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Prognostic Significance of p27^KIP1 Protein and Ki-67 Growth Fraction in Non-small Cell Lung Cancers

First Department of Medicine [F. H., H. D-A., T. M., T. K., H. H., S. O., Y. K.] and Second Department of Surgery [H. K.], Hokkaido University School of Medicine; Department of Surgical Pathology, Hokkaido University Medical Hospital [M. S.]; and Department of Public Health, Sapporo Medical University [M. N.], Sapporo 060-8638, Japan

	ABSTRACT

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We immunohistochemically examined specimens of 215 surgically resected non-small cell lung cancers (NSCLCs) for p27^KIP1 protein (p27) expression and the growth fraction determined by the Ki-67 labeling index (LI). The NSCLCs analyzed showed considerable heterogeneity in both p27 and Ki-67 LIs; 25 of 207 (13%) lacked p27 expression (p27 LI < 5%), and 116 of 215 (54%) showed a high Ki-67 LI (>30%). The p27 LI was not significantly associated with the Ki-67 LI. A ² test showed that loss of p27 expression was inversely correlated with smoking (P = 0.01) and that a high Ki-67 LI was significantly associated with male gender, squamous cell carcinoma histology, and smoking (P < 0.0001 each). Prognostic values of p27 and Ki-67 expression were evaluated in 109 tumors of postsurgical pathological stages I and II. Patients with tumors lacking p27 expression survived for a significantly shorter time than patients with tumors expressing p27 (5-year survival rates, 38% and 68%, respectively; P = 0.02). Patients with tumors having a high Ki-67 LI survived for a significantly shorter time than patients with tumors having a low Ki-67 LI (5-year survival rates, 48% and 78%, respectively; P = 0.005). Multivariate analysis showed that loss of p27 expression tended to be an unfavorable prognostic factor (P = 0.054), whereas a high Ki-67 LI was a significant and independent unfavorable prognostic factor (P = 0.004). When analyzed by cell types, loss of p27 expression was a significant and independent unfavorable prognostic factor in squamous cell carcinomas (P = 0.01), whereas a high Ki-67 LI was a significant and independent unfavorable prognostic factor in nonsquamous cell carcinomas (P = 0.007). We further evaluated the importance of p27 expression in clinical outcome in combination with the Ki-67 LI and ras p21 protein (ras) expression, which we previously reported as an important prognostic factor in NSCLCs. Patients with tumors lacking p27 expression and having a high Ki-67 LI survived for a significantly shorter time than those with tumors expressing p27 and having a high Ki-67 LI (5-year survival rates, 17% and 52%, respectively; P = 0.003). Patients with p27-negative and ras-positive tumors survived for a significantly shorter time than those with both p27- and ras-positive tumors (5-year survival rates, 0% and 38%, respectively; P < 0.0001). These results indicate the pivotal roles of p27 and Ki-67 expression in the clinical outcome of NSCLCs.

	INTRODUCTION

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NSCLCs² represent a heterogeneous group of cancers in terms of biology, histology, and clinical behavior (1) . Recent progress in the molecular biology of cancer has demonstrated that multiple molecular genetic changes are involved in the development, progression, and, presumably, clinical behavior of NSCLCs (2) . On the other hand, the prognosis of patients with NSCLCs has improved only minimally despite major advances in cancer treatment in the past two decades (3) , and there are disparities in their clinical outcomes within the same tumor-node-metastasis (TNM) subsets (1) . Under such circumstances, it may be possible to identify patients with a good or poor prognosis using molecular biological alterations as clinical biomarkers, including alterations of cell cycle regulators.

Altered regulation of the cell cycle is a hallmark of human cancers (4 , 5) . The cell cycle is governed by cdks, the activity of which is regulated by the binding of positive effectors, the cyclins (6 , 7) , and by negative regulators, the cdk inhibitors (8 , 9) . The cdks integrate mitogenic and growth inhibitory signals and coordinate cell cycle transitions (6 , 7) .

p27, a member of the cip/kip family of cdk inhibitors, plays a pivotal role in cell cycle regulation from the G₁ to S phase by inhibiting cdk4/6-cyclin D1 and cdk2-cyclin E (8) . Regulation of this protein occurs primarily at the posttranslational level by proteasomal degradation (10) . A decrease or lack of p27 has been reported to correlate with shorter survival or to be a negative prognostic factor in many kinds of tumors, including NSCLCs (see Ref. 11 for a review). The proliferation index is a potent biological marker that estimates the growth of neoplasms quantitatively and has been reported to aid in determining the prognosis of patients with NSCLCs (12, 13, 14) . Therefore, it is intriguing to examine primary NSCLCs for loss of p27 expression and the growth fraction determined by the Ki-67 LI.

We have previously reported alterations of regulator molecules of the G₁-S-phase transition, including p53, RB, p16^INK4A, and cyclin D1, and their biological and clinical importance in resected NSCLCs (15, 16, 17, 18, 19) . Moreover, we have recently demonstrated by immunohistochemistry that patients with curatively resected NSCLCs having high-level expression of cyclin E survived for a significantly shorter time than those with tumors having a low level of cyclin E expression (20) . In a previous study, we also showed that altered ras expression was an independent unfavorable prognostic factor in surgically resected NSCLCs (21) . It has been shown that ras function is required as an upstream regulator for down-regulation of p27 in vitro (22) .

The purpose of the present study is to evaluate the importance of p27 expression alone and in combination with Ki-67 LI or ras expression in the clinical outcome of NSCLCs and to identify NSCLC patients at high risk of early disease recurrence after surgery to select patients who may benefit from intensive adjuvant therapy.

	MATERIALS AND METHODS

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Tumor Specimens and Survival Data.
Primary tumor specimens from 215 patients with NSCLC were obtained consecutively during surgery at the Hokkaido University Medical Hospital between 1976 and 1994. The patients with NSCLCs consisted of 144 men and 71 women (average age at diagnosis, 63.3 years). According to the 1981 WHO classification (23) , tumor specimens were histopathologically diagnosed as adenocarcinoma (n = 106), squamous cell carcinoma (n = 91), large cell carcinoma (n = 10), and adenosquamous cell carcinoma (n = 8). They represented 120 p-Stage I, 17 p-Stage II, 70 p-Stage IIIa, 1 p-Stage IIIb, and 7 p-Stage IV tumors. p-Stage was determined according to the guidelines of the American Joint Committee on Cancer (24) .

Of the 215 NSCLCs, 128 were p-Stage I and II tumors resected with curative intent (112 patients with p-Stage I tumors and 16 patients with p-Stage II tumors). The prognostic value of p27 and Ki-67 expression was evaluated for the 109 patients with p-Stage I and II tumors who: (a) survived for more than 3 months after surgery; (b) did not die of causes other than lung cancer within 5 years after surgery; and (c) were followed for more than 2 years after surgery (for patients who remained alive). Ten patients who did not meet the above-mentioned criteria (three died within 3 months after surgery and seven died of causes other than lung cancer within 5 years) were excluded from the survival analysis. Nine patients for whom no survival records after surgery were obtained were also excluded from the survival analysis. Seventy-five patients received chemotherapy as a postsurgical treatment. Radiation therapy was not performed before or after surgery for any patients. Because all of the patients enrolled in the current study were coded, they could not be individually identified. We previously reported on the prognostic significance of ras expression in NSCLCs for 46 of the 109 patients (17 , 21) .

Immunohistochemistry for p27, Ki-67, and ras Proteins.
p27 expression and Ki-67 expression were analyzed by immunohistochemistry. The labeled streptavidin biotin method was used on 4-µm sections of formalin-fixed, paraffin-embedded tissues after deparaffinization. Briefly, deparaffinized tissue sections were treated with an autoclave in 10 mM citrate buffer (pH 6.0) for 20 min at 121°C to retrieve the antigenicity. The sections were then immersed in methanol containing 1.5% hydrogen peroxide for 20 min to block endogenous peroxidase activity and incubated with normal rabbit serum to block the nonspecific antibody binding sites. The sections were reacted consecutively with mouse monoclonal anti-p27 antibody clone 1B4 (Novocastra, Newcastle, United Kingdom) at a dilution of 1:30 or with mouse monoclonal antibody MIB-1 (Immunotech, Marseilles, France) at a dilution of 1:50 at 4°C overnight. Immunostaining was performed by the biotin-streptavidin immunoperoxidase method with 3,3'-diaminobenzidine as a chromogen (SAB-PO kit; Nichirei, Tokyo, Japan). Methyl green was used as the counterstain. p27 expression was detected in nonneoplastic bronchial epithelia, glands, fibroblasts, and infiltrating lymphocytes, which served as positive internal controls. Tumor cells were judged as p27 positive when the staining intensity of those nuclei was similar to that of surrounding normal lymphocytes, whereas tumor cells exhibiting a readily noticeable reduction in staining intensity compared with normal lymphocytes were judged as p27 negative. For Ki-67 staining, tumor cells were considered positive if any nuclear staining was present.

The p27 and Ki-67 LIs were defined as the percentage of tumor cells displaying nuclear immunoreactivity and calculated by counting the number of nuclear p27- or Ki-67-stained tumor cells in 1000 tumor cells in each section. A single representative tissue section from each tumor was surveyed microscopically at x100 for at least two or three areas at random for p27 and Ki-67. Cell counts were performed at x400 in at least seven fields in these areas, using a Videomicrometer (Model VM-30; Olympus, Tokyo, Japan) equipped with a light microscope. The p27 and Ki-67 LIs were reliably and reproducibly obtained using this Videomicrometer system.

For the ras staining of 101 of the 215 tumor specimens, slides and results that had been reported previously (17 , 21) were used for the current study. ras staining was scored as positive (+) and negative (-) for the current study, as described in the previous study (17) .

Statistical Analysis.
The relationship between p27 LI and Ki-67 LI was analyzed by the Spearman rank correlation coefficient. The associations between p27 or Ki-67 expression and categorical variables were analyzed by the ² test or Fisher’s exact test, as appropriate. The associations between p27 or Ki-67 expression and age were analyzed by Student’s t test. To simultaneously examine the effect of more than one factor on Ki-67 expression, multivariate logistic regression analysis was used (25) . The survival curves were estimated using the Kaplan-Meier method, and differences in survival distributions were evaluated by the generalized Wilcoxon test. Cox’s proportional hazards modeling of factors potentially related to survival was performed to identify which factors might have a significant influence on survival. The significance level chosen was P < 0.05, and all tests were two-sided.

	RESULTS

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Typical immunostaining patterns for p27 in NSCLCs are shown in Fig. 1 . Of the 215 NSCLC tumors analyzed, 8 tumors were uninterpretable for p27 expression because there were no tumor cells or they lacked nuclear staining of p27 in infiltrating lymphocytes (positive internal controls). Therefore, p27 expression was analyzed for the remaining 207 tumors. NSCLCs showed considerable heterogeneity in the p27 LI, and the mean value of the p27 LI was 28.6% (SD, 23.6; range, 0–96%). p27 LIs (mean ± SD) of 89 squamous and 118 nonsquamous cell carcinomas were 27.6 ± 23.4% and 29.3 ± 23.9%, respectively. The distributions of p27 expression in both cell types were unimodal, with peaks at 5–15% (squamous cell carcinomas) and 0–10% (nonsquamous cell carcinomas). To define an appropriate cutoff level for p27 LIs in the subsequent statistical analysis, we referred to the previously reported and well prespecified criteria on immunohistochemical expression of p27 in various types of cancer (26, 27, 28, 29) and divided tumors into a group having p27 expression with a LI 5% and a group lacking p27 with a LI < 5%. Of 207 tumors, 25 (13%) lacked p27 expression (LI < 5%).

View larger version (115K): [in this window] [in a new window]   Fig. 1. Immunohistochemical staining patterns for p27KIP1 protein (p27) in primary NSCLCs. A and B, an adenocarcinoma (A) and a squamous cell carcinoma (B) having p27 expression (LIs, 93% and 89%, respectively). C, a squamous cell carcinoma lacking p27 expression with a 4% LI. Note that admixed lymphocytes (arrows) show distinct nuclear staining of p27 in all specimens, which provide positive internal controls. A–C, x400. Scale bar, 20 µm.

View larger version (112K): [in this window] [in a new window]   Fig. 2. Immunohistochemical staining patterns for Ki-67 protein in NSCLCs. A, a squamous cell carcinoma having a high Ki-67 LI of 81%. B, a poorly differentiated adenocarcinoma having a high Ki-67 LI of 50%. C, a well-differentiated adenocarcinoma having a low Ki-67 LI of 0%. A–C, x400. Scale bar, 20 µm.

View larger version (31K): [in this window] [in a new window]   Fig. 3. Kaplan-Meier survival curves of patients with p-Stage I and II NSCLCs, stratified by p27KIP1 protein (A for overall NSCLCs and C for squamous cell carcinomas) and Ki-67 protein (B for overall NSCLCs and D for nonsquamous cell carcinomas).

View larger version (41K): [in this window] [in a new window]   Fig. 4. Kaplan-Meier survival curves of patients with p-Stage I and II NSCLCs stratified by ras p21 protein (ras; A), p27KIP1 in combination with Ki-67 protein (B), p27KIP1 in combination with ras status (C), and Ki-67 protein in combination with ras status (D).

	DISCUSSION

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The present study demonstrated large variations in p27 and Ki-67 expression among surgically resected NSCLCs. p27 and Ki-67 expression, both alone and in combination, exhibited pivotal roles in the clinical outcome of patients with p-Stage I and II NSCLCs, all of whom were operated on at a single institute and had long enough postsurgical observation periods for survival analysis.

We showed that patients with tumors lacking p27 expression survived for a significantly shorter time than patients with tumors expressing p27 and that loss of p27 expression tended to be an unfavorable prognostic factor in multivariate analysis. These findings are in agreement with previous studies (26 , 27 , 30) , where decreased p27 expression in NSCLCs was correlated with poor survival. Furthermore, lack or retention of p27 expression in combination with a high or low Ki-67 LI indicated potential synergistic effects on survival and prognosis. p27 or Ki-67 status in combination with ras protein expression also showed potential synergistic effects on clinical outcome.

The lack of an association between p27 and Ki-67 LIs in this study may have been because the cell growth fraction indicated by Ki-67 LI is not determined only by p27 but also by other cell cycle regulators, including RB, p16^INK4A, cyclin D1, and cyclin E (4, 5, 6, 7, 8, 9) . In fact, a lack of association between p27 and tumor cell proliferation was also reported in colorectal and breast cancers (31 , 32) . In addition, increased expression of p27 has been shown to correlate with cyclin D1 expression in some highly proliferative human breast cancer cell lines, esophageal cancer cell lines, and some colorectal carcinomas, indicating the existence of a mechanism by which some growing tumors may tolerate p27 (33, 34, 35) . Sui et al. (36) also presented evidence that borderline ovarian tumors with high-level p27 expression also had enhanced cyclin E and cdk2 expression. Furthermore, p27 may have pivotal roles in promoting apoptosis (37 , 38) and as a cell cycle regulator, as a consequence making its loss of expression an unfavorable prognostic factor of NSCLCs. However, the relationship between p27 and Ki-67 LIs remains to be determined because it has been reported recently that the p27 LI was inversely related to the Ki-67 LI in NSCLCs (13) .

In this study, p27 expression was lost significantly frequently in squamous cell carcinomas from nonsmokers. The inverse association between loss of p27 and smoking may indicate (a) that p27 is not a target for tobacco-induced carcinogenesis and (b) that p27 expression is retained to overcome cyclin E expression, which has been shown to be frequently found in NSCLCs from smokers (20) , because it has been shown that high-level p27 expression is associated with enhanced cyclin E and cdk2 expression (36 , 39 , 40) .

An investigation in vitro showed that ras function was required in late G₁ for down-regulation of p27 in NIH3T3 fibroblasts (22) . However, Catzavelos et al. (30) found no relationship between K-ras mutations and p27 levels in surgically resected NSCLCs. In the present study as well, p27 was not associated with ras protein expression. It remains to be determined whether ras protein is involved in the down-regulation of p27 in NSCLCs.

We showed that a high Ki-67 LI was found in squamous cell carcinomas compared with nonsquamous cell carcinomas and was associated with an unfavorable clinical outcome. Consistent with these findings, there have been several studies on Ki-67 in NSCLCs (12 , 14 , 41) . Viberti et al. (12) reported that in both biopsy and surgical specimens, a high Ki-67 LI was associated with squamous cell carcinoma and poorly differentiated tumors and that patients with tumors having a high Ki-67 LI had a significantly shorter disease-free interval. Moreover, Mehdi et al. (14) investigated 260 patients with stage I and II NSCLCs and reported that Ki-67 expression was higher in squamous cell cancers than in nonsquamous cell cancers and that there was a trend for shorter overall and disease-free survival in patients with tumors having high Ki-67 expression.

Molecular prognostic markers have not yet emerged clearly for NSCLCs (1) , although many have been reported. Therefore, as some investigators, including ourselves, have proposed, two or more biological markers in addition to disease stage could be necessary to coherently stratify the survival or prognosis of NSCLC patients (15 , 17 , 41 , 42 , 43) . In fact, in this study, combinations of two markers (p27/Ki-67, p27/ras, and Ki-67/ras) were able to stratify NSCLC patients of p-Stage I and II into better or worse prognostic groups. It is noteworthy that all four patients with p27-negative and ras-positive tumors died within 2 years, having a poorer survival rate than those with both p27- and ras-positive tumors (Fig. 4 C). Moreover, among patients with ras-negative tumors, those with low Ki-67 LIs had a better survival rate than those with high Ki-67 LIs (Fig. 4 D).

In conclusion, we have demonstrated the considerable heterogeneity in p27 and Ki-67 expression in individual NSCLCs, which is likely to be reflected in the biological behavior of the tumors, and then identified pivotal roles of p27 and Ki-67 in the clinical outcome of resected NSCLCs. The status of these proteins may have great value in combination or in combination with ras expression in identifying NSCLC patients at high risk of early disease recurrence after surgery and thus in selecting patients who will benefit from intensive adjuvant therapy.

	ACKNOWLEDGMENTS

 
We thank Dr. Takashi Nishizaka (Clinical Research Laboratory, Hiroshima Prefectural Hospital, Hiroshima, Japan) for the technical information on p27 immunohistochemical staining and Prof. Kazuo Nagashima (Laboratory of Molecular and Cellular Pathology, Hokkaido University School of Medicine, Sapporo, Japan) for the use of the Videomicrometer.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom requests for reprints should be addressed, at First Department of Medicine, Hokkaido University School of Medicine, North 15, West 7, Kita-ku, Sapporo 060-8638, Japan. Phone: 81-11-716-1161; Fax: 81-11-706-7899; E-mail: hdakita{at}med.hokudai.ac.jp

² The abbreviations used are: NSCLC, non-small cell lung cancer; cdk, cyclin-dependent kinase; p-Stage, postsurgical pathological tumor-node-metastasis (TNM) stage; LI, labeling index.

Received 2/28/00; revised 7/17/00; accepted 7/17/00.

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