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Paradoxical Correlations of Cyclin-dependent Kinase Inhibitors p21waf1/cip1 and p27kip1 in Metastatic Colorectal Carcinoma

Divisions of Medical Science [J. D. C., N. J. M.] and Population Science [J. S. B., N. J. M.], Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, and Division of Pathology, Roswell Park Cancer Institute, Buffalo, NY 14263 [B. A. W.]

	ABSTRACT

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The cyclin-dependent kinase inhibitors (CDIs) p27kip1 and p21waf1/cip1 are key cell cycle-negative regulatory enzymes. The objective of this study was to correlate expression of p27kip1 and p21waf1/cip1 with survival, chemotherapy responsiveness, and expression of the proliferation marker Ki-67 in patients with advanced colorectal cancer.

Immunohistochemistry was performed with antibodies to p27kip1, p21waf1/cip1, and Ki-67 on samples from 66 patients with metastatic colorectal carcinoma. Interpretation was performed by visual inspection and automated image analysis.

Patients who obtained a response to chemotherapy had greater p21waf1/cip1 tumor staining with a mean of 10.0 positive cells/high-powered field, compared with 4.5 positive cells/high-powered field for nonresponders (P = 0.03). A positive Spearman correlation was seen between Ki-67 and p27kip1 (r = 0.48; P = 0.0001), as well as between Ki-67 and p21waf1/cip1 (r = 0.48; P = 0.0001). A trend toward shorter survival was seen in patients with positive specimens (median survival of 10 months for patients with both p27kip1- and p21waf1/cip1-positive specimens, compared with 22 months for patients with neither p27kip1- nor p21waf1/cip1-positive specimens).

In contrast to that previously reported in normal colonic mucosa or early-stage colorectal cancer, we observed positive correlations of Ki-67 with both p27kip1 and p21waf1/cip1, a trend toward greater CDI staining indicating worse prognosis, and greater p21waf1/cip1 staining in tumors that were chemosensitive. These findings suggest that in the metastatic setting, CDIs may show altered function, compared with their role in the normal cell cycle.

	INTRODUCTION

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CDKs² are key regulatory enzymes of the cell cycle. Precise activation and inactivation of CDKs at specific points in the cell cycle are required for orderly cell division. CDIs are involved in the negative regulation of CDK activities, playing a role in control of cell cycle progression and, thus, potentially acting as tumor suppressors (1) . p21waf1/cip1 is a nuclear protein with at least two functions, as a CDI (2, 3, 4) and as a proliferating cell nuclear antigen inhibitor (5 , 6) . p21waf1/cip1 is induced by the tumor suppressor gene p53 (7) and acts as a downstream effector of p53 in inhibiting mitotic progression. p21waf1/cip1 can also be induced independent of p53 through various growth regulators, such as platelet-derived growth factor, fibroblast growth factor, and epidermal growth factor (8) . p21waf1/cip1 has a wide range of actions on cell cycle control, including induction of G₁ arrest (9) . p27kip1 is another CDI that shares partial homology with p21waf1/cip1 and binds to a number of CDK complexes (1) . p27kip1 is abundant in quiescent cells, with growth factor stimulation causing p27kip1 protein levels to fall. p27kip1 may, therefore, provide a threshold for the G₁ cyclin/CDK complexes to overcome for cell proliferation (10) .

Ki-67 is a nuclear antigen of unknown function that is expressed in all stages of the cell cycle, except resting cells in G₀ (11 , 12) . It correlates well with cell proliferation, as measured by other methods such as flow cytometry and [³H]thymidine uptake (13) . Ki-67 provides a reliable method of assessing proliferative activity, and its expression correlates with responsiveness to chemotherapy (14) .

We hypothesized that nuclear expression of CDIs would predict for improved survival in patients with metastatic colorectal carcinoma, given their role as inhibitors of cell cycle progression. We also hypothesized that CDI expression would predict for resistance to 5-FU-based chemotherapy because fewer malignant cells would be undergoing mitosis and synthesizing DNA at any given time. We also anticipated an inverse relationship between the CDIs and the proliferation marker Ki-67. This study was conducted to assess the correlation of p27kip1 and p21waf1/cip1 expression with cell proliferation and clinical outcome in patients with metastatic colorectal cancer. The results reported herein describe an unexpected paradoxical relation of CDI expression and tumor natural history.

	PATIENTS AND METHODS

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Patient Samples.
The Roswell Park Cancer Institute Tumor Registry was used to identify all patients with metastatic colorectal cancer who had undergone their surgical resection or biopsy at Roswell Park Cancer Institute between 1990 and 1995 (n = 97). Of these patients, 80 had formalin-fixed, paraffin-embedded blocks available for IHC analysis. After the IHC staining process, 10 samples had no tissue present on the slide after microwave antigen retrieval, and 4 samples had no carcinoma present in the tissue specimen stained. The remaining 66 samples were used for all further analyses. Clinical data from these 66 consecutive patients were obtained by retrospective chart review. Thirty-nine patients received 5-FU-based systemic chemotherapy. Thirty patients were evaluable for response. Nine patients were not evaluable due to concurrent radiation therapy given for local recurrence. Complete response was defined as radiographic disappearance of all measurable disease. Partial response was defined as a reduction of >50% of the sum of the products of the perpendicular diameters of all measurable metastatic lesions. Overall survival was determined from the time of biopsy-proven diagnosis until the time of death or last known follow-up. Disease-free survival was determined from the time of initial resection until the time of first radiographic appearance of subsequent biopsy-proven metastasis. Patients who did not undergo an initial resection of their tumor were excluded from disease-free survival analysis (n = 39).

Immunohistochemical Analysis.
IHC was performed on deparaffinized 4-µm sections. Sections were subject to heat-induced antigen retrieval for 10 min at 100°C in 10 mM citrate buffer before addition of the primary antibody. The anti-p27kip1 antibody (Transduction Laboratories, Lexington, KY) was used at a dilution of 1:400, anti-p21waf1/cip1 (Waf-1; Ab-1; Oncogene Science, Uniondale, NY) was used at a dilution of 1:20, and anti-Ki-67 (MIB-1; Immunotech Inc., Westbrook, ME) was used at a dilution of 1:50 with overnight incubation at 25°C. The avidin-biotin detection method was used on a 320 Ventana Automated System (Ventana Medical System, Tucson, AZ). Di-amino-benzidine was used as the chromogen. Substitution of isotypic mouse IgG for the primary antibody served as a negative control. Lymph node specimens served as the positive control.

IHC Scoring.
Two methods were used to score immunohistochemical staining. First, Ki-67-, p27kip1-, and p21waf1/cip1-stained slides were assessed in a semiquantitative method by consensus of two observers without prior knowledge of patient clinical features. Ki-67 and p27kip1 slides were graded according to the extent of nuclear staining on a scale of 0–4: 0, no staining; 1, <1% staining; 2, 1–5% staining; 3, 6–50% staining; and 4, >50% staining. Staining scores of 0, 1, and 2 were considered negative, and staining scores of 3 and 4 were considered positive. p21waf1/cip1 slides were graded according to the extent of strong nuclear staining on a scale of 0–3: 0, no staining; 1, scattered positive nuclei; 2, focal strong positivity; and 3, diffuse strong positivity. A staining score of 0 was considered negative, and staining scores of 1, 2, and 3 were considered positive. Objective validation of IHC interpretation was obtained using a three-chip color video camera (Sony 3CCD-IRIS) and the Samba 4000 Cell Image Analysis System (Imaging Products International Inc., Chantilly, VA) and immunolabeling analysis software, according to the manufacturer‘s instructions. The number of positive staining cells/HPF (x40) were counted with the segmentation threshold set at 50 and the RGB color bar set at 200, based on positive and negative controls. The field of greatest positive staining was selected for each sample.

Statistical Considerations.
Sample size calculations indicated that a minimum of 63 patients were required to detect a survival risk ratio of 2.0 with an error of 0.05 and 80% power, assuming that 30–70% of the pathological specimens would stain IHC positive (15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25) . Spearman rank correlation analysis was used to assess the association between selected measurements. ² and Mann-Whitney tests were used to examine differences among patient subgroups with respect to the degree of positive staining determined semiquantitatively and the number of positive cells/HPF, respectively. Kaplan-Meier product-limit estimators were used for median survival times, and log rank tests were conducted to assess the statistical significance of differences in survival curves across predefined patient strata.

	RESULTS

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Patient Characteristics.
Patient characteristics are shown in Table 1 . The mean age at diagnosis was 61.3 years, with a slight female preponderance (53%). Although all patients eventually developed metastatic disease, 62% presented with metastatic disease. Fifty-nine percent of patients had a primary site in the left colon, most commonly in the rectum. Seventy percent of patients had documented liver metastases, with 38% having metastatic disease confined only to the liver.

View larger version (11K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Kaplan-Meier estimate of overall survival (months). Survival calculated from time of diagnosis.

Association of IHC Staining with Response to Chemotherapy and Survival.
Positive p21waf1/cip1 IHC staining showed a greater probability of response to systemic chemotherapy. The mean number of p21waf1/cip1-positive cells for patients who obtained a partial response to systemic chemotherapy (10.0/HPF), as evaluated by the Samba system, was higher than the mean number of positive cells for patients who did not respond to chemotherapy (4.5/HPF; P = 0.03). The site of biopsy of the 30 patients evaluable for chemotherapy response were from the primary site in 19 cases and the metastatic site in 11 cases. Analysis of covariance to take into account the potential confounding factor of the site of biopsy showed a lack of interaction (P = 0.87) between tumor response to chemotherapy and specimen site of origin. Analysis of p21waf1/cip1 and response to chemotherapy adjusting for interaction with site of biopsy gave the same result, with a similar statistical significance (P = 0.04). There was no significant difference in the mean number of positively staining cells/HPF for p27kip1 (20 versus 18.7) or Ki-67 (20.2 versus 27.1) comparing patients who obtained a partial response to those who did not respond to chemotherapy.

No correlation was seen between the individual IHC stains and overall survival (Table 3) . Similarly, when the median survivals were evaluated based on dichotomized positive or negative IHC stains for Ki-67, p27kip1, or p21waf1/cip1, no significant differences in overall survival were observed. However, there was a trend toward inferior survival with CDI-positive specimens. This survival trend was more pronounced when the CDIs were analyzed together. The median survival was 10 months for patients with both p27kip1 and p21waf1/cip1 positivity, 15 months for patients with either p27kip1 or p21waf1/cip1 positivity, and 22 months for patients with neither p27kip1 nor p21waf1/cip1 positivity; but, these differences were not statistically significant (P = 0.33). The survival data were reanalyzed after censoring the seven patients who were alive and disease-free after potentially curative hepatic resection, given that these patients probably have a significantly different natural history from the majority of patients in the study. The trend of inferior survival with CDI-positive specimens reached statistical significance for p27kip1 (median survival 10 months positive stain versus 21 months negative stain, P = 0.01). The median survival when the CDIs were analyzed together was 10 months for patients with both p27kip1 and p21waf1/cip1 positivity, 13.5 months for patients with either p27kip1 or p21waf1/cip1 positivity, and 21.5 months for patients with neither p27kip1 nor p21waf1/cip positivity (P = 0.10).

	DISCUSSION

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Previous studies have assessed the correlation of CDIs with survival in early-stage colorectal cancer. Our study differs in that we evaluated the role of CDIs in the metastatic setting, not only in predicting survival, but also response to chemotherapy. The median survival in our group of patients was 17 months, which, as expected, is worse than median survivals of 95–180 months (15 , 16) in other studies evaluating CDIs in earlier-stage disease. We postulate that this more aggressive tumor biology in our patient population accounts for the paradoxical findings of this study.

The degree of positive IHC staining in our samples was 56% for Ki-67, 51% for p27kip1, and 36% for p21waf1/cip1. This is consistent with other studies in colorectal cancer showing 29–62% positive staining for Ki-67 (16, 17, 18) , 30–56% strong positive staining for p27kip1 (15 , 19 , 20) , and 38–69% positive staining for p21waf1/cip1 (21, 22, 23, 24, 25) . The semiquantitative observer method IHC scoring in our study was validated by the strong correlation with the Samba Cell Image Analysis System. Correlation coefficients of r = 0.91 for Ki-67, r = 0.86 for p27kip1, and r = 0.88 for p21waf1/cip1 were seen between these two methods.

Previous studies have found an association between p27kip1 expression and improved survival (15) , as well as lower-stage disease (19) in colorectal carcinoma. p27kip1 expression also has been associated with improved survival in breast cancer (26 , 27) , as well as non-small cell lung cancers (28) . Other studies of colorectal carcinoma have found p21waf1/cip1 and p27kip1 expression to have no relationship to overall survival (22) . No correlation has been seen between Ki-67 and overall survival (16) , disease-free survival (29) , or other prognostic parameters such as stage or grade (17 , 18 , 30) . In the current study, we found no correlation of overall or disease-free survival (data not shown) with individual IHC expression of Ki-67, p27kip1, or p21waf1/cip1 in metastatic colorectal carcinomas (Table 4) . However, by combining the CDI p27kip1 and p21waf1/cip1, a trend toward worse survival was observed (22 months for double negative versus 10 months for double positive). When patients who were alive and disease-free after hepatic metastatic resection were censored, positive p27kip1 staining indicated a worse survival (21 months negative stain versus 10 months positive stain; P = 0.01). A smaller study evaluating p27kip1 in metastatic colorectal carcinoma found a similar survival trend (31) . The reason for this paradoxical correlation of improved survival with negative CDI staining is unexplored in the metastatic setting because most previous studies have focused on earlier-stage disease. We hypothesize that the growth-inhibitory activity of the CDIs may be modulated in metastatic tumors, possibly through CDI inactivation, as has been suggested for p27kip1 (32) . The worse prognosis seen with high CDI expression may then be a result of these more aggressive tumors with an up-regulated, but functionally inactive, CDI. This would also be consistent with the positive correlation seen between Ki-67 and the CDIs (r = 0.48, P = 0.0001 for p27kip1; r = 0.48, P = 0.0001 for p21waf1/cip1) in our study.

Correlating response to chemotherapy with Ki-67 or the CDIs has not been extensively studied in colorectal carcinomas. CDIs may regulate chemotherapy resistance (33) and are important downstream effectors of p53-dependent growth arrest in response to DNA-damaging agents (3 , 34) . 5-FU, which inhibits DNA synthesis, may, therefore, be sensitive to differences in CDI expression. Expression of p21waf1/cip1 in ovarian cancer has been shown to decrease the cytotoxic effects of chemotherapy (35) . We, however, found no difference in Ki-67 or p27kip1 expression when comparing responders to nonresponders to 5-FU chemotherapy, which is consistent with a previously published abstract (31) . However, patients with partial responses had greater p21waf1/cip1 tumor expression compared with patients with stable or progressive disease (10 versus 4.5 positive cells/HPF; P = 0.03). The reason for this paradoxical finding is unknown. If cells up-regulate CDIs in an attempt to control proliferation only to later be inactivated, then the CDIs may function as surrogate markers of cellular proliferation in metastatic disease.

Our data also suggest a paradoxical correlation not previously described in colorectal cancer between Ki-67 and the CDIs. A positive correlation was seen between the number of Ki-67-positive cells and the number of positively staining cells for both p27kip1 and p21waf1/cip1. This correlation was consistent when analyzing different tumor sample sites, as well as different patient survival subgroups (Table 4) . Previous studies have shown the expected inverse relationship of Ki-67 with CDI in normal tissue (19 , 23 , 36) . But this inverse correlation may be lost as tumors exhibit a more aggressive growth pattern (19 , 20 , 37 , 38) . Paradoxical correlations of CDIs with cell cycle proliferation and tumor biology have been reported in other malignancies, including gastric (39) and breast (40) carcinomas. Our study demonstrates a positive correlation between Ki-67 and both CDI p27kip1 and p21waf1/cip1 in metastatic colorectal carcinoma. The inverse relationship between Ki-67 and CDI expression seen in normal tissue may convert to a positive correlation in the metastatic setting.

Several theories have been postulated to explain the dysregulation of CDIs in malignant tissue. Mutations in CDI genes are rare, so hypotheses have focused on the feedback mechanisms of the CDI induction pathway. They include: (a) escape of terminal differentiation and growth arrest by tumor cells becoming refractory to inhibitory signals from CDIs (39) ; (b) amplification of other cell cycle regulators, such as cyclin D or cyclin E, which may induce p27kip1 expression (19) ; (c) induction of wild-type p53 expression with subsequent p21waf1/cip1 induction, followed by selection of a p53 mutant cell line (38) ; (d) p53-independent induction of p21waf1/cip1 (41) , possibly through transforming growth factor ß (40) or other growth factors (8) ; and (e) altered down-regulation of the ubiquitin-mediated p27kip1 degradation mechanism (41) .

In conclusion, we found IHC patterns of the CDI p27kip1 and p21waf1/cip1 different from those commonly observed in normal tissue or lower-stage malignancy. We observed greater p21waf1/cip1 staining in patients with chemosensitive tumors, a trend toward greater CDI staining indicating worse survival, and a positive correlation of Ki-67 to both p27kip1 and p21waf1/cip1. These observations suggest that cell cycle regulatory processes may differ in those colorectal tumors destined to become metastatic, compared with more indolent early-stage cancers.

Our study is limited by the heterogeneous population of our samples because tumor blocks were obtained from primary, as well as metastatic, sites. Our finding of decreased p27kip1 expression in metastatic samples confirms a recently reported study (42) . However, our findings of p21waf1 predicting a greater likelihood of chemotherapy response and correlation between Ki-67 and the CDIs were consistent regardless of the sample location. The roles of other mediators that may help explain the mechanism behind our paradoxical findings in the CDI regulation pathway remain to be examined. These include the role of mutant p53, ubiquitin degradation of CDIs, p53-independent induction of factors such as transforming growth factor ß or other growth factors, and other unknown mediators that may render CDI functionally inactive. Confirmation of the predictive value of these IHC markers in a larger homogeneous colorectal cancer population will help define the role of cell cycle regulators in the life cycle of colorectal cancer and the potential use of these IHC markers in establishing prognosis and making treatment decisions.

	ACKNOWLEDGMENTS

 
We thank Leslie E. Blumenson for valuable biostatistical suggestions.

	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 Divisions of Medical Science and Population Science, Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 19111. Phone: (215) 728-2450; Fax: (215) 728-3639; E-mail: nj_meropol{at}fccc.edu

² The abbreviations used are: CDK, cyclin-dependent kinase; CDI, CDK inhibitor; 5-FU, 5-fluorouracil; IHC, immunohistochemistry; HPF, high-powered field.

Received 10/20/98; revised 2/ 8/99; accepted 2/ 9/99.

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