/cbr.2018.2589)
Johansson N, Ahonen M & Kähäri VM 2000 Matrix metalloproteinases in tumor invasion. Cell Mol Life Sci 57 5–15. (https://doi.org/10.1007/S000180050495)
Jordan RC, Lingen MW, Perez-Ordonez B, et al. 2012 Validation of methods for oropharyngeal cancer HPV status determination in US cooperative group trials. Am J Surg Pathol 36 945–954. (https://doi.org/10.1097/PAS.0B013E318253A2D1)
Kawamoto H, Kawamoto K, Mizoue T, et al. 1996a Matrix metalloproteinase-9 secretion by human pituitary adenomas detected by cell immunoblot analysis. Acta Neurochir 138 1442–1448. (https://doi.org/10.1007/BF01411124)
Kawamoto H, Uozumi T, Kawamoto K, et al. 1996b Type IV collagenase activity and cavernous sinus invasion in human pituitary adenomas. Acta Neurochir 138 390–395. (https://doi.org/10.1007/BF01420300)
Knappe UJ, Hagel C, Lisboa BW, et al. 2003 Expression of serine proteases and metalloproteinases in human pituitary adenomas and anterior pituitary lobe tissue. Acta Neuropathol 106 471–478. (https://doi.org/10.1007/S00401-003-0747-5)
Knosp E, Steiner E, Kitz K, et al. 1993 Pituitary adenomas with invasion of the cavernous sinus space. Neurosurgery 33 610–618. (https://doi.org/10.1097/00006123-199310000-00008)
Langenskiöld M, Holmdahl L, Falk P, et al. 2005 Increased plasma MMP-2 protein expression in lymph node-positive patients with colorectal cancer. Int J Colorectal Dis 20 245–252. (https://doi.org/10.1007/s00384-004-0667-4)
Lefevre E, Chasseloup F, Hage M, et al. 2024 Clinical and therapeutic implications of cavernous sinus invasion in pituitary adenomas. Endocrine 85 1058–1065. (https://doi.org/10.1007/s12020-024-03877-2)
Liu W, Kunishio K, Matsumoto Y, et al. 2005a Matrix metalloproteinase-2 expression correlates with cavernous sinus invasion in pituitary adenomas. J Clin Neurosci 12 791–794. (https://doi.org/10.1016/j.jocn.2005.03.010)
Liu W, Matsumoto Y, Okada M, et al. 2005b Matrix metalloproteinase 2 and 9 expression correlated with cavernous sinus invasion of pituitary adenomas. J Med Invest 52 151–158. (https://doi.org/10.2152/jmi.52.151)
Liu HY, Gu WJ, Wang CZ, et al. 2016 Matrix metalloproteinase-9 and-2 and tissue inhibitor of matrix metalloproteinase-2 in invasive pituitary adenomas: a systematic review and meta-analysis of case-control trials. Medicine 95 e3904. (https://doi.org/10.1097/MD.0000000000003904)
Liu X, Feng M, Zhang Y, et al. 2018 Expression of matrix metalloproteinase-9, pituitary tumor transforming gene, high mobility group A 2, and Ki-67 in adrenocorticotropic hormone–secreting pituitary tumors and their association with tumor recurrence. World Neurosurg 113 e213–e221. (https://doi.org/10.1016/J.WNEU.2018.01.214)
Lopes MBS 2020 World Health Ozganization 2017 classification of pituitary tumors. Endocrinol Metab Clin North Am 49 375–386. (https://doi.org/10.1016/j.ecl.2020.05.001)
Mastronardi L, Guiducci A, Spera C, et al. 1999 Ki-67 labelling index and invasiveness among anterior pituitary adenomas: analysis of 103 cases using the MIB-1 monoclonal antibody. J Clin Pathol 52 107–111. (https://doi.org/10.1136/jcp.52.2.107)
Mete O, Ezzat S & Asa SL 2012 Biomarkers of aggressive pituitary adenomas. J Mol Endocrinol 49 R69–R78. (https://doi.org/10.1530/JME-12-0113)
Micko ASG, Wöhrer A, Wolfsberger S, et al. 2015 Invasion of the cavernous sinus space in pituitary adenomas: endoscopic verification and its correlation with an MRI-based classification. J Neurosurg 122 803–811. (https://doi.org/10.3171/2014.12.JNS141083)
Molitch ME 2008 Nonfunctioning pituitary tumors and pituitary incidentalomas. Endocrinol Metab Clin North Am 37 151–171. (https://doi.org/10.1016/j.ecl.2007.10.011)
Moreno CS, Evans CO, Zhan X, et al. 2005 Novel molecular signaling and classification of human clinically nonfunctional pituitary adenomas identified by gene expression profiling and proteomic analyses. Cancer Res 65 10214–10222. (https://doi.org/10.1158/0008-5472.CAN-05-0884)
Pan LX, Chen ZP, Liu YS, et al. 2005 Magnetic resonance imaging and biological markers in pituitary adenomas with invasion of the cavernous sinus space. J Neurooncol 74 71–76. (https://doi.org/10.1007/s11060-004-6150-9)
Qiu L, He D, Fan X, et al. 2011 The expression of interleukin (IL)-17 and IL-17 receptor and MMP-9 in human pituitary adenomas. Pituitary 14 266–275. (https://doi.org/10.1007/S11102-011-0292-5)
Sounni NE, Rozanov DV, Remacle AG, et al. 2010 Timp-2 binding with cellular MT1-MMP stimulates invasion-promoting MEK/ERK signaling in cancer cells. Int J Cancer 126 1067–1078. (https://doi.org/10.1002/IJC.24690)
Strongin AY 2010 Proteolytic and non-proteolytic roles of membrane type-1 matrix metalloproteinase in malignancy. Biochim Biophys Acta 1803 133–141. (https://doi.org/10.1016/j.bbamcr.2009.04.009)
Thapar K, Kovacs K, Scheithauer BW, et al. 1996 Proliferative activity and invasiveness among pituitary adenomas and carcinomas: an analysis using the MIB-1 antibody. Neurosurgery 38 99–107. (https://doi.org/10.1097/00006123-199601000-00024)
Trouillas J, Roy P, Sturm N, et al. 2013 A new prognostic clinicopathological classification of pituitary adenomas: a multicentric case-control study of 410 patients with 8 years post-operative follow-up. Acta Neuropathol 126 123–135. (https://doi.org/10.1007/s00401-013-1084-y)
Turner HE, Nagy Z, Esiri MM, et al. 2000 Role of matrix metalloproteinase 9 in pituitary tumor behavior. J Clin Endocrinol Metab 85 2931–2935. (https://doi.org/10.1210/JCEM.85.8.6754)
Vasaturo F, Solai F, Malacrino C, et al. 2013 Plasma levels of matrix metalloproteinases 2 and 9 correlate with histological grade in breast cancer patients. Oncol Lett 5 316–320. (https://doi.org/10.3892/ol.2012.977)
Wu CY, Wu MS, Chiang EP, et al. 2007 Plasma matrix metalloproteinase-9 level is better than serum matrix metalloproteinase-9 level to predict gastric cancer evolution. Clin Cancer Res 13 2054–2060. (https://doi.org/10.1158/1078-0432.CCR-06-2299)
Zucker S, Lysik RM, Zarrabi MH, et al. 1993 Mr 92,000 type IV collagenase is increased in plasma of patients with colon cancer and breast cancer. Cancer Res 53 140–146.
