Summary

The Ki-67 antigen is a protein expressed in cell nuclei throughout the entire cell cycle. It is related to the cell proliferation in a variety of pituitary tumors. Pituitary tumors are usually benign, but may be aggressive and invade surrounding tissues in about one third of the cases. The aim of this study is to determine the proliferative index of the pituitary adenomas, using MIB-1 monoclonal antibody in paraffin embedded sections and also to correlate this index with clinical parameters and radiological evidence of invasiveness. Twenty-eight patients (mean age, 46.46±13.14; range, 13-80 yr) who underwent surgery for pituitary adenomas were enrolled in this study. Immunohistochemical staining for anti-Ki-67 monoclonal antibody (MIB-1) was performed and the proliferative activity was determined as the percentage of MIB-1 labeled nuclei (MIB-1 index). The overall mean Ki-67 labeling index was 2.04±1.17 (range 1-5). This index was not associated with gender and age of the patients and functional status of pituitary adenomas. Although Ki-67 index was higher in suprasellar adenomas than in sellar adenomas, the difference was not statistically significant (2.20±1.15 for suprasellar adenomas vs. 1.85±1.21 for sellar adenomas; p= 0.316). Ki-67 index was distributed almost the same amount between the groups of adenomas with (2±1.51) or without cavernous sinus invasion (2.05±1.03) (p= 0.498). Unexpectedly, no significant relationship was identified between proliferation index and the invasiveness of pituitary adenomas in this study, but this raises the question whether proliferation markers in pituitary adenomas are useful or not?
Keywords: Ki-67 index and pituitary adenomas

Summary

The Ki-67 antigen is a protein expressed in cell nuclei throughout the entire cell cycle. It is related to the cell proliferation in a variety of pituitary tumors. Pituitary tumors are usually benign, but may be aggressive and invade surrounding tissues in about one third of the cases. The aim of this study is to determine the proliferative index of the pituitary adenomas, using MIB-1 monoclonal antibody in paraffin embedded sections and also to correlate this index with clinical parameters and radiological evidence of invasiveness. Twenty-eight patients (mean age, 46.46±13.14; range, 13-80 yr) who underwent surgery for pituitary adenomas were enrolled in this study. Immunohistochemical staining for anti-Ki-67 monoclonal antibody (MIB-1) was performed and the proliferative activity was determined as the percentage of MIB-1 labeled nuclei (MIB-1 index). The overall mean Ki-67 labeling index was 2.04±1.17 (range 1-5). This index was not associated with gender and age of the patients and functional status of pituitary adenomas. Although Ki-67 index was higher in suprasellar adenomas than in sellar adenomas, the difference was not statistically significant (2.20±1.15 for suprasellar adenomas vs. 1.85±1.21 for sellar adenomas; p= 0.316). Ki-67 index was distributed almost the same amount between the groups of adenomas with (2±1.51) or without cavernous sinus invasion (2.05±1.03) (p= 0.498). Unexpectedly, no significant relationship was identified between proliferation index and the invasiveness of pituitary adenomas in this study, but this raises the question whether proliferation markers in pituitary adenomas are useful or not?
Keywords: Ki-67 index and pituitary adenomas

Introduction

Several proliferation markers can be used for differentiating aggressive or rapidly growing tumors from those with slower growth. MIB-1 is the most reliable marker among them for tumorigenesis of the cell [1].
Ki-67 is a nuclear antigen expressed in the G1, S, G2, and M phases of cellular cycle. The monoclonal antibody MIB-1 detects this nuclear antigen expressed by proliferating cells during the entire cell cycle [2]. The percentage of immunopositive cells is referred to as the MIB-1 labeling index (L1) [3].
Pituitary adenomas comprise 10-15% of all primary brain tumors [4,5]. The prevalence of pituitary incidentaloma finding in computerized tomography (CT) ranges from 3.7% to 20%, in magnetic resonance imaging (MRI) is 10% and in autopsy studies ranges from 1.5% to 26.7% [6]. Most of them are microadenomas (<10mm). Pituitary adenomas are usually asymptomatic. They are symptomatic as a result of either hormonal hypersecretion or mass effect [6]. Both of them exhibit benign behavior but they can occasionally become aggressive and infiltrate adjacent structures such as the sphenoid sinus, diaphragma sellae and cavernous sinus (CS) [4,7]. The incidence of invasion by pituitary tumors ranges from 10 to 85% according to the method of diagnosis [8]. Even if they are benign histologically in most cases, they behave as aggressive tumors biologically. So its preoperative diagnosis is important for the prognosis of these tumors [9].
Ki-67 is a marker associated with invasiveness and prognosis of pituitary adenomas. Adenomas showing a threshold Ki-67 labeling index of >3% immunoreactivity should be termed "atypical adenomas" suggesting aggressive potential [10]. The first report on the use of MIB-1 antibody was published by Landolt et al in 1987 [11]. The mean percentage of Ki-67 positive cells in immunohistochemistry ranges from 0.1% to 3.7%. This value is lower (0.1% to 1.0%) in nonfunctional adenomas and (0.60%) in noninvasive adenomas or higher (1.1% to 1.5%) in acromegalic patients and (1.15%) in invasive adenomas [11]. Several studies have documented the proliferating cell index as measured by antibody MIB-1 with varying frequency ranging from 0.1% to 23.37 % [11,12] in pituitary adenomas.
So, under these circumstances, there is a wide discrepancy related to Ki-67 labeling index measurement in pituitary adenomas.
The purpose of this study was to investigate the correlation between the proliferative activity of pituitary adenomas measured by Ki-67 labeling index and clinical characteristics and invasiveness.

Methods

Twenty-eight patients who underwent surgery for pituitary adenomas between 2003 and 2005 were included in this study. Mean age of the patients was 46.46±13.14 (range, 13-80 yr). Medical records of these patients were reviewed for clinical presentation, hormone evaluation, pituitary images, medications used and paraffin blocks. Male to female ratio was 2:1.5 (16 males and 12 females). Functioning adenomas was present in 11 (39.2%) cases. Hormonal disturbances were related to high levels of serum prolactine in 6 cases, of serum growth hormone in 4 cases and of serum ACTH in only one case. Neuro-radiological diagnosis with magnetic resonance imaging (MRI) was used to define tumor diameter and invasion. Tumors were microadenomas in 3 (10.7%) and macroadenomas in 25 (89.7%) cases. Supra-sellar growth was considered to be an extension rather than an invasion. Pituitary adenomas were sellar in 13 (46.4%) and suprasellar in 15 (53.5%) cases. Maximal tumor diameter on MRI was classified into five groups. <1 cm, 1.1- 2 cm, 2.1- 3 cm, 3.1- 4 cm, 4- 5 cm. Invasion was defined as radiological invasion into cavernous or sphenoid sinuses. The demographic and clinical data of the patients (adenomas) are summarized in Table 1.
Table 1: Demographic and clinical data of the patients (adenomas)
The study was approved by the Ethics Committee of Dr. Lütfi Kirdar Kartal Training and Research Hospital.

Ki-67 Immunostaining
MIB-1 antibody was used to identify Ki-67 antigen. Paraffin blocks were cut into 3-µm sections and mounted on glass slides prepared with organosilane. Immunostaining was performed using the avidin-biotin-peroxidase method. Antigen retrieval was performed using pressure cooker. They were incubated with MIB-1 (NeoMarkers, 1/400) antibody for one hour. The regions with highest concentrations of MIB-1 positive nuclei were selected and analyzed at high power magnification (X400). On the basis of 1000 neoplastic nuclei, Ki-67 labeling index was calculated in each slide as the percentage of immunopositive nuclei.

Statistical Analysis

Computer-assisted data analysis was performed by GraphPad Prisma V.3 program. Data was given as mean ± SD. Mann-Whitney-U test was used in the comparison of the two groups. Kruskal-Wallis test was also used to compare more than two groups. P<0.05 was accepted as significant.
Table 2: Correlation of the clinical/ radiological data and Ki-67 labeling index

Results

The overall mean Ki-67 labeling index was 2.04±1.17 (range 1-5), in consistency with the literature. The distribution of Ki-67 index was almost the same between female and male groups (2.06±1.12 for males and 2±1.28 for females; p=0.732). For statistical analysis patient’s age at surgery was classified into two groups: <50 y (n: 17) and ≥50 y (n: 11). Although the mean Ki-67 index was higher in ≥50 y group (2.18±1.08) vs. <50 y group (1.94±1.25), the difference was not significant (p=0.378). The Ki-67 index was higher in macroadenomas than in microadenomas, but without a statistical significance (2.08±1.19 versus 1.67±1.15; p=0.529). No statistical difference was observed in the Ki-67 index in relation to functional status of pituitary adenomas (2.24±1.35 for nonfunctional adenomas vs. 1.73±0.79 for functional adenomas; p= 0.458). For the extension of pituitary adenomas they are evaluated in two groups: sellar or suprasellar tumors. Although Ki-67 index was higher in suprasellar adenomas than in sellar adenomas, the difference was not significant (2.20±1.15 vs. 1.85±1.21; p= 0.316). Ki-67 index was distributed almost evenly, between the groups of adenomas with (2±1.51) or without cavernous sinus invasion (2.05±1.03) (p= 0.498).
Maximum tumor diameter in MR was classified in five groups. <1 cm, 1.1- 2cm, 2.1- 3cm, 3.1- 4cm, 4- 5cm. The mean Ki-67 index was found as 1.67±1.15, 2.20±1.32, 1.78±0.83, 2±0.82, 3±2.83, respectively, in these groups. Although the mean Ki-67 index was higher in group IV, the difference was not statistically significant (p=0.92).

Discussion

In the present study; the overall mean Ki-67 index was found to be 2.04±1.17 (range 1-5) in agreement with the literature. But no statistically differences were observed in the Ki-67 labeling index in relation to sex and age of the patients, maximal tumor diameter, functional status, exten-sion and invasion of the pituitary adenomas.
Wide variation exists when considering all the data published to date on the MIB-1 labeling index. This might be explained by several factors:
First, MIB- 1 labeling index (LI) was found in different values related with age. Pituitary ade-nomas in the elderly have a higher proliferation index than in younger patients. The mean Ki-67 LI was 4.06±6.73 versus 2.35±2.54% of younger patients [13]. In another study the mean LI was found as 1.24±1.59% and it decreased with patient's age (P=0.025, r=0.28) [3]. In spite of these studies a significantly high Ki-67 index was also reported in younger patients versus in older patients [14]. No statistical differences were observed in the Ki-67 labeling index in relation to age in the other studies [5,8,15]. In the present study patients older than 50 years tend to show higher values (2.18±1.08) but this was not statistically significant (p=0.378).
Figure 1: The samples of immunostaining for Ki-67 labelling index in pituitary adenomas. A and B: The Ki-67 labelling index in pituitary adenomas (5%). C, D and E: The Ki-67 labelling index in pituitary adenomas (2%) F: The Ki-67 labelling index in normal pituitary tissue (original magnification, x400).
Second MIB-1 labeling index was found in different values related with sex. Prolactinomas in men have been suggested to have a higher proliferation activity because they are usually larger than those in women. A higher indexes of proliferating cells by Ki-67 immunoreactivity (2.6 ± 1.1% of positive nuclei) was exhibited in males than female patients (0.4 ± 0.2%; P = 0.08) [16]. Prolactinomas in men also exhibited higher the mean Ki-67 (MIB-1) positive cell index (p = 0.0138) than those in women in another study [17]. On the other hand no statistical difference was observed in relation to sex in the other study [5]. In this study the distribution of Ki-67 labeling index was almost the same (males; 2.06±1.12 and females: 2±1.28, p=0.732) between female and males without a statistically significant (P=0.732).
Third statistical difference in Ki-67 immunoreactivity was observed in relation to functional status of pituitary adenomas. Significantly higher Ki-67 expression was found in clinically fun-ctioning tumors when compared to nonfunctioning tumors (P < .03) [18]. Mean MIB-1 labeling index was significantly higher in functional adenomas (2.06±2.39) than nonfunctional adenomas (1.05±1.12) [3]. Significantly higher Ki-67 index was also found in functional adenomas in some studies [11,19]. On the other hand no statistical difference was observed in the Ki-67 index in relation to functional status of pituitary adenomas in the other studies [5,8,20-22]. We did not also observe statistically significant index in relation functional status of the pituitary adenomas. More-over, Ki-67 labeling index was higher in non-functioning adenomas than in functioning ade-nomas.
Fourth invasion of the pituitary adenomas might cause to the variation in the prevalence of Ki-67 labeling index. Pituitary tumors are classified as microadenomas and macroadenomas [23]. They are divided in "enclosed" (intrasellar adenomas) and "invasive" (intrasellar and intra-extrasellar adenomas) according to Hardy’s classification. Enclosed adenomas are subdivided in Grade 0 (microadenomas), Grade I (macroadenoma with slight lowering of the floor), and Grade II (macroadenoma enlarging the sella, with intact floor). Invasive adenomas are subdivided in Grade III (macroadenomas eroding the floor) and Grade IV (macroadenoma destroying the floor) [23]. On the other hand pituitary adenomas with a supra-sellar extension are classified in five types: A) tumor bulging into the chiasmatic cistern; B) tumor reaching the anterior third ventricle; C) huge suprasellar extension filling entirely the third ventricle; D) parasellar extension into the temporal, frontal, or posterior fossa; and E) lateral expansion towards the cavernous sinus [23]. Invasion may be defined radiologically, operatively, or histologically. On the basis of surgery, gross invasion by pituitary adenomas is approximately 35%, but it tends to increase about 90% histologically [2]. Invasive adenomas demonstrated significantly higher Ki-67 values (average 1.15%) than noninvasive adenomas (average 0.60%) [11]. A statistically significant Ki-67 labeling index (LI) was also found in invasive adenomas respect to noninvasive adenomas in the other studies [2,13,21,24-30]. A threshold Ki-67 labeling index of 3% was established to distinguish invasive from noninvasive adenomas with 97% specificity and 73% sensitivity [27]. On the other hand no significant difference was found in the frequency of MIB1-positive cells between invasive and noninvasive adenomas in some studies [5,12,22,31]. They suggest that invasion and proliferation markers are independent biological factors. Therefore, invasion may be due to chemical factors produced by the tumor itself rather than as a result of rapid tumor growth [12]. In the present study Ki-67 index was found to be higher in suprasellar adenomas than in sellar adenomas, but it was not significant (2.20±1.15 vs. 1.85±1.21; p= 0.316). Similarly although Ki-67 index was a little higher in invasive adenomas to cavernous sinus than in noninvasive adenomas, it was also not significant (p= 0.498).
Most studies agree on the lack of association between tumor volume and proliferation activity. Mastronadi reported the mean Ki-67 index of 2.59±1.81 in microadenomas, 2.63±3.45 in intrasellar macroadenomas, 1.91±2.11 in intra-suprasellar macroadenomas, and 3.29±5.45 in intrasupra-parasellar macroadenomas (p = 0.27) [2]. No statistical difference was also reported in the Ki-67 labeling index in relation to maximal tumor diameter by Paek et al [5]. In the present study although the index was higher in macroadenomas than in microadenomas, the difference was not significant. The same relation was also found when the maximal tumor diameter was considered. The mean Ki-67 index was found as ( Group 1: 1.67±1.15, Group II: 2.20±1.32, Group III: 1.78±0.83, Group IV: 2±0.82 and Group V: 3±2.83) in agreement with the literature [5]. No statistical difference was found between the groups. However a significantly higher Ki-67 index was found in macro (9.3 ± 2.7%) vs. micro (2.8 ±0.5%; P < 0.002)-ACTH secreting adenomas by Losa et al [32]. A similar index was also found in macro-prolactinomas versus micro-prolactinomas by Turner et al [33].
In conclusion, Ki-67 labeling index was 2.04±1.17, in agreement with the literature. This index was not associated with gender and age of the patients and functional status, invasion and maximum tumor diameter of pituitary adenomas. When all the studies in the literature were eva-luated all together, the relationship between Ki-67 labeling index and clinical parameters is con-flicting. Although studies on the Ki-67 labeling index from long term follow-up series are re-appraised and limited, the index appears to provide valuable prognostic information. High Ki-67 labeling index might suggest the need for careful clinical and radiological follow-up. Thus, long term follow up studies with higher number of subjects are needed for better demonstration of this relationship

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