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Journal of Clinical Oncology, Vol 24, No 36 (December 20), 2006: pp. 5672-5679 © 2006 American Society of Clinical Oncology. DOI: 10.1200/JCO.2006.07.5499 Raf Kinase Inhibitor Protein Expression in a Survival Analysis of Colorectal Cancer Patients
From the Kuwait University, Faculty of Medicine, Safat, Kuwait; Proteomics and Signalling Networks Group, Beatson Institute for Cancer Research; Department of Pathology, University of Glasgow; Centre for Oncology and Applied Pharmacology, Cancer Research UK, Beatson Laboratories, Glasgow; and the Department of Pathology, University of Aberdeen, Aberdeen, United Kingdom Address reprint requests to Walter Kolch, MD, The Beatson Institute for Cancer Research, Garscube Estate, Switchback Rd, Glasgow G61 1BD, United Kingdom; e-mail: wkolch{at}beatson.gla.ac.uk; and Fahd Al-Mulla, MB, ChB, PhD, Department of Pathology, Faculty of Medicine, Kuwait University, PO Box 24923, Safat 13110, Kuwait; email: fahd{at}al-mulla.org
PURPOSE: Raf kinase inhibitor protein (RKIP) inhibits the Raf and nuclear factor kappa B signaling pathways, and suppresses metastasis in animal models. We examined whether RKIP expression in primary colorectal cancers (CRCs) correlates with the risk of metastasis and overall survival. PATIENTS AND METHODS: RKIP expression was examined immunohistochemically in three separate cohorts: a tissue microarray containing 276 samples from human tumors and normal tissues, and retrospective studies of 268 CRC patients and 65 early-stage CRCs. Overall and metastasis-free survival rates were measured. RESULTS: RKIP was expressed in normal epithelia but was reduced in metastatic tumors. RKIP expression in primary CRC was an independent prognostic marker for survival using multivariate Cox regression analysis (hazard ratio, 2.808; 95% CI, 1.58 to 4.96; P = .0002), independent of Dukes' stage. Patients with Dukes' C RKIP-positive tumors had similar 5-year survival rates as early-stage patients if tumors had equivalent RKIP expression levels. An independent study of early-stage CRCs confirmed that reduced RKIP expression predicted metastatic recurrence and reduced disease-free survival (hazard ratio, 4.5; 95% CI, 1.7 to 12.3; P = .003). RKIP expression was independent of sex, age, mitotic index, lymphatic and vascular invasion, depth of invasion, and tumor site, but correlated positively with apoptotic index (P = .024). Weak or loss of RKIP expression was the most significant and independent prognostic marker using a multivariate regression equation (hazard ratio, 4.5; 95% CI, 1.7 to 12.3; P = .003). CONCLUSION: RKIP expression in primary CRCs correlates with overall and disease-free survival, and can be useful for identifying early-stage CRC patients at risk of relapse.
Raf kinase inhibitor protein (RKIP) was originally identified as an endogenous inhibitor of the Raf–MAPK kinase (MEK) –ERK pathway, which interfered with the phosphorylation and activation of MEK by Raf-1.1,2 Subsequently, RKIP was shown also to suppress the activation of the nuclear factor kappa B (NF  B) transcription factor by blocking the inactivation of the inhibitor of NFB, IB.3 Both pathways play an important role in cancer and invasion.4,5 Recently, it was suggested that RKIP suppresses metastasis in prostate cancer,6,7 breast cancer,6 and melanoma.8 These studies demonstrated a reduction or loss of RKIP expression in metastatic cell lines or metastatic lesions. Reconstitution of RKIP levels in metastatic cell lines by exogenous expression impaired in vitro invasiveness8 and the ability to form metastases in mouse models.7 In contrast, downregulation of RKIP expression by antisense RNA promoted invasiveness. In an orthotopic mouse model for prostate cancer, RKIP expression did not affect primary tumor growth, even though it prevented the tumor from metastasizing.7 These results were obtained in model systems and by analysis of small numbers of cell lines and tumors. To address whether RKIP expression in primary human tumors is related to metastatic behavior, we investigated RKIP expression using tissue microarrays (TMAs) of normal and cancerous human tissues. Pursuing the finding that RKIP expression was reduced in metastatic colorectal cancer (CRC), we evaluated the hypothesis that RKIP expression may predict the risk of metastatic relapse and overall survival in two independent cohorts of CRC patients. CRC is a prevalent cancer in the industrialized world, with unique challenges to clinical management. Patients presenting with limited disease or local lymph node metastases have high rates of tumor recurrence (10% to 55%), but a measurable fraction of these patients (up to 15%) may be rendered permanently disease free by adjuvant chemotherapy.9-13 However, thousands of patients are treated needlessly with adjuvant chemotherapy every year because there are no reliable means to identify the small fraction who will benefit from the treatment.14 Dukes' staging and lymph node status are the best available clinicopathologic markers, but there is an urgent need to define markers that can stratify patients better and earlier according to their risk of CRC recurrence and overall survival.10,15
Study Population and Specimens Three independent patient cohorts were studied. For the initial survey of RKIP expression, Landmark High-Density Cancer Survey Tissue MicroArrays (catalog No. 3161, lot No. 030102; Ambion [Europe], Huntingdon, United Kingdom) were used. These contain 279 paraffin-embedded tissue specimens from 190 individuals, including 242 specimens from tumors with matched normal tissue from the same patient and organ (where available), and 37 independent normal specimens (Table 1). The second cohort consisted of a TMA prepared from 268 CRC patients selected from the Aberdeen Colorectal Tumor Bank in Scotland (Table 2). All of these patients had undergone surgery for CRC at Aberdeen Royal Infirmary between 1994 and 2003. The TMA was produced as described previously.16,17 The third cohort comprised 65 patients from Glasgow, Scotland, and Kuwait: 25 patients with early-stage CRC who had no evidence of metastatic disease at the time of surgery, but subsequently experienced relapse with metastasis (group 1), and 40 patients who remained disease free after treatment.
All patients were treated surgically; 14 Dukes' B2 patients also received six cycles of standard chemotherapy and were followed up prospectively (group 2). The patients in this cohort were followed up for a minimum period of 2 years (range, 2 to 9 years), with a median follow-up of 5.5 years for survivors, and 84% of survivors were observed for more than 3.5 years. They were clinically assessed for signs of metastatic recurrence, which were confirmed radiologically, histologically, or postmortem. Four patients were lost to follow-up. For 10 patients, who had no recurrence time recorded but died as a result of metastatic disease, the date of death was used as disease-free survival. For assessing disease-free survival, patients who died as a result of causes unrelated to cancer but had no evidence of metastatic recurrence at the time of death were censored. Fourteen patients received chemotherapy (all from Kuwait) and were staged as Dukes' B2. Eleven of the 14 had survival data only. All work with patients' specimens and clinical data was carried out following the ethical guidelines and approval procedures implemented at the Universities of Glasgow, Aberdeen, and Kuwait.
Immunohistochemistry To quantify tumor cell apoptosis, a subset of sections from cohort group 2 were stained using the In Situ Cell Death Detection Kit (Roche Diagnostics Ltd, Lewes, United Kingdom), according to the manufacturer's instructions. Briefly, paraffin-embedded sections were dewaxed, rehydrated to water, and incubated with 20 µg/mL proteinase K for 20 minutes at room temperature. Endogenous peroxidase activity was quenched with 1% H2O2 and the terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate-biotin nick-end labeling reaction mixture was applied to sections for 1 hour at 37°C. After several phosphate-buffered saline washes, slides were incubated with 50 µL converter-peroxidase for 30 minutes. DNA breakpoints were visualized using diaminobenzidine, which stained apoptotic cell nuclei dark brown. The apoptotic index is the number of nuclei per 1,000 nuclei that stain positive with terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate-biotin nick-end labeling.
Statistical Analysis
To assay RKIP expression, we validated the specificity of our RKIP antibody rigorously for both Western blot and IHC applications (Appendix Fig A2, online only). On Western blots, the antibody only detected RKIP and no other bands. Importantly, preadsorbing the RKIP antibody with cognate antigen (ie, purified recombinant RKIP produced in E coli) strongly reduced its reactivity on sections of paraffin-embedded cell lines and human tissues. Having established the specificity of our RKIP antiserum, we examined the expression of RKIP in human tissues. To survey the expression of RKIP in humans, we used a TMA featuring 279 samples taken from different tumors and matched normal tissues, where available. Excluding samples with missing data, or those that had lifted off the slide (61 samples) or showed metastases of uncertain origin (one sample), 217 samples were evaluated (Table 1). RKIP expression was low in connective tissue and lymphocytes. In contrast, RKIP was expressed in epithelial and endothelial cells of all tissues, including glandular epithelia of breast, pancreas, and salivary glands; tubular epithelia and glomeruli of the kidney; transitional epithelium of the bladder; and endothelia of lymph and blood vessel. High RKIP expression also was observed in neuronal cells, hepatocytes, and muscle cells. Tumors derived from these tissues exhibited variable RKIP levels, often with reduced RKIP expression. In tumors of the kidney and pancreas, this trend toward reduced RKIP expression came close to statistical significance, with P values of .053 and .054, respectively. In CRC, the reduction of RKIP levels was statistically significant, with a P value of .01 (Table 1), prompting us to perform a more thorough study in a large cohort of CRC patients. First, we examined the expression of RKIP in normal colon in more detail (Appendix Fig A1, online only). RKIP expression was almost undetectable in the crypts, but steadily increased as cells move upward and differentiate. RKIP was also strongly expressed in the ganglia of Auerbach's myenteric plexus, and in chromogranin A–positive neuroendocrine cells in the crypts. To assess RKIP expression in CRC, a TMA containing samples from 268 patients with corresponding clinical data, including Dukes'grade, sex, age, tumor site, and differentiation grade, was used (Table 2). The proportions of Dukes' grades were A, 19.8%; B, 38.8%; and C, 41.4%. This TMA underwent immunohistochemical staining for RKIP, and samples that could not be scored because they detached during staining or contained mostly nontumor tissue were excluded. As expected from previous studies,6,7 RKIP expression was downregulated in lymph node metastases. Of 79 lymph node metastases on the array, 67 (85%) had no or weak RKIP expression compared with 12 (15%) that expressed RKIP, confirming that the metastatic process in CRC also involves a reduction or loss of RKIP expression. More interestingly, however, 202 primary tumor samples eligible for analysis showed a statistically significant positive correlation (P < .001) between RKIP expression and overall survival (Fig 1A). Patients whose primary CRC scored positive for RKIP expression had a mean survival time of 93 months, whereas low or negative RKIP expression correlated with a shortened mean survival time of 61 months. Given that the survival curve of RKIP-positive patients never decreased below 50%, no median survival could be calculated. RKIP expression was independent of p53 status, tumor differentiation, tumor site (proximal colon, distal colon, or rectum), and B-Raf expression according to multivariate Cox proportional hazards model (Appendix Table A1, online only). This analysis also demonstrated that negative or weak RKIP expression was associated with a significant hazard ratio of 2.84, a value comparable to the risk associated with an advanced Dukes' stage.
Given that Dukes' staging is still a clinical gold standard for CRC risk prediction, we stratified the cohort according to Dukes' stage and RKIP expression. This analysis showed that within Dukes' stage C tumors, patients whose CRCs were positive for RKIP expression had a mean survival of 78 months, which was statistically not significantly different from the overall survival of patients with RKIP-positive Dukes' A and B tumors, with mean survival periods of 92 and 85 months, respectively (Fig 1B). In contrast, weak or negative RKIP expression reduced the overall survival in all Dukes' stages (Fig 1C). For instance, the mean survival of Dukes' A versus Dukes' B and Dukes' C patients was 59, 70, and 49 months, respectively. These data suggest that RKIP expression in the primary tumor can predict patients' overall survival independent of Dukes' stage and may be useful as prognostic marker to delineate the high-risk patient population. Therefore, we examined RKIP expression in 65 early stage (Dukes' A and B) CRC patients for whom data on disease-free survival (measured as time to metastatic relapse) were available (Table 3). Metastatic relapse did not correlate significantly with sex, age, tumor site, tumor differentiation, mitotic and apoptotic indexes, lymphatic and vascular invasion, or the depth of invasion (Appendix Table A2), confirming the limited usefulness of current clinical parameters in identifying patients at risk from metastatic relapse in early CRC. In contrast, RKIP expression exhibited a statistically significant correlation with disease-free survival in Dukes' stage A and B patients (Table 3). Multivariate analysis of RKIP expression as covariates in the multiple regression model showed that weak or loss of RKIP expression was the most significant and independent prognostic factor when all variables were included in the multivariate regression equation (hazard ratio, 4.5; 95% CI, 1.7 to 12.3; P = .003).
The median survival for patients with no or weak RKIP expression was 4.57 and 3.46 years, respectively, compared with more than 8 years in patients with positive RKIP expression (P = .004). Similarly, 5-year disease-free survival was 47% for patients with RKIP-negative tumors, 31% for patients with weakly RKIP-positive tumors, and 79% for patients with RKIP-positive tumors. Kaplan-Meier plots (Fig 2) showed that patients with RKIP-positive tumors had stable disease-free survival rates of  90% between years 2 and 4 after surgery, and disease-free survival rates of 80% after year 4. In contrast, patients with reduced or no RKIP expression experienced a steady decline in disease-free survival during the entire observation period. This pattern was similar in the nonstratified patient population (P < .001) and in Dukes' A and B1 patients treated with surgery alone (P < .001) or in Dukes' B1 patients alone (P < .001) Thus, the level of RKIP expression in primary CRCs is significantly and inversely associated with metastatic disease, and can predict the risk of metastatic relapse in CRC patients with nonmetastatic disease. The association between RKIP expression and metastatic recurrence was independent of sex, age, tumor site, mitotic index, lymphatic or vascular invasion, Dukes' stage, and pT stage (Table 3).
To investigate the association of metastatic phenotype and reduced RKIP expression further, we also examined mitotic index, tumor differentiation, vascular endothelial growth factor production, tumor blood vessel counts, p53 expression, and apoptotic index in the primary tumors (Appendix Table A2). There was a positive correlation (P = .003) between RKIP expression and tumor differentiation (Table 3), mainly due to the prevalence of RKIP positivity (68%) in moderately differentiated tumors. Conversely, 52% of patients with well-differentiated tumors had lost RKIP expression, and the correlation between RKIP expression and tumor differentiation was not significant in the Aberdeen cohort. Thus, we cannot conclude that RKIP is simply a differentiation marker. However, this does not impinge on the usefulness of RKIP as prognostic marker, given that differentiation was not linked significantly to survival in either cohort. This latter finding is consistent with observations in the literature that differentiation status may have little prognostic bearing.19
More interestingly, the apoptotic index correlated positively (P = .024) with RKIP expression (Table 3). Tumors with negative or weak RKIP expression had mean apoptotic indexes of 6.3 and 11.3, respectively, compared with a mean apoptotic index of 15 in tumors expressing RKIP (Table 3 and Appendix Fig A3, online only). Given that paraffin sections only capture a snapshot of the steady-state situation, these differences are likely to reflect much larger rates of apoptosis in RKIP-expressing tumors. Additional investigations into the molecular mechanism of this phenomenon may be guided by the observation that RKIP can promote apoptosis by counteracting NF
In developed countries CRC is the third most frequent cancer and the second most common cause of cancer death.20 Patients with Dukes' C or D metastatic CRC have 5-year survival rates of 37% and 11%, respectively. Patients diagnosed at an early stage (Dukes' A and B) with no evidence of metastatic disease at time of surgery have a significantly better prognosis, with 5-year survival rates of 85% and 67%, respectively.20 However, a significant proportion of these patients (10% to 45%) experience relapse with metastatic disease.10-13 Prognostic diagnosis is still based predominantly on histopathologic evaluation, although its prognostic reliability is being questioned.15,19 Tumor markers such as p53 mutation, endothelial growth factor receptor status, or ß-catenin, claudin, Ki-67, or thymidylate synthase expression have some prognostic value,15,21-24 but are not widely used. The most routinely used marker is carcinoembryonic antigen, which can be useful as part of close post-treatment monitoring schemes.15 However, the required high frequency of monitoring is expensive and requires high compliance by patients. Another parameter by which CRCs can be classified involves analysis of microsatellite instability, in which a proportion of CRC patients (15%) display methylation of the hMLH1 promotor, resulting in ineffective DNA mismatch repair. However, our cohorts showed no correlation between microsatellite instability and RKIP expression (Appendix Tables A3-A9 [online only]). The ability to predict the risk of metastatic relapse is of paramount importance because it may allow the identification of patients who should be monitored frequently and who could benefit from adjuvant chemotherapy, given that newer studies indicate a benefit from chemotherapy given to selected patients with early CRC who are at risk of metastatic relapse.14 This study did not address the relationship between RKIP expression and patients' response to chemotherapy. Such a follow-up study could be of interest given the recent data showing that chemotherapeutic drug treatments induce RKIP expression, which in turn sensitizes cells to apoptosis.6 Our data support this positive correlation between RKIP expression and apoptosis (Table 3 and Appendix Fig A3). Attempts to identify this high-risk group ranged from investigating histopathologic features, tumor markers, and metastasis suppressor genes (including nm23, E-cadherins, CD44, and KAI1) to molecular methods such as counting alleles and detection of micrometastases by reverse transcriptase polymerase chain reaction.25-31 However, their value in predicting metastatic relapse in early CRCs remains disappointing. For instance, the expression of the CD44(v8-10) splice variant could be associated with an elevated risk of metastases, but its discriminatory power was limited to 5- and 10-year survival rates between patients with CD44(v8-10)-negative and -positive tumors that only differed by 18% and 32%, respectively.32,33 We have shown that RKIP expression was downregulated in lymph node metastases. This is consistent with recent data published on melanoma, prostate, and breast cancers and cancerous cell lines.34-36 Our results show a significant relationship among reduced RKIP expression, metastatic recurrence, and overall survival. Notably, this relationship was independent of Dukes' staging, in that Dukes' A and C patients with no or low RKIP expression had equivalent overall survival and vice versa. We excluded misclassification as a reason for this observation by revisiting the Aberdeen slides, confirming the staging, and adding another independent cohort of early-stage CRC, and noted a significant association between reduced RKIP and metastatic relapse. Thus, we believe that the Dukes' A patients in the Aberdeen cohort with no or weak RKIP expression could have had a higher rate of metastatic recurrences. Unfortunately, the Aberdeen cohort had no data on recurrences; only overall survival was registered. We believe that this observation can be explained by recent data demonstrating that RKIP regulates the spindle checkpoints in cells and that its loss could lead to chromosomal instability, which in turn could influence tumor aggressiveness and its response to therapy irrespective of Dukes' stage.37,38 Nevertheless, the direct relationship between RKIP loss and chromosomal instability remains to be elaborated. Regardless of the mechanism, RKIP expression seems to be a suitable and easily determinable marker in the primary tumor that could predict the risk of early CRC to metastasize, and hence guide strategies for monitoring and therapy. However, it is worth noting that 12 patients (33%) who did not suffer any metastatic relapse had no or low RKIP expression in their primary tumors. Although the follow-up period, in our opinion, was sufficient enough, it is likely that other factors or metastasis suppressors than RKIP are of importance here. Nevertheless, the relationship between RKIP expression in primary tumors and metastatic relapse gains in significance given the high prevalence of CRC and the lack of effective markers of metastasis risk, especially in Dukes' A and B cancers. In the absence of such markers it is unclear which early-stage CRC patients should receive adjuvant chemotherapy, or how closely CRC patients should be monitored after primary treatment.39 A simple test such as RKIP expression in the primary tumor could provide an economically viable and immediately available decision aid.
Aberdeen Colorectal Tumour Bank cohort. In this cohort there is no relationship between MSI and RKIP expression. The MSI status of the tumors was determined by immunohistochemistry, as described (Wright CL, Stewart ID: Am J Surg Pathol 27:1393-1406, 2003; Kumarakulasingham M, Rooney PH, Dundas SR, et al: Clinical Cancer Res, 11:3758-3765, 2005). Sections were immunostained with monoclonal antibodies to hMLH1 (clone G168-728; BD Biosciences, Oxford, United Kingdom) used at a dilution of 1:50 and hMSH2 (clone Fe11; Oncogene, Merck Biosciences, Nottingham, United Kingdom) also used at a dilution of 1:50. Immunohistochemistry, including an antigen retrieval step for each antibody, was performed as described (Kumarakulasingham M, Rooney PH, Dundas SR, et al: Clinical Cancer Res, 11:3758-3765, 2005). Loss of expression when none of the tumor nuclei stained with either hMLH1 or hMSH2 was regarded as mismatch repair defective (mmr-d) or microsatellite instability (MSI) high, whereas staining of tumor nuclei for either hMLH1 or hMSH2 was considered as mismatch repair intact (mmr-i) or MSI low (Wright CL, Stewart ID: Am J Surg Pathol 27:1393-1406, 2003; Kumarakulasingham M, Rooney PH, Dundas SR, et al: Clinical Cancer Res, 11:3758-3765, 2005; Appendix Tables A3 to A8, online only)
Glasgow and Kuwait cohort.
The authors indicated no potential conflicts of interest.
Supported by Grant No. 99-07-07 from Kuwait Foundation for the Advancement of Sciences, Shared Facility Grant No. GM/0101 from Kuwait University, the Association for International Cancer Research (Grant No. 02-141), the European Union (FP6 STREP: COSBICS), the Chief Scientist Office of the Scottish Executive Health Department, and Cancer Research UK. The funding organizations did not have any role in the design or conduct of the study; collection, analysis, and interpretation of the data; and preparation and review of the manuscript. Authors contributed equally to this work. Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.
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Galizia G, Lieto E, Ferraraccio F, et al: Determination of molecular marker expression can predict clinical outcome in colon carcinomas. Clin Cancer Res 10:3490-3499, 2004 Submitted May 23, 2006; accepted October 5, 2006. This article has been cited by other articles:
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ABSTRACT
INTRODUCTION
2 or Fisher's exact tests were used for significance testing of contingency tables containing categoric variables. Mann-Whitney U test was used to compare medians of pairs of independent samples from continuous variables, and Kruskal-Wallis test was used to compare means of more than two independent samples from continuous variables. Follow-up to event outcomes were analyzed by Kaplan-Meier survival curves and compared by log-rank tests. All reported P values are two-sided. Data were analyzed using the SPSS version 11.0 software package (SPSS Inc, Chicago, IL). 
30% of the analyzed loci were classified as MSI high (one patient who was excluded from the survival analysis was not tested), allelic instability in one or two of the 13 microsatellites or 
30% of the loci analyzed was considered as MSI low (13 patients). Samples with no MSI were classified as stable (50 patients). In six patients, microsatellite stability could not be assessed. Thus, most of our patients are microsatellite stable or MSI low. This is consistent with the older presentation of our cohort and the exclusion of patients with family history of CRC. MSI and RKIP expression were correlated in 59 patients from whom both RKIP and MSI data were available. Forty-seven patients were microsatellite stable, and 12 showed low MSI (Appendix Table A9, online only). 
RKIP+comp) before use on a section of the same Western blot. Note that RKIP, even when expressed exogenously from a cDNA vector, migrates as two bands on Western blots. (B) The cells lines were embedded in paraffin in order to test the RKIP antibody under conditions of immunohistochemical staining. (C) Paraffin embedded sections of human colon were stained with RKIP antibody, unrelated IgG, or RKIP antibody preabsorbed with cognate antigen (
