Lack of correlation between Ki-67 labelling index and tumor size of anterior pituitary adenomas

Aims and background The Ki-67 is a nuclear antigen detected by the monoclonal antibody MIB-1 and its Labeling Index (LI) is considered a marker of normal and abnormal cell proliferation. Pituitary adenomas are generally well differentiated neoplasms, even if in about one third of cases they are invasive of surrounding tissues. The aim of this study is to evaluate the correlation between Ki-67 labelling index and tumor size of pituitary adenomas extimated by means CT and MRI and confirmed at operation. Methods Using the monoclonal antibody MIB-1, we evaluated the expression of Ki-67 in 121 anterior pituitary adenomas consecutively operated on in a 48-month period. Results In relation to neuroradiological (CT and MRI) and surgically verified tumor size, we identified 24 microadenomas, 27 intrasellar macroadenomas, 34 intra-suprasellar macroadenomas, and 36 intra-supra-parasellar macroadenomas. The adenomas were non-infiltrating (76 cases) and infiltrating (45 cases) adenomas. The wall of the cavernous sinus (CS) was infiltrated in 18 cases. Forty-eight adenomas were non-functioning and 73 functioning. The overall mean ± SD Ki-67 LI was 2.72 ± 2.49% (median 1.6). It was 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 intra-supra-parasellar macroadenomas (p = 0.27). It was 3.73 ± 5.13% in infiltrating and 2.03 ± 2.41% in non-infiltrating adenomas (p = 0.02), and 5.61 ± 7.19% in CS-infiltrating versus 2.09 ± 2.37% in CS-non-infiltrating adenomas (p = 0.0005). Conclusions Our preliminary results seem to exclude significative correlations between Ki-67 LI and tumor size of anterior pituitary adenomas, even if this index can be considered a useful marker in the determination of the infiltrative behaviour of these tumors.

The aim of this study is to investigate the relationship between proliferative activity of anterior pituitary adenomas, quantified with Ki-67 LI, and their size neuroradiologically (CT and MRI) and surgically verified.

Patient characteristics
From July, 1994, to July, 1998, 121 patients suffering from an anterior pituitary adenoma were consecutively operated on. In all cases an evaluation of the the Ki-67 LI of surgical specimens was performed by means the monoclonal antibody MIB-1 [21]. Plurihormonal adenomas were classified according to the predominant endocrine symptoms (in 4 cases GHand in 3 PRL-related disturbances). Twenty out of 29 patients (68.9%) with prolactinomas or with mixed adenoma with prevalent PRL expression received preoperative medical therapy (bromocriptine).

Determination of the Ki-67 Labeling Index (MIB-1 immunostaining)
The surgical specimens were routinely processed, fixed in neutral buffered formalin, and embebbed in paraffin.
In order to evaluate the Ki-67 antigen staining, 5-micrometer sections, previously mounted onto glass slides and dried, were incubated overnight at 4°C in the MIB-1 antibody (Immunotech, Marseille, France) [21]. Immunostaining was performed using the avidin-biotin-peroxidase method. Ten fields were selected in regions with highest concentrations of MIB-1-positive nuclei and were examinated at high power magnification (x400). Each field corresponded to a total number of cells ranging from 700 to 1000, in relation with the cellularity of the tumor specimen ( Fig. 1). Areas of necrosis, normal adenohypophysial cells, and endothelial cells were exluded from the evaluation. On considering 1000 cells with "manual" counting, the Ki-67 Labeling Index has been defined as the percentage of MIB-1 positive cells (dense brown precipitate restricted to the nuclei).

Statistical analysis
Computer-assisted data analysis was performed with a commercially available software (SPSS 6.0, SPSS Inc., Chicago, IL). The normal distribution of Ki-67LI values was verified by using normal Q-Q plots, with the Blom's method. The Χ 2 (1 or 2 df, continuity correction) and ANOVA tests were used to identify the statistical significance of differences of Ki-67 LI observed in relation to functioning vs non functioning adenomas, presence or not of pre-operative visual disturbances, neuroradiological and surgical extimated volume of tumor, surgical invasiveness, and infiltration of CS. Values are expressed as the mean ± standard deviation (SD) of the mean; for each comparison, a P value was obtained (significance, ≤ 0.05).

Discussion
Pituitary tumors are usually classified in microadenomas and macroadenomas [10,11]. In the first group the tumor consists of a well circumscribed nodule smaller than 10 mm in diameter [9], whereas macroadenomas produce enlargement of the contour of sella turcica [10,11]. Jules Hardy [10] proposed a schematic radiological classification of anterior pituitary adenomas as outlined by axial and coronal views of CT scan, today appliable to MRI. He divided pituitary tumors in "enclosed" (intrasellar adenomas) and "invasive" (intrasellar and intra-extrasellar adenomas). 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).
Pituitary adenomas may be associated with a suprasellar extension, as well outlined by coronal CT scan and coronal and sagittal MR images. Hardy [10] classified them 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 In order to simplify these classifications and to reduce the number of subgroups for correlations with Ki-67 LI values, we preferred to subdivide the tumors of our series into microadenomas, intrasellar macroadenomas, intrasuprasellar macroadenomas, and intra-supra-parasellar adenomas, including in the last group also pituitary adenomas infiltrating the wall of cavernous sinus.
Several cell cycle-specific nuclear antigens have been recognized with various immunohistochemical methods, allowing a reliable evaluation of tumor growth fraction.
In particular, Ki-67 is a nuclear antigen nowadays simply recognized by monoclonal antibody MIB-1 [21], typically expressed in proliferating cells during the G 1 , S, G 2 , and M phases of the cell-cycle [17,21,22,24,29,21,34]. It turned out to be useful in a number of human neoplasms [3,8,13,33], providing information about cellular proliferation rate and, thus, about long-term prognosis. It revealed to be a practical method that can be used in the routine histological evaluation of brain tumors and, also, of anterior pituitary adenomas. A high growth fraction expressed by a high Ki-67 LI should suggest a high proliferative rate and, thus, tight clinical-radiological follow-up, in relation to possible post-operative relapse of adenoma [14]. With te aim to establish a correlation between the growth fraction of pituitary adenomas and their neuroradiological and surgical extimated tumor size, we evaluated the Ki67 LI by means te monoclonal antibody MIB-1 in 121 patients. As summarized in table 1, the values of mean index were similar among microadenomas, intrasellar macroadenomas, and intra-suprasellar macroadenomas. The mean LI of intra-supra-parasellar macroadenomas (3.29%) was higher than others subgroups and than the overall mean value (2.72%); this difference was not statistically significant (p = 0.27) and presumably reflects the higher incidence of invasive tumors among intra-supra-parasellar adenomas. Therefore, with the exception of invasive adenomas, the different tumor size of pituitary adenoma seems to be correlated to the time of growth rather than to a different growth fraction

Conclusion
The evaluation of growth fraction of pituitary tumors, identified by the Ki-67 nuclear antigen detected with the MIB-1 monoclonal antibody, presumably gives further information about their infiltrative behaviour. In our series of 121 cases, the analysis of proliferative rate seems to exclude a definite correlation between the mean value of Ki67 LI and the neuroradiological and surgical extimated tumor size of anterior pituitary adenomas.