Purpose: Although the decreased expression of p27kip-1, a cyclin-dependent kinase inhibitor, has been correlated with advanced tumor stage and short survival of patients with colorectal adenocarcinomas (CRCs), its prognostic value based on the tumor site, tumor stage, and patient ethnicity was not assessed. Therefore, in this study, we investigated whether the prognostic value of p27kip-1 expression varies with the tumor site, tumor stage and patient ethnicity.

Experimental Design: We evaluated 206 (85 African Americans and 121 Caucasians) archival tissue specimens of first primary CRCs for immunohistochemical expression of p27kip-1, and its prognostic significance was analyzed using univariate Kaplan-Meier and multivariate Cox regression survival methods.

Results: Although, similar proportion of CRCs with decreased p27kip-1 expression was observed in all stages (range, 26–36%), the decreased p27kip-1 expression has been shown as a marker of poor prognosis only for patients with stage III tumors both in univariate (log-rank test, P = 0.014) and multivariate (hazard ratio = 3.2, 95% confidence interval = 1.3–7.7; P = 0.01) survival analyses. The decreased expression of p27kip-1 was associated with a high histologic grade (P = 0.016) in stage II CRCs, and with distal tumors (P = 0.001), tumor invasion (P = 0.044), and with local recurrence (P = 0.008) in stage III CRCs.

Conclusions: No prognostic significance was found for p27kip-1 expression in stages I, II, or IV CRCs, and its prognostic value was not associated with either ethnicity or tumor location. These studies suggest that decreased expression of p27kip-1 is an indicator of poor prognosis and aids in identifying a subset of patients with aggressive forms of stage III CRCs.

Colorectal adenocarcinoma (CRC) is the third leading cause of cancer associated deaths in both men and women in the United States (1). The stage of the tumor at the time of diagnosis remains one of the powerful indicators of aggressiveness of this disease and aids in predicting the survival of patients. However, pathological stage of CRC may not be the best indicator of clinical outcome because groups of patients with tumors of identical stage have different treatment responses and clinical outcomes. Recent studies, however, have reported that the molecular changes that are associated with the aggressiveness of CRC have different biological consequences based on patient age, race, and ethnicity, as well as the anatomical location of the tumor in the colorectum even after controlling for the tumor stage (2, 3, 4, 5, 6, 7, 8).

Aberrations in the molecular components of cell cycle checkpoints are a common feature of many human malignancies, and several of these molecules are known to have prognostic significance in CRC. Of these, p27kip-1, a cyclin-dependent kinase inhibitor that regulates progression of cells from G1 into S phase in a cell cycle is being increasingly recognized as an important factor for determining the biological behavior of invasive tumors. Decreased expression of p27kip-1 has been correlated with advanced tumor stage and short patient survival in several human cancers, including CRC (9, 10, 11). Both biochemical and immunohistochemical evaluation of p27kip-1 expression in human CRCs have shown diverse results in relation to its prognostic value (Table 1).

In a recent study, it was noted that both benign (histologically normal) and neoplastic tissues expressed variable amounts of the p27kip-1 protein, as assessed by Western blot analyses; however, these differences were not statistically significant (12). In another study, no relationships were found between p27kip-1 expression and gender, age, tumor location, growth pattern, and expression of other molecular markers, including p53, p73, and DCC (13); however, in a subset of patients, it was reported that the lack of p27kip-1 expression is an indicator of short survival for patients with early-stage CRCs (Duke Stage B) located in the proximal colon (13, 14). Additional larger studies are required to evaluate the biological consequences of expression of p27kip-1 in CRCs in relation to tumor stage, anatomical location of the tumor, and patient ethnicity.

In this study, we evaluated a large CRC patient population [total = 206 (85 African Americans and 121 Caucasians)] for phenotypic expression of p27kip-1 to assess its prognostic significance based on tumor stage, anatomical location, and patient ethnicity.

Formalin-fixed archival tissue blocks of CRCs from 504 patients (204 African Americans and 300 non-Hispanic Caucasians) were collected randomly from archives of the University of Alabama at Birmingham and the Veterans Administration Hospitals. All these patients underwent surgery for adenocarcinoma of the colorectum with curative or palliative intent. The selection of archival tissues was restricted to patients with first primary CRCs resected between 1981 and 1993. Because of limitations of the resources, for the current study, subsets of 206 patients with CRCs (85 African Americans and 121 Caucasians) were selected randomly from the initial group of 504 without the knowledge of clinical outcome. The pathological characteristics of CRCs, patient demographics, and patient follow-up information were collected from the pathology reports, patient charts, and tumor registries, respectively. Tissue sections stained with H&E were evaluated to determine the grades of the tumors, which were categorized as either well, moderately, or poorly differentiated as suggested by WHO (15). Of these CRCs, 41 were classified as well, 134 as moderately, and 31 as poorly differentiated. Tumor stage was based on the classification of the American Joint Committee on Cancer classification (16, 17). There were 42 stage I, 69 stage II, 53 stage III, and 42 stage IV patients in this study. The anatomical locations of the tumors and their grouping into proximal and distal colonic and the rectal tumors were performed as described in earlier studies (18, 19).

During our initial process of selection, patients with multiple primaries, with multiple malignancies, or with family or personal histories of cancer were excluded; therefore, our study population consists only patients with sporadic first primary CRCs. We included patients who have undergone surgery alone as a therapeutic intervention, but we excluded patients who received pre- or postsurgical chemo- or radiation therapy in our study to control for treatment bias.

Immunohistochemical Staining.

Paraffin sections (5-μm thickness) of blocks representative of both tumor and benign tissues of each case were mounted on Superfrost/Plus slides (Fisher Scientific, Pittsburgh, PA). The immunohistochemical staining procedure was carried out as described earlier (2, 3, 4). Specifically, the tissues were incubated for 1 h at room temperature with monoclonal antibody to p27kip-1 (clone 1B4; Novacostra, Newcastle upon Tyne, United Kingdom) after antigen recovery by microwave boiling in citrate buffer (pH 7.4) for 10 min. The remainder of the staining procedure and the protocol for the antigen retrieval were described in detail elsewhere (2, 3, 4). Hematoxylin was used as a counterstain. A multitissue control block with colonic lymph nodes and benign colonic epithelium was used as the positive control.

Assessment of p27kip-1 Staining.

The staining assessment was performed by two authors (U. M. and N. J.) independently but together to ensure consistency in evaluation. The proportion of p27kip-1-expressing cells varied from 0 to 100%, and the intensity of nuclear staining also varied from weak to strong. Therefore, the percentage of cells at each intensity of staining were recorded on a scale of 0 (no staining) to +4 (strongest staining) by two authors independently. If there was a disagreement in their assessment, they resolved before combining the individual scores. The immunostaining scores of the two authors were combined to obtain an average percent positive score as well as staining intensity for each case. Using this method of staining assessment, we evaluated expression of p27kip-1 in benign colonic epithelium away and adjacent to invasive malignant lesion. A cutoff value of immunostaining staining score of ≥0.7 plus at least 50% of malignant cells immunostaining was used to classify tumors with increased expression (≥50% cells positive plus ≥0.7 staining intensity score) or with decreased expression (<50% positivity or <0.7 intensity score) of p27kip-1 antigen. This cutoff value was the median value of p27kip-1 staining in the benign epithelium (average of away and adjacent to invasive lesion). Staining in lymphocytes and uninvolved colonic epithelium within the tissue sections served as internal positive controls for the expression of p27kip-1.

Statistical Analyses.

The association between p27kip-1 and clinicopathological or biological characteristics was analyzed using the χ2 test (20). Ps were calculated, and significance was assessed at an α level of 0.05. The median follow-up period of the complete study population of 206 patients was 4.7 years (range, <1–18 years). The period from the date of resection to the date of death or last contact (if alive) was used for survival analyses. Outcome analyses were based on patients who were alive or had died of CRC as described previously (2, 3, 4, 5, 6). Univariate overall survival was obtained using Kaplan-Meier estimates (21). The log-rank test was used to compare Kaplan-Meier survival curves based on the status of p27kip-1 expression. Separate multivariate Cox regression models (22) were built for patients with stages I, II, III, and IV CRCs, and the survival of patients was compared with and without p27kip-1 expression after adjustments for confounding variables. The clinical confounding variables of CRC used in these analyses were pT, pN, and M components of Tumor-Node-Metastasis stage, age, sex, ethnicity, tumor location, tumor size, and differentiation. A stepwise model-building procedure was used to determine the significant factors in predicting survival related to CRC. The interactions between p27kip-1 expression with other significant variables were tested for significance. Hazard ratios and 95% confidence intervals were calculated to identify the risk factors.

Demographic and Clinicopathological Characteristics of the Patient Population.

The clinical, pathological, and biological features of the 206 patients are reported in Table 2. Patients with different tumor stages (I, II, III, and IV) were evenly distributed in this population. The distribution of CRCs in the colorectum was 43, 37, and 20% in the proximal colon, the distal colon, and the rectum, respectively. Mean age at the time of surgery was 65.4 years (range, 26.0–70.0 years), and the median survival was 5.58 years (95% confidence interval = 3.59–9.0). There was a predominance of males in our study (124 of 206, 60%) because the majority of patients treated at the Veterans Administration Hospital of Birmingham were males. At the last follow-up, the proportion of patients alive was 49% (101 of 206), dead because of colorectal neoplasia was 32% (66 of 206), and died because of other causes was 19% (Table 2).

p27kip-1 Expression and Its Association with Different Clinicopathological Parameters.

As observed in several other studies (Table 1), the expression of p27kip-1 was predominantly in the nucleus; however, in a small proportion of cells (∼20%) in a tumor tissue section p27kip-1 was localized in the cytoplasm. For this study, we considered only distinct nuclear expression of p27kip-1. Increased phenotypic expression of p27kip-1 was detected in 70% (145 of 206) of CRCs (Table 2).

The analysis of correlation between the proportions of CRCs with increased expression of p27kip-1 with different clinicopathological parameters in each tumor stage group was shown in Table 3. The incidence of CRCs with decreased p27kip-1 expression was 36, 28, 30, and 26% in stages I, II, III, and IV, respectively. There was a significant association between p27kip-1 expression and the degree of histological tumor differentiation in patients with stage II CRCs, which is that its expression was gradually decreased from well to poorly differentiated CRCs (χ2, P = 0.016; Table 3). In stage III patient group, the expression of p27kip-1 was significantly associated with the anatomical location of CRCs, the increased p27kip-1 was observed in CRCs located in the proximal colon (91%), and in the rectum (77%) as compared with the distal colon CRCs (39%; χ2, P = 0.001). In this patient group, the extent of expression of p27kip-1 decreased as the extent of invasion of tumor into the bowel increased (pT1 to pT4, χ2, P = 0.044). Also in the stage III cases, the decreased expression of p27kip-1 was significantly associated with an increasing incidence of recurrence (χ2P = 0.008) and with clinical outcome; specifically, a higher proportion of patients with increased p27kip-1 expression were alive at the time of last follow-up (χ2, P = 0.018; Table 3).

We also used a multiple logistic regression model to simultaneously evaluate the association of each parameter with p27kip-1 in all four tumor stage groups (stages I, II, III, and IV). This multivariate logistic analysis will address the issue of multiple testing for each individual parameter, and only one overall model is used to evaluate the clinical parameters simultaneously. In this analysis of association of variables with p27kip-1 positivity, we found similar results as reported in Table 3; specifically, tumor differentiation (P = 0.008) remained significantly associated with increased expression of p27kip-1 in patients with stage II CRCs (data not shown). Despite a small number of patients in stage III group, we still observed significance or an indication of trend of the association of p27kip-1 expression with patient outcome (P = 0.008), tumor invasion (pT; P = 0.062), and tumor location (P = 0.095; data not shown).

Survival Analyses.

Kaplan-Meier univariate survival analysis on the complete patient population (n = 206) demonstrated no significant differences in their overall survival between the patient groups with decreased or increased levels of phenotypic p27kip-1 expression (log rank, P = 0.152; data not shown). Because, this study population has a considerable number of African American patients, we also conducted survival analyses to assess the significance of p27kip-1 expression based on patient race; the analyses of clinical outcome demonstrated that in either of the racial groups, the cumulative survival curves of low and increased levels of p27kip-1 expression did not show statistically significant differences (log rank, P = 0.273 and P = 0.183, for African Americans or Caucasians, respectively; data not shown). Univariate survival analyses based on p27kip-1 expression and on tumor location also did not show significant differences for all three anatomical sites (log rank, P = 0.377, P = 0.248 and P = 0.631, for proximal, distal, and rectal tumors, respectively; data not shown).

We also analyzed the significance of p27kip-1 expression in the complete study population based on the tumor stage and found that patients with tumors exhibiting decreased p27kip-1 expression had significantly shorter overall survival only in the stage III patient-group (log rank, P = 0.014; Fig. 1,C). Similar analyses in stage I, stage II, and stage IV groups did not show statistically significant differences between patients with decreased or increased levels of p27kip-1 expression (Fig. 1 A, B, and D).

The multivariate regression models built separately for each tumor stage demonstrated that decreased expression of p27kip-1 was an independent indicator of poor prognosis only for patients with stage III CRCs. The hazard ratios and 95% confidence interval for stage III was 3.2 and 1.3–7.7 (P = 0.01), respectively (Table 4). No prognostic significance was found for p27kip-1 expression in stages I, II, or IV CRCs (Table 4). In multivariate regression model, which included all groups of patients (stage I–IV together), the decreased p27kip-1 expression was an independent marker of poor overall survival; however, it was only marginally significant (hazard ratio 0.7, 95% confidence interval, 0.4–1.0; P = 0.058). In this overall model, older age, African American race, high tumor grade, bowel wall invasion, regional lymph node invasion, and distant metastasis were independent predictors of overall survival (Table 4).

Our study population had similar proportions of CRCs with the decreased levels of p27kip-1 expression in all stages (stages I–IV); however, decreased expression of p27kip-1 was associated significantly with short patient survival of patients with stage III tumors only. Our findings also demonstrated that stage III CRCs, which were located in the distal colon, are less likely to express p27kip-1 at high levels and that low levels of p27kip-1 expression were correlated significantly with the extent of invasion of tumor to other adjacent organs (pT4) and with local recurrence. These results suggest that p27kip-1 expression may be used to identify more aggressive phenotypes of stage III CRCs.

Several abnormalities in the molecular features of cell cycle checkpoints have been implicated in several human malignancies, including CRC; however, the prognostic values of many of these cell cycle antigens are controversial in CRC. For example, wild-type p53 eliminates genetically unstable cells that enter the cell cycle, and a mutant p53 will allow these cells to progress to a malignant phenotype. The role of p53 in colorectal neoplasia has been well studied; however, its value in predicting the clinical outcome has been controversial. Nevertheless, some recent studies from others and our laboratories evaluated its prognostic value based on tumor location and ethnicity and have demonstrated that nuclear accumulation of p53 is an independent prognostic indicator of patients with proximal tumors (23, 24, 25), specifically, for Caucasian patients (3, 7, 8). These novel strategies of assessing the predictive value of a molecular marker of colorectal neoplasia would yield valuable information, which will be useful in identifying aggressive phenotypes.

Increased expression of p27kip-1in mammalian cells induces a G1 block of the cell cycle (26, 27) and high levels of p27kip-1 found in quiescent cells suggest that expression of p27kip-1 also plays a role in maintaining cells in G0(28, 29). As with other Kip proteins, p27kip-1 has a nuclear localization signal in its -COOH terminus. Loss of p27kip-1 expression may result in the development and/or progression of tumors; however, this loss of expression does not appear to result from gene mutation (30, 31, 32). p27kip-1 is posttranslationally regulated by an ubiquitin-proteasome-dependent degradation pathway, and p27kip-1 also is regulated by cell-cell contacts (33). p27kip-1 expression can also be regulated by transcriptional mechanism. In CRCs, reduced expression of p27kip-1 in the metachronous metastases compared with the corresponding primary tumor suggests that the down-regulation of p27kip-1 in circulating malignant cells may accentuate the ability of these cells to metastasize to liver (34). Also, accumulation of p27kip-1 has been implicated in the differentiation of different varieties of cells (35, 36). In CRCs, Loda et al. (9) demonstrated that absent or low levels of p27kip-1 are caused by increased protease-mediated degradation rather than from altered gene expression.

Variable levels of p27kip-1 expression have been observed in benign and malignant epithelial components of the colorectum (9). The role of this molecular marker in assessing the aggressiveness of CRCs and in predicting the clinical outcome of patients with colorectal neoplasia has been examined by several investigators and the majority of these studies, which are cited in the Medline or PubMed (until the end of July 2003) are listed in Table 1. This review table clearly indicates discrepancy of prognostic significance of p27kip-1 expression in colorectal neoplasia. Several studies reported that the lack of p27kip-1 expression was associated with short patient survival (9, 11, 13, 14, 37, 38, 39, 40); however, such an association was not found in some other studies (41, 42, 43, 44, 45). In some studies, it was suggested that p27kip-1 was an independent predictor of patients specifically with early stage CRCs (stages I and II; Refs. 9, 13, 39), whereas, studies by Tenjo et al. (11) observed that p27kip-1 expression as an independent prognostic marker for patients with stage III CRCs.

In our study, all patients have received a uniform treatment of surgical resection either with curative or palliative intents and none of them were treated with chemotherapeutic agents such as 5-fluorouracil or with any other neoadjuvant or adjuvant therapies. Adjuvant chemotherapies were not common practices at the beginning of our study in 1981, but use of adjuvant therapy for CRC increased in 1988. Although, the Food and Drug Administration has approved leucovorin for use in combination with 5-fluorouracil to prolong survival in the palliative treatment of patients only with advanced CRC (stage IV) in 1952, the adjuvant treatment in combination with 5-fluorouracil after surgical resection in patients with Dukes’ Stage C colon cancer was approved only in 1990.3 Therefore, we did not identify many patients who received chemotherapy during our study period and as stated in methods section, we excluded patients who received pre- or postsurgical treatment. Thus, using our study population, we evaluated the prognostic value of p27kip-1 expression without treatment biases. Also, we assessed its prognostic value based on race, the tumor stage, and tumor site.

In this study, similar proportions of CRCs with the decreased levels of p27kip-1 expression were noted in all stages (26–36%). Similar to studies by Palmqvist et al. (14), we also did not see a significant association between p27kip-1 expression and the tumor differentiation; however, when stratified by tumor stage, our study found that in stage II CRCs, the decreased expression of p27kip-1 was associated with poorer histological differentiation. This association, however, did not impact overall survival of patients with stage II CRCs. In stage III CRCs, p27kip-1 expression was significantly correlated with tumor location, i.e., distal tumors exhibited decreased levels of p27kip-1 expression and the extent of p27kip-1 expression decreased as the invasion of tumor into the lower layers of the bowel wall increased (pT1 to pT4). Similar to our findings, Ciaparrone et al. (46) also found a significant correlation between p27kip-1 expression and tumor grade with well and moderately differentiated CRCs expressing higher p27kip-1, whereas the poorly differentiated CRCs had significantly lower p27kip-1 expression. Furthermore, we observed that tumors, which exhibited decreased expression of p27kip-1, had higher incidence of local recurrence in patients with stage III CRCs, and such an association also was reported in prostatic carcinoma (47).

Our study demonstrated in both univariate and multivariate analyses that the increased expression of p27kip-1 was associated with improved survival of patients with stage III CRCs but not with stages I, II, or IV tumors. These results are in contrast to some of the previously published studies where it was shown that decreased p27kip-1 expression was associated with poor prognosis of patients with stage II tumors (9, 13) tumors without nodal involvement (stage I plus II; Ref. 39). However, similar to our findings, studies by Tenjo et al. (11) observed that p27kip-1 is an independent prognostic marker for patients with stage III CRCs. Although, the exact reasons for these contradictory results are not known, the admixture of our patient population for race and for the tumor site may be affecting these findings. Also, it is possible that in our study, we used monoclonal antibody clone 1B4 and established rigorous standards of evaluation as well as cutoff values (≥50% cells with immunostaining score value ≥ 0.7), and only nuclear p27kip-1 expression was considered in staining evaluation. Whereas, studies by Zhang et al. (13) used a different antibody clone (SX53G8), different cutoff values for categorizing tumors as high expressors of p27kip-1 (>10%), considered only cytoplasmic p27kip-1 staining for survival analyses and suggested that decreased expression of p27kip-1 was associated with poor survival of patients with Dukes B tumors in univariate analysis. Unlike studies of Zhang et al. (13) and Kobayashi et al. (42), ours as well as several other studies (Table 1) have used nuclear p27kip-1 staining for survival analyses. The higher cutoff values used in our study to categorize CRCs into high and low expressors are similar to other investigators; additionally, we used more rigorous standards of staining evaluation that included two independent observers to reduce individual bias. Besides these technical variations, other confounders of patient survival, including the adjuvant treatment therapies received and racial admixture specifically in studies of the United States, were not clearly described in the majority of earlier studies. Moreover, these contradictory findings also suggest that the future larger studies should consider the tumor stage and the anatomical location of tumor in the colorectum, race, and ethnicity in evaluating the prognostic value of p27kip-1 expression in colorectal neoplasia. This has led us to propose that molecular and demographic features should be added to the staging of colorectal tumors (7, 8).

It is well established in the literature that patients with higher tumor stage (stages III and IV) have a poorer prognosis than stage I or II and staging is used in clinical practice for therapeutic decision making. To add to this concept, multivariate survival analyses of our study also noted significant differences in patient outcome based on age (≥65 years), tumor differentiation (poor differentiation), and ethnicity (African Americans) even after controlling for pathological stage of the tumor (Table 4).

In this study, we have shown both in univariate as well as in multivariate analyses that decreased expression of p27kip-1 is a significant predictor of poor clinical outcome of stage III patients who received surgery alone as a treatment regimen. Our findings also suggest that in stage III tumors, p27kip-1 expression will determine local recurrence. Also, these higher stage tumors exhibit underlying molecular heterogeneity that might determine the behavior of selected tumors. In our studies, the prognostic value of p27kip-1 expression was not associated with race or the anatomical location of the tumor. As demonstrated in our prior studies (2, 3, 4, 5, 6, 7, 8), the use of multiple molecular markers, including p27kip-1 expression, may help in identifying more aggressive tumors and in predicting the clinical outcome, thus, might help clinical oncologists in designing or selecting more aggressive therapies for subgroups of patients with colorectal cancer.

Grant support: National Cancer Institute Grants 1RO1 CA98932-01, 1RO3 CA097542-01, and CA86359-04, which are funded by NIH.

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.

Requests for reprints: Upender Manne, Assistant Professor, Department of Pathology, University of Alabama at Birmingham, 565-LHRB, Building 701, 19th Street South, Birmingham, AL, 35294-0007. Phone: (205) 934-4276; Fax: (205) 975-9927; E-mail: manne@path.uab.edu

3

Internet address: http://www.fda.gov/cder/cancer/druglistframe.htm.

Fig. 1.

Kaplan-Meier analysis showing the overall survival of colorectal adenocarcinoma patients categorized according to the stage of the tumor and status of p27kip-1 expression. The statistical significance of the difference between curves of p27kip-1 high expressors (increased expression) p27kip-1 low expressors (decreased expression) was compared in stage I (A), stage II (B), stage III (C), and stage IV (D) patient groups. Ps were calculated by the log-rank test.

Fig. 1.

Kaplan-Meier analysis showing the overall survival of colorectal adenocarcinoma patients categorized according to the stage of the tumor and status of p27kip-1 expression. The statistical significance of the difference between curves of p27kip-1 high expressors (increased expression) p27kip-1 low expressors (decreased expression) was compared in stage I (A), stage II (B), stage III (C), and stage IV (D) patient groups. Ps were calculated by the log-rank test.

Close modal
Table 1

Review of description of p27kip-1 expression and its prognostic value in colorectal adenocarcinomas

Reference/country of study populationTumor stages and size of study populationAnatomic location of tumorTissue/fixativeARaMonoclonal antibody clone analyzed & dilutionStaining localization & cut-off
Present studyn = 206ColorectalParaffin/Yes1B4Nu
 United States (AL) All stages  Formalin  1:30 0.7 ISS+ ≥50% 
M. Loda et al (1997) n = 149 Colorectal Paraffin/ Yes 57 Nu & Cy 
 United States (MA) All stages  Formalin  1:200 >50% = High 
      ≤50% = Low 
      0% = NE 
      Present = >0% 
J.D. Cheng et al (1999) n = 66 Colorectal Paraffin/ Yes 57 Nu 
 United States (NY) All stages  Formalin  1:400 6–100% = positive 
      0–5% = negative 
R. Palmqvist et al (1999) n = 89 Colorectal Paraffin/ Yes 57 Nu 
 Sweden (Umea) Duke’s A, B, C  Formalin  1:200 <50% = +, ++ 
      ≥50% = +++, ++++ 
C. Belluco et al (1999) n = 124 Colorectal Paraffin/ Yes 57 Nu 
 Italy (Padova) Stages I, II, III  Formalin  1:600 >50% = High 
      ≤50% = Low 
      0% = Absent 
K. Gunther et al (2000) n = 164 Rectal Paraffin/ Yes 57 Nu & Cy 
 Germany (Magdeburg) All stages  Formalin  1:200 −, +, ++ = NE-moderate 
      +++ = strong 
J. Yao et al (2000) n = 136 Colorectal Paraffin/ Yes 57 Nu & Cy 
 Singapore All stages  Formalin  1:200 0–3 = No/Week 
      4–6 = strong positive 
T. Tenjo et al (2000) n = 171 Colorectal Paraffin/ Yes 57 Nu 
 Japan (Osaka) All stages  Formalin  1:200 ≥46.8% = positive 
      <46.8% = negative 
H. Zhang et al (2001) n = 178 Colorectal Paraffin/ Yes SX53G8 Cy 
 Sweden (Linkoping) All stages  Formalin  1:50 >10% = positive 
M. Kobayashi et al (2002) n = 221 Colorectal Paraffin/ Yes SX53G8 Cy 
 Japan (Mie) All stages  Formalin  1:200 >11.7% = High 
      ≤11.7% = Low 
H.A. Rossi et al (2002) n = 187 Colorectal Paraffin/ Yes 57 Nu & Cy 
 United States (MA) Stages I, II, III  Formalin  1:50 <25% = Low 
      ≥25% = High 
J.A. McKay et al (2002) n = 249 Colorectal Paraffin/ Yes 1B4 Nu & Cy 
 UK (Scotland) All stages  Formalin  1:40 >50% = High 
      ≤50% = Low 
O. Schwandner et al (2002) n = 160 Rectal Paraffin/ Yes SX53G8 Nu 
 Germany (Lubeck) Stages I, II, III  Formalin  1:35 >10% = positive 
Hoos et al (2002) n = 97 Rectal Paraffin/ Yes DCS72 Nu 
 United States (NY) T2 = 48  Formalin  1:500 >20% = positive 
 T3 = 49     <20% = negative 
      Cyto 
      Normal, abnormal 
Reference/country of study populationTumor stages and size of study populationAnatomic location of tumorTissue/fixativeARaMonoclonal antibody clone analyzed & dilutionStaining localization & cut-off
Present studyn = 206ColorectalParaffin/Yes1B4Nu
 United States (AL) All stages  Formalin  1:30 0.7 ISS+ ≥50% 
M. Loda et al (1997) n = 149 Colorectal Paraffin/ Yes 57 Nu & Cy 
 United States (MA) All stages  Formalin  1:200 >50% = High 
      ≤50% = Low 
      0% = NE 
      Present = >0% 
J.D. Cheng et al (1999) n = 66 Colorectal Paraffin/ Yes 57 Nu 
 United States (NY) All stages  Formalin  1:400 6–100% = positive 
      0–5% = negative 
R. Palmqvist et al (1999) n = 89 Colorectal Paraffin/ Yes 57 Nu 
 Sweden (Umea) Duke’s A, B, C  Formalin  1:200 <50% = +, ++ 
      ≥50% = +++, ++++ 
C. Belluco et al (1999) n = 124 Colorectal Paraffin/ Yes 57 Nu 
 Italy (Padova) Stages I, II, III  Formalin  1:600 >50% = High 
      ≤50% = Low 
      0% = Absent 
K. Gunther et al (2000) n = 164 Rectal Paraffin/ Yes 57 Nu & Cy 
 Germany (Magdeburg) All stages  Formalin  1:200 −, +, ++ = NE-moderate 
      +++ = strong 
J. Yao et al (2000) n = 136 Colorectal Paraffin/ Yes 57 Nu & Cy 
 Singapore All stages  Formalin  1:200 0–3 = No/Week 
      4–6 = strong positive 
T. Tenjo et al (2000) n = 171 Colorectal Paraffin/ Yes 57 Nu 
 Japan (Osaka) All stages  Formalin  1:200 ≥46.8% = positive 
      <46.8% = negative 
H. Zhang et al (2001) n = 178 Colorectal Paraffin/ Yes SX53G8 Cy 
 Sweden (Linkoping) All stages  Formalin  1:50 >10% = positive 
M. Kobayashi et al (2002) n = 221 Colorectal Paraffin/ Yes SX53G8 Cy 
 Japan (Mie) All stages  Formalin  1:200 >11.7% = High 
      ≤11.7% = Low 
H.A. Rossi et al (2002) n = 187 Colorectal Paraffin/ Yes 57 Nu & Cy 
 United States (MA) Stages I, II, III  Formalin  1:50 <25% = Low 
      ≥25% = High 
J.A. McKay et al (2002) n = 249 Colorectal Paraffin/ Yes 1B4 Nu & Cy 
 UK (Scotland) All stages  Formalin  1:40 >50% = High 
      ≤50% = Low 
O. Schwandner et al (2002) n = 160 Rectal Paraffin/ Yes SX53G8 Nu 
 Germany (Lubeck) Stages I, II, III  Formalin  1:35 >10% = positive 
Hoos et al (2002) n = 97 Rectal Paraffin/ Yes DCS72 Nu 
 United States (NY) T2 = 48  Formalin  1:500 >20% = positive 
 T3 = 49     <20% = negative 
      Cyto 
      Normal, abnormal 
Table 1A

Continued

Percent positivity (considered for survival analysis)Follow-upPrognostic value of p27Kip-1Independent prognostic markers
UnivariateMultivariate
I = 64 4.7 yrs All P = NS All P = 0.058 p27Kip-1, pT, pN, 
II = 72 (median) III P = 0.014 III P = 0.010 Distant metastasis, 
III = 70    Differentiation, 
IV = 74    Ethnicity, age, 
   All-II 9 yrs All P = 0.0048 P = 0.003 p27Kip-1 
>50% = 29 7 (median) II P = 0.0002   
Present = 34 32  All P = 0.0014   
  II P = 0.0017   
Positive = 51 3.2 yrs (median) NS NP  
≥50% = 56 NA P = 0.0069 P = 0.01 p27Kip-1, gender, 
    Tumor type, 
    Location, 
    Growth pattern 
Present = 86.3 4.6 yrs DFS P = 0.005 p27Kip-1
 (median) All P < 0.000  Tumor-Node- 
  I–II P = 0.014  Metastasis stage 
Cy +++ = 25.6 6.2 yrs Cy P = 0.0185 Cy P = 0.0552 Cytoplasmic 
Nu +++ = 51.8 (median) Nu-NS  p27Kip-1 
I/II = 85.4 5 Years All P = 0.04 NP  
III/IV = 76.1  (5-yr Overall)   
  III/IV-NS   
0 = 57.7b 5.6 yrs P < 0.0001 All P = 0.0146 p27Kip-1
I = 48.1b (median) (5-yr Overall) II,III P = 0.0375 Tumor stage 
II = 47.2b     
III = 42.9b     
IV = 41.6b     
Normal = 35 5.3 yrs All P = 0.08 NP  
Primary = 49 (mean) Duke’s B P = 0.03   
Metastatic = 32  Proximal colon P = 0.05   
High = 52 3.4 yrs (median) P = 0.0577 NP  
High = 33 3.9 yrs P = 0.02 P = 0.01 p27Kip-1, p53, 
 (median)   Tumor stage, 
    Location, 
    Post-op chemotherapy 
High = 21.3 2.9 yrs NS NS p53, 
 (median)   Dukes Stage 
Positive = 44 3.2 yrs NS NS p21Waf1/Cip1
 (mean) (Overall)  p53, 
  P = 0.0016  Union International 
  (5-Yr DFS)  Contre Cancer stage 
Nu positive = 34.1 6.2 yrs Nu-NS NS  
Cy positive = 23.3 (median) Cy-NS NS  
Percent positivity (considered for survival analysis)Follow-upPrognostic value of p27Kip-1Independent prognostic markers
UnivariateMultivariate
I = 64 4.7 yrs All P = NS All P = 0.058 p27Kip-1, pT, pN, 
II = 72 (median) III P = 0.014 III P = 0.010 Distant metastasis, 
III = 70    Differentiation, 
IV = 74    Ethnicity, age, 
   All-II 9 yrs All P = 0.0048 P = 0.003 p27Kip-1 
>50% = 29 7 (median) II P = 0.0002   
Present = 34 32  All P = 0.0014   
  II P = 0.0017   
Positive = 51 3.2 yrs (median) NS NP  
≥50% = 56 NA P = 0.0069 P = 0.01 p27Kip-1, gender, 
    Tumor type, 
    Location, 
    Growth pattern 
Present = 86.3 4.6 yrs DFS P = 0.005 p27Kip-1
 (median) All P < 0.000  Tumor-Node- 
  I–II P = 0.014  Metastasis stage 
Cy +++ = 25.6 6.2 yrs Cy P = 0.0185 Cy P = 0.0552 Cytoplasmic 
Nu +++ = 51.8 (median) Nu-NS  p27Kip-1 
I/II = 85.4 5 Years All P = 0.04 NP  
III/IV = 76.1  (5-yr Overall)   
  III/IV-NS   
0 = 57.7b 5.6 yrs P < 0.0001 All P = 0.0146 p27Kip-1
I = 48.1b (median) (5-yr Overall) II,III P = 0.0375 Tumor stage 
II = 47.2b     
III = 42.9b     
IV = 41.6b     
Normal = 35 5.3 yrs All P = 0.08 NP  
Primary = 49 (mean) Duke’s B P = 0.03   
Metastatic = 32  Proximal colon P = 0.05   
High = 52 3.4 yrs (median) P = 0.0577 NP  
High = 33 3.9 yrs P = 0.02 P = 0.01 p27Kip-1, p53, 
 (median)   Tumor stage, 
    Location, 
    Post-op chemotherapy 
High = 21.3 2.9 yrs NS NS p53, 
 (median)   Dukes Stage 
Positive = 44 3.2 yrs NS NS p21Waf1/Cip1
 (mean) (Overall)  p53, 
  P = 0.0016  Union International 
  (5-Yr DFS)  Contre Cancer stage 
Nu positive = 34.1 6.2 yrs Nu-NS NS  
Cy positive = 23.3 (median) Cy-NS NS  
a

AR, antigen retrieval; ISS, immunostaining score; Nu, nucleus; Cy, cytoplasm; DFS, disease-free survival; OS, overall survival; n, sample size; NE, nonexpressors; NP, not performed; NS, not significant; NA, not available.

b

Presented as mean expression of p27Kip-1/tumor stage.

Table 2

Clinicopathological characteristics and expression of p27Kip-1 in colorectal adenocarcinomas

No.(%)
Sex   
 Male 124 (60) 
 Female 82 (39) 
Age (years)   
 <65 90 (44) 
 ≥65 116 (56) 
Ethnicity   
 African Americans 85 (59) 
 Caucasians 121 (41) 
Tumor location   
 Proximal colon 89 (43) 
 Distal colon 76 (37) 
 Rectum 41 (20) 
Tumor size (cm)a   
 ≤5 128 (63) 
 >5 74 (37) 
Tumor differentiation   
 Well 41 (20) 
 Moderate 134 (65) 
 Poor 31 (15) 
Tumor stage (Union International Contre Cancer)   
 I 42 (20) 
 II 69 (34) 
 III 53 (26) 
 IV 42 (20) 
Tumor type   
 Mucinous 25 (12) 
 Nonmucinous 181 (88) 
pT component of stage (bowel wall invasion)   
 pTx (<1) 
 pT1 12 (6) 
 pT2 44 (21) 
 pT3 111 (54) 
 pT4 38 (18) 
pN component of stage (regional lymph node invasion)   
 pNx (4) 
 pN0 113 (55) 
 pN1 50 (24) 
 pN2 25 (12) 
 pN3 11 (5) 
M component of stage (distant metastasis)   
 M0 164 (80) 
 M1 42 (20) 
Cancer relapse (at follow-up)   
 Absent 68 (33) 
 Present 94 (46) 
 Unknown 44 (21) 
Vital status (at follow-up)   
 Alive 101 (49) 
 Deaths because of colorectal cancer 66 (32) 
 Deaths because of unknown cause or other than colorectal cancer 39 (19) 
Mean age at surgery 65.4 ± 11.9  
 Years ± SD (range) (26.0–70.0)  
Median follow-up time in years (range) 4.7  
 (<1.0–18.0)  
Median survival in years 5.58  
 (95% confidence interval) (3.59–9.0)  
Expression of p27kip-1   
 Negative 61 (30) 
 Positive 145 (70) 
No.(%)
Sex   
 Male 124 (60) 
 Female 82 (39) 
Age (years)   
 <65 90 (44) 
 ≥65 116 (56) 
Ethnicity   
 African Americans 85 (59) 
 Caucasians 121 (41) 
Tumor location   
 Proximal colon 89 (43) 
 Distal colon 76 (37) 
 Rectum 41 (20) 
Tumor size (cm)a   
 ≤5 128 (63) 
 >5 74 (37) 
Tumor differentiation   
 Well 41 (20) 
 Moderate 134 (65) 
 Poor 31 (15) 
Tumor stage (Union International Contre Cancer)   
 I 42 (20) 
 II 69 (34) 
 III 53 (26) 
 IV 42 (20) 
Tumor type   
 Mucinous 25 (12) 
 Nonmucinous 181 (88) 
pT component of stage (bowel wall invasion)   
 pTx (<1) 
 pT1 12 (6) 
 pT2 44 (21) 
 pT3 111 (54) 
 pT4 38 (18) 
pN component of stage (regional lymph node invasion)   
 pNx (4) 
 pN0 113 (55) 
 pN1 50 (24) 
 pN2 25 (12) 
 pN3 11 (5) 
M component of stage (distant metastasis)   
 M0 164 (80) 
 M1 42 (20) 
Cancer relapse (at follow-up)   
 Absent 68 (33) 
 Present 94 (46) 
 Unknown 44 (21) 
Vital status (at follow-up)   
 Alive 101 (49) 
 Deaths because of colorectal cancer 66 (32) 
 Deaths because of unknown cause or other than colorectal cancer 39 (19) 
Mean age at surgery 65.4 ± 11.9  
 Years ± SD (range) (26.0–70.0)  
Median follow-up time in years (range) 4.7  
 (<1.0–18.0)  
Median survival in years 5.58  
 (95% confidence interval) (3.59–9.0)  
Expression of p27kip-1   
 Negative 61 (30) 
 Positive 145 (70) 
a

Four tumors were not evaluated for tumor size because of nonavailability of tumor measurements in the surgical pathology report.

Table 3

Association of p27kip-1 expression with selected clinicopathological characteristics of patients with colorectal adenocarcinomas in relation to their tumor stage

Stage I (n = 42)Stage II (n = 67)Stage III (n = 49)Stage IV (n = 35)
No. of tumors positive (27)% (64%)PNo. of tumors positive (50)% (72%)PNo. of tumors positive (37)% (70%)PNo. of tumors positive (31)% (74%)P
Age (yrs)             
 <65 12 57  14 61  21 81  15 75  
 ≥65 15 71 0.334 36 78 0.127 16 59 0.088 16 73 0.867 
Sex             
 Male 17 57  28 72  24 75  15 68  
 Female 10 83 0.080 22 73 0.887 13 62 0.309 16 80 0.384 
Ethnicity             
 Caucasians 20 71  28 70  23 74  18 75  
 African Americans 53 0.076 22 76 0.590 14 64 0.409 13 72 0.839 
Tumor location             
 Proximal colon 10 63  22 71  20 91  14 70  
 Distal colon 64  18 69  39  14 78  
 Rectum 67 0.974 10 83 0.644 10 77 0.001 75 0.861 
Tumor size (cm)             
 ≤5 24 64  23 72  24 72  18 69  
 >5 60 0.361a 25 71 0.967 11 61 0.393 13 81 0.390 
Tumor type             
 Mucinous  80  71  50  
 Nonmucinous 27 71 0.012a 42 71 0.564 32 70 0.920 29 76 0.255 
Tumor differentiation             
 Well 25  100  12 78  61  
 Moderate 16 73  38 73  25 69  19 83  
 Poor 10 63 0.183 38 0.016 0.240 67 0.351 
pT component of stage (bowel wall invasion)             
 pT1 73       
 pT2 19 63 0.341    80  50  
 pT3    41 71  25 78  15 68  
 pT4    82 0.473 40 0.044 14 82 0.459 
pN component of stage (lymph node invasion)             
 pN0 27 64  50 72     50  
 pN1       26 70  62  
 pN2       70  11 73  
 pN3       67 0.984 100 0.470 
M. (component of stage distant metastasis)             
 M0 27 64  50 72  37 70     
 M1          31 74  
Cancer status (at follow-up)             
 Absent 17 61  19 73  12 86   
 Present 63  16 67  13 50  25 70  
 Unknown 83 0.573 15 79 0.667 12 92 0.008 100 0.115 
Outcome             
 Alive 67  13 57  15 83  27 71  
 Colorectal cancer deaths 13 68  23 79  15 54   
 Death because of unknown cause or other than cancer 56 0.731 14 82 0.123 100 0.018 100 0.210 
p53nac             
 Negative 17 68  25 66  22 69  13 65  
 Positive 10 59 0.542 25 81 0.169 15 71 0.835 18 82 0.216 
Stage I (n = 42)Stage II (n = 67)Stage III (n = 49)Stage IV (n = 35)
No. of tumors positive (27)% (64%)PNo. of tumors positive (50)% (72%)PNo. of tumors positive (37)% (70%)PNo. of tumors positive (31)% (74%)P
Age (yrs)             
 <65 12 57  14 61  21 81  15 75  
 ≥65 15 71 0.334 36 78 0.127 16 59 0.088 16 73 0.867 
Sex             
 Male 17 57  28 72  24 75  15 68  
 Female 10 83 0.080 22 73 0.887 13 62 0.309 16 80 0.384 
Ethnicity             
 Caucasians 20 71  28 70  23 74  18 75  
 African Americans 53 0.076 22 76 0.590 14 64 0.409 13 72 0.839 
Tumor location             
 Proximal colon 10 63  22 71  20 91  14 70  
 Distal colon 64  18 69  39  14 78  
 Rectum 67 0.974 10 83 0.644 10 77 0.001 75 0.861 
Tumor size (cm)             
 ≤5 24 64  23 72  24 72  18 69  
 >5 60 0.361a 25 71 0.967 11 61 0.393 13 81 0.390 
Tumor type             
 Mucinous  80  71  50  
 Nonmucinous 27 71 0.012a 42 71 0.564 32 70 0.920 29 76 0.255 
Tumor differentiation             
 Well 25  100  12 78  61  
 Moderate 16 73  38 73  25 69  19 83  
 Poor 10 63 0.183 38 0.016 0.240 67 0.351 
pT component of stage (bowel wall invasion)             
 pT1 73       
 pT2 19 63 0.341    80  50  
 pT3    41 71  25 78  15 68  
 pT4    82 0.473 40 0.044 14 82 0.459 
pN component of stage (lymph node invasion)             
 pN0 27 64  50 72     50  
 pN1       26 70  62  
 pN2       70  11 73  
 pN3       67 0.984 100 0.470 
M. (component of stage distant metastasis)             
 M0 27 64  50 72  37 70     
 M1          31 74  
Cancer status (at follow-up)             
 Absent 17 61  19 73  12 86   
 Present 63  16 67  13 50  25 70  
 Unknown 83 0.573 15 79 0.667 12 92 0.008 100 0.115 
Outcome             
 Alive 67  13 57  15 83  27 71  
 Colorectal cancer deaths 13 68  23 79  15 54   
 Death because of unknown cause or other than cancer 56 0.731 14 82 0.123 100 0.018 100 0.210 
p53nac             
 Negative 17 68  25 66  22 69  13 65  
 Positive 10 59 0.542 25 81 0.169 15 71 0.835 18 82 0.216 
a

Fisher’s exact test P.

Table 4

Cox regression multivariate analysis to evaluate independent prognostic value of p27kip-1 expression in colorectal adenocarcinomas in relation to tumor stage

VariableIndicator of poor prognosisHazard ratio (95% confidence interval)P
Overall    
 p27kip-1 expression (positive versus negative) p27kip-1 negative 0.7 (0.4–1.0) 0.058 
 Age (<65 versus ≥65 years) ≥65 years 1.8 (1.2–2.8) 0.002 
 Ethnicity    
  (African American versus Caucasian) African American 1.8 (1.2–2.6) 0.003 
 Tumor differentiation    
  (Moderate versus well) Moderate 1.7 (1.1–2.0)  
  (Poor versus well) Poor 2.4 (1.3–4.8) 0.009 
 pT component of stage (bowel wall invasion)    
  (pT2versus pT1pT2 1.4 (1.2–1.9)  
  (pT3versus pT1pT3 1.9 (1.0–3.4)  
  (pT4versus pT1pT4 2.6 (1.1–6.3) 0.039 
 pN component of stage (regional nodal invasion)    
  (pN1versus pN0pN1 1.9 (1.3–2.9)  
  (pN2versus pN0pN2 3.8 (2.6–5.1)  
  (pN3versus pN0pN3 5.9 (3.4–9.6) 0.0001 
 Distant metastasis    
  (M1versus M0M1 8.9 (5.4–14.7) <0.0001 
Stage I    
 p27kip-1 expression p27kip-1 negative 1.3 (0.4–4.5) 0.680 
  (positive versus negative)    
 Age    
  (<65 versus ≥65 years) ≥65 years 5.7 (1.5–21.5) 0.011 
Stage II    
 p27kip-1 expression p27kip-1 negative 0.6 (0.2–1.3) 0.180 
  (positive versus negative)    
 Ethnicity African American 3.1 (1.3–7.4) 0.012 
  (African American versus Caucasian)    
Stage III    
 p27kip-1 expression p27kip-1 negative 3.2 (1.3–7.7) 0.010 
  (positive versus negative)    
 Tumor differentiation    
  (Moderate versus well) Moderate 3.0 (1.2–7.4)  
  (Poor versus well) Poor 9.1 (1.5–55.1) 0.028 
Stage IV    
 p27kip-1 expression p27kip-1 negative 1.3 (0.6–2.7) 0.555 
  (positive versus negative)    
VariableIndicator of poor prognosisHazard ratio (95% confidence interval)P
Overall    
 p27kip-1 expression (positive versus negative) p27kip-1 negative 0.7 (0.4–1.0) 0.058 
 Age (<65 versus ≥65 years) ≥65 years 1.8 (1.2–2.8) 0.002 
 Ethnicity    
  (African American versus Caucasian) African American 1.8 (1.2–2.6) 0.003 
 Tumor differentiation    
  (Moderate versus well) Moderate 1.7 (1.1–2.0)  
  (Poor versus well) Poor 2.4 (1.3–4.8) 0.009 
 pT component of stage (bowel wall invasion)    
  (pT2versus pT1pT2 1.4 (1.2–1.9)  
  (pT3versus pT1pT3 1.9 (1.0–3.4)  
  (pT4versus pT1pT4 2.6 (1.1–6.3) 0.039 
 pN component of stage (regional nodal invasion)    
  (pN1versus pN0pN1 1.9 (1.3–2.9)  
  (pN2versus pN0pN2 3.8 (2.6–5.1)  
  (pN3versus pN0pN3 5.9 (3.4–9.6) 0.0001 
 Distant metastasis    
  (M1versus M0M1 8.9 (5.4–14.7) <0.0001 
Stage I    
 p27kip-1 expression p27kip-1 negative 1.3 (0.4–4.5) 0.680 
  (positive versus negative)    
 Age    
  (<65 versus ≥65 years) ≥65 years 5.7 (1.5–21.5) 0.011 
Stage II    
 p27kip-1 expression p27kip-1 negative 0.6 (0.2–1.3) 0.180 
  (positive versus negative)    
 Ethnicity African American 3.1 (1.3–7.4) 0.012 
  (African American versus Caucasian)    
Stage III    
 p27kip-1 expression p27kip-1 negative 3.2 (1.3–7.7) 0.010 
  (positive versus negative)    
 Tumor differentiation    
  (Moderate versus well) Moderate 3.0 (1.2–7.4)  
  (Poor versus well) Poor 9.1 (1.5–55.1) 0.028 
Stage IV    
 p27kip-1 expression p27kip-1 negative 1.3 (0.6–2.7) 0.555 
  (positive versus negative)    

We thank the staff of the Tissue Procurement Facility at University of Alabama at Birmingham for their technical help, and we also thank Dr. Sameera Vohra for her assistance in manuscript preparation. We thank Libby Chambers, Cindy Legge, and Lisa Jinright for their secretarial support.

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