Immunohistochemical Analyses of Cell Cycle-related Proteins,Apoptosis,and Proliferation in Pituitary Adenomas

Abstract

To analyze the cell cycle regulatory mechanisms in the growth of pituitary adenomas, we investigated immunohistochemically the expression of the cell cycle-related proteins cyclin A and p27 in 48 pituitary adenomas. The frequency of apoptosis and the proliferative potential were also examined. The percentage of apoptotic cells was evaluated by immunohistochemical analysis using the anti-single-strand DNA antibody. The proliferative potential was assessed using the anti-Ki-67 antibody. The mean cyclin A labeling index (LI) for the non-recurrent group was 1.03% and for the recurrent group 2.31%. A positive linear correlation between cyclin A LI and Ki-67 LI was found. The mean p27 LI for the non-recurrent group was 67.4% and for the recurrent group 47.0%. There were significant differences in cyclin A LI and p27 LI between the non-recurrent group and the recurrent group. The mean apoptotic rate for the non-recurrent group was 0.87% and for the recurrent group 1.05%. There was no significant difference. Multivariate regression analysis revealed that high cyclin A LI and high Ki-67 LI were significant factors for shorter progression-free survival. The results suggest that the cyclin A LI is a useful prognostic factor in pituitary adenomas. (J Histochem Cytochem 49:1193–1194, 2001)

Keywords

cyclin A p27 single-strand DNA apoptosis pituitary adenoma

Cell cycle-related proteins control the cell cycle and are closely related to tumorigenesis of the cell. On the other, several studies revealed that apoptosis also plays an important role in neoplastic development. To analyze the cell cycle-regulatory mechanisms in the growth of pituitary adenomas, we investigated the expression of cyclin A and p27. Cyclin A is an S-phase and G₂M-phase regulatory protein, and p27 is a cyclin-dependent kinase inhibitor that regulates the progression of the cell cycle from G₁- to S-phase. We also examined the frequency of apoptosis and the proliferative potential to analyze growth characteristics of pituitary adenomas.

A total of 48 histologically confirmed pituitary adenomas, obtained at Osaka City University, were studied. All cases were divided into the recurrent group and the non-recurrent group. Ten tumors showed recurrence. All cases were followed for more than 5 years after initial surgery.

Tumor tissues were fixed in 10% buffered formalin and embedded in paraffin. The expression of the cell cycle-related proteins cyclin A and p27 was evaluated by immunostaining with monoclonal antibodies to cyclin A and p27. The immunostaining was performed using the Dako Envision⁺ (Dako; Carpinteria, CA) system. After antigen retrieval, endogeneous peroxidase activity was blocked by 0.3% hydrogen peroxidase in methanol. Then the sections were incubated with the primary antibodies (cyclin A ×100; p27 ×50) for 1 hr at room temperature. Then they were incubated with the peroxidase-labeled polymer for 1 hr. Finally, they were incubated with DAB. The percentage of cells showing positive nuclear staining was assessed in each case to determine the cyclin A and p27-labeling index (LI). The percentage of apoptotic cells (apoptotic rate) was evaluated by immunohistochemical analysis using the anti-single-strand (ss)DNA antibody. The immunostaining was also performed using the Dako Envision⁺ system. The proliferative potential was assessed using the anti-Ki-67 antibody (MIB 1).

Thirty of the pituitary adenomas were in women and 18 were in men: The mean age of patients was 40.7 years. There were 32 gross total removals and 16 subtotal removals.

In cyclin A immunostaining, the staining pattern was nuclear (Figure 1). The mean cyclin A LI for the non-recurrent group was 1.03 ± 0.50% and for the the recurrent group 2.31 ± 0.27%. A statistically significant difference in cyclin A LI was observed between the two groups (p < 0.005). A significant correlation was seen between the cyclin A LI and the Ki-67 LI (r = 0.91; p < 0.005). The cyclin A LI showed a tendency to increase as the Ki-67 LI increased. In p27 immunostaining, p27 was expressed in the nuclei (Figure 1). The non-recurrent group had a significantly higher mean p27 LI (67.4 ± 10.8%) than that of the recurrent group (47.0 ± 10.2%) (p < 0.005). Apoptotic cell were observed in 38 of 48 cases. The apoptotic rate was higher in the recurrent group (1.05 ± 0.47%) than in the non-recurrent group (0.87 ± 0.05%), but there was no significant difference (p = 0.489). The recurrent group had significantly higher numbers of macroadenomas (chi-square test p < 0.05) than the non-recurrent group (Table 1). Subtotal removal was also more common in the recurrent group than in the non-recurrent group (chi-square test p < 0.05). Type of tumor and extrasellar extension also did not correlate with recurrence. Multivariate regression analysis revealed that high cyclin A LI, high Ki-67 LI, and subtotal removal were significant factors for shorter progression-free survival.

This study showed that cyclin A expression was associated with tumor recurrence and correlated with Ki-67 LI. These results were generally in agreement with recent studies that reported the correlation of cyclin A expression with cell proliferation and tumor progression in various tumors (Paterlini 1993; Huuhtanen 1999). To our knowledge, there are no reports concerning cyclin A expression in pituitary adenomas. Our results suggest that cyclin A may be positively involved in the increased proliferation of pituitary adenomas. We also found that p27 expression was higher in the non-recurrent than in the recurrent pituitary adenomas. Recent studies have shown that p27 reactivity evaluated immunohistochemically correlates with an unfavorable prognosis in several cancers (Sanchez–Beato 1996; Jordan 1998). Our study showed that, similar to cancers, p27 is downregulated and is associated with recurrence in pituitary adenomas. Several studies have documented that higher level of apoptosis are a negative prognostic index in cancers (Mattern 1998; Evertsson 1999). In this study the recurrent group had a higher apoptotic rate than the non-recurrent group, but there was no significant difference. In conclusion, our results suggest that high cyclin A LI together with low p27 LI is associated with an unfavorable prognosis for pituitary adenomas.

Figure 1

Sections of pituitary adenoma immunostained with cyclin A (A) and p27 (B). Original magnification ×400.

Table 1

Factors correlated with the recurrence of pituitary adenomas

Factors	Non-recurrent group	Recurrent group	p value
Cyclin A LI (%)	103+050	2.31 ±0 27	<0.005
p27 LI (%)	67.4 ± 10.8	47.0 ± 10.2	<0.005
Ki-67 LI (%)	1.42 ±0.64	3.21 ±0.41	<0005
Apoptotic rate (%)	0.87 ±0.05	1.05 ±0.47	0.489
Tumor size (%) Macroadenoma	63.1	100	0.024
Macroadenoma	36.9	0
Extrasellar extension (%) With	63.1	90	0 139
Without	36.9	10
Operation (%) Gross total removal	737	30	0.022
Subtotal removal	26 3	70

References

Evertsson

(1999) Apoptosis in relation to proliferating cell nuclear antigen and Dukes' stage in colorectal adenocarcinoma. Int J Oncol 15:53–58

Huuhtanen

(1999) Expression of cyclin A in soft tissue sarcoma correlates with tumor aggressiveness. Cancer Res 15:2885–2890

Jordan

(1998) Reduced level of the cell-cycle inhibitor p27Kip1 in epithelial dysplasia and carcinoma of the oral cavity. Am J Pathol 152:585–590

Mattern

(1998) Spontaneous apoptosis in ovarian cancer: an unfavorable prognostic factor. Int J Oncol 12:351–354

Paterlini

(1993) Cyclin A expression in human hematological malignancies: a new marker of cell proliferation. Cancer Res 15:235–238

Sanchez–Beato

(1996) Cyclin-dependent kinase inhibitor p27Kip1 in lymphoid tissue: p27Kip1 expression in inversely proportional to the proliferative index. Am J Pathol 151:151–160