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© 1999 American Society for Clinical Oncology Analysis of the p53/BAX Pathway in Colorectal Cancer: Low BAX Is a Negative Prognostic Factor in Patients With Resected Liver MetastasesFrom the Department of Hematology, Oncology, and Tumor Immunology, Charité-Campus Berlin-Buch, Humboldt University; Max Delbrück Center for Molecular Medicine; InViTek GmbH, Berlin-Buch; Institute of Pathology, Charité-Campus Berlin-Mitte, Berlin; and Department of General and Vascular Surgery, University Hospital, Johann Wolfgang Goethe University, Frankfurt, Germany. Address reprint requests to Peter Daniel, MD, Department of Hematology, Oncology, and Tumor Immunology, Charité-Campus Berlin-Buch, Humboldt-University, Lindenberger Weg 80, 13125 Berlin-Buch, Germany; email pdaniel{at}mdc-berlin.de
PURPOSE: To determine the prognostic value of the central downstream apoptosis effector BAX in relation to its upstream regulator p53 in R0-resected hepatic metastases of colorectal cancer. PATIENTS AND METHODS: Retrospective analysis of 41 patients who underwent potentially curative resection of liver metastases from colorectal cancer was performed. Tumor DNA was screened for p53 mutations by single-stranded conformational polymorphism polymerase chain reaction and for BAX frameshift mutations by fragment length analysis. Protein expression of BAX, p21, and p53 was investigated by immunohistochemistry. RESULTS: Overall median survival was 40.2 months. Tumors with BAX frameshift mutations were considered microsatellite mutator phenotype–positive and were excluded from further prognostic analyses. Patients with high BAX protein expression had a median survival of 53.6 months compared with 35.4 months for patients with low BAX expression (P < .05). The negative prognostic value of low BAX expression was more evident in those patients with wild-type p53 (median survival, 54.0 v 23.3 months for BAX-negative tumors; P < .01). Low BAX expression was an independent negative prognostic marker in multivariate regression analysis for all patients independent of the p53 status (relative risk, 3.03, P = .03), especially for p53 wild-type tumors (relative risk, 8.21; P = .0095). CONCLUSION: We conclude that low BAX expression is an independent negative prognostic marker in patients with hepatic metastases of colorectal cancer. The best survival was seen in patients with an intact p53-to-BAX pathway; ie, wild-type p53- and BAX-positive tumors. Thus, analysis of apoptosis signaling pathways (here, p53 in concert with its downstream death effector, BAX) might yield more prognostic power in future studies as compared with analysis of single genes such as p53 alone.
THE BCL-2 FAMILY, with its antiapoptotic members BCL-2, BCL-XL, MCL-1, and A1, and death-promoting members BAX, BCL-XS, BAK, BAD, and BIK, plays a central role in the regulation of cell death. The overexpression of antiapoptotic factors of this gene family, such as BCL-2, contributes to cancer pathogenesis as in the case of follicular lymphoma1,2 and may be involved in the resistance to cancer therapy.3-5 In breast cancer, we previously described a defect in expression of the BAX protein, a key promoter of apoptosis.6 Restoration of BAX expression in breast cancer cell lines inhibited tumorigenicity7 and increased sensitivity to cytotoxic drug therapy.8,9 In breast cancer patients, reduced BAX expression correlates with a poor response to chemotherapy and shorter overall survival.10 To further investigate the role of BAX in carcinoma cells, we analyzed the BAX mutational status and expression in patients with hepatic metastases of colorectal cancer. Metastatic disease in colorectal cancer is frequently restricted to the liver. Surgical excision is a potentially curative treatment for patients with solitary or limited focal lesions. However, in the majority of patients (70%), tumor relapses occur either in the liver or at extrahepatic sites, and the 5-year survival rate is reported to be between 20% and 39%.11 Clinical prognostic factors were retrospectively investigated from a French group12 that collected information on 1,568 cases. This group identified seven clinical risk factors (age, extension into serosa, lymphatic spread of primary cancer, time interval from primary cancer to metastases, size of largest metastases, number of metastases, and surgical margin clearance), which were nevertheless of minor prognostic value. Stronger prognostic factors for the improvement of individual decision making and postsurgical management (ie, chemotherapy or no therapy) would therefore be desirable. In the present study, BAX was found to be differentially expressed in colorectal metastases. p53 is known to be a transcriptional regulator of the BAX gene, and a portion of the tumor suppressor properties of the p53 gene may be mediated by transcriptional activation of the BAX gene.13 We therefore assessed whether the differential expression of BAX is correlated to the p53 mutational status or the p53 expression level. Thus our study provides an example for a pathway analysis in which p53 alterations are analyzed and compared with expression and mutation of the downstream death effector BAX and with the expression of the p53-induced cell cycle inhibitor p21. Absence of BAX protein expression has been demonstrated in colorectal cancer specimens that are microsatellite mutator phenotype–positive (MMP+) (due to defects in mismatch repair). As many as 15% of nonselected ("sporadic") gastrointestinal tumors belong to the MMP pathway of tumorigenesis.14-16 In these tumors, single base insertions or deletions in a polyguanine G8 tract within the BAX open reading frame result in premature termination of translation, which results in absent BAX protein expression.17-19 Thus absence of BAX protein could be due to the presence of a frameshift mutation. It is known that colorectal MMP+ and MMP- tumors exhibit fundamental differences in genotype and phenotype.17,18,20 Consequently, we decided to exclude tumors with BAX frameshift mutation from the survival analysis.
Patients Between 1985 and 1995, 116 patients with hepatic metastases from colorectal cancer underwent liver resection with potential curative intention at a single institution (University Hospital at Johann Wolfgang Goethe University, Frankfurt, Germany). To be eligible for this study, patients were required to have the following: (1) resectable hepatic lesion, (2) no evidence of extrahepatic disease, and (3) tumor-free resection margins. Patients with microscopically detected tumor in the resection margin were excluded from the study. Considering the availability of paraffin-embedded material and clinical data in addition to these criteria, 41 patients were included in the study: 18 patients were male and 23 were female, with a median age of 61 years (range, 20 to 75 years). In all patients, the liver (ie, presence of liver metastases) was the only site with evidence of disease. Site and stage of primary tumor as well as number of hepatic lesions resected and time of occurrence of liver metastasis in relation to the primary tumor are listed in Table 1. Postoperative treatment was individualized; 29 patients (71%) received local or systemic chemotherapy with fluorouracil after R0 resection of hepatic metastases, five patients (12%) underwent repeated resection of recurrent hepatic metastases, and 11 patients (27%) did not receive any further antitumor therapy.
Mutation Analysis for p53 For detection of p53 mutations in the DNA binding region, single-stranded conformational polymorphism (SSCP) polymerase chain reaction (PCR) analysis was performed. Genomic DNA was subjected to PCR using oligonucleotide primers for amplification of exons 5 to 8 of the p53 gene; primer sequences are as follows21: exon 5a, CCA GTT GCT TTA TCT GTT CA and TGT GGA ATC AAC CCA CAG (codons 126 to 150, 139 base pairs [bp]); exon 5b, CAA CTG GCC AAG ACC TGC and AAC CAG CCC TGT CGT CTC T (codons 134 to 186, 191 bp); exon 6, CTC TGA TTC CTC ACT GAT TGC and GAG ACC CCA GTT GCA AAC CA (codons 187 to 224, 163 bp); exon 7, TTG CCA CAG GTC TCC CCA A and AGG GTG GCA AGT GGC TCC (codons 225 to 261, 190 bp); and exon 8, CCT TAC TGC CTC TTG CTT C and CGC TTC TTG TCC TGC TTG C (codons 262 to 306, 199 bp). The PCR was carried out according to the following protocol, with a final extension time of 7 minutes at 72°C using a model 2400 thermocycler (Perkin-Elmer/Cetus, Weiterstadt, Germany): exon 5a, 94°C for 5 minutes followed by 35 cycles at 94°C for 30 seconds, 55°C for 20 seconds, and 72°C for 15 seconds; exon 5b, 94°C for 5 minutes followed by 35 cycles at 94°C for 30 seconds, 65°C for 20 seconds, and 72°C for 15 seconds; exon 6, 94°C for 5 minutes followed by 35 cycles at 94°C for 30 seconds, 60°C for 20 seconds, and 72°C for 15 seconds; exon 7, 94°C for 5 minutes followed by 35 cycles at 94°C for 30 seconds, 58°C for 20 seconds, and 72°C for 20 seconds; exon 8, 94°C for 5 minutes followed by 35 cycles at 94°C for 30 seconds, 60°C for 20 seconds, and 72°C for 15 seconds. Reaction mixture contained 100µmol/L of each dNTP, 1µmol/L of each 5' and 3' primer, 1 mmol/L MgCl2 (except exon 7: 0.8 mmol/L MgCl2), 16 mmol/L (NH4)2SO4, 50 mmol/L Tris-HCl (pH 8.8 at 25 °C), 0.01% Tween-20, and 1.5 U Taq DNA polymerase (InViTek) in a total volume of 50µL. For SSCP analysis, 5µL of the amplified fragments were diluted in 7µL of loading buffer (82% formamide, 10 mmol/L NaOH, 50 mmol/L EDTA, bromophenol blue, xylene xyanole dye). The samples were denatured at 95°C for 5 minutes and cooled on ice. The denatured fragments were analyzed on a 10% nondenaturing polyacrylamide gel at 500 V and 50 mA for 2 hours at 10°C in a Genephor electrophoresis chamber (Pharmacia, Freiburg, Germany) and subsequently visualized by silver staining.
Detection of BAX Frameshift Mutations As a positive control, the human colon carcinoma cell line LoVo was used, which carries mutations in both BAX alleles: one shows an insertion (G9) and the other a deletion (G7) in the G8 tract.17 The human colon carcinoma cell line SW 620 served as a wild-type control. In dilution experiments, the sensitivity of the fragment length analysis was confirmed, and in blinded experiments, the specificity was confirmed.
Immunohistochemistry for BAX, p21, and p53 Blinded analysis of slides was performed by two observers who were without knowledge of clinicopathologic data. Four high-power fields (400x) were evaluated for localization, percentage of positive cells (0% to 100% in 5% steps), and staining intensity (0 to +++). Protein expression levels of BAX, p21, and p53 (product of the percentage of positive cells and staining intensity) were correlated to the mutational p53 status. For all other analyses, the percentage of positive cells was used.
Statistical Analysis
For univariate and multivariate assessment, the following variables were selected in accordance with the prognostic parameters established by two publications with large case numbers that were shown to be of prognostic value11,12: number of hepatic lesions, nodal positivity, size of the primary lesion, grading, disease-free interval between resection of the primary tumor and the detection of hepatic metastases, and the application of chemotherapy for metastatic colorectal disease. In addition, BAX expression and p53 mutation were included in the multivariate analysis of survival with determination of relative risk ratios. The data fitted to the Cox proportional hazards model and relative risk ratios were calculated according to this model. For intervariable assessment, the unpaired t test and
Analysis of BAX Frameshift Mutations In spiking experiments using different dilution ratios of BAX wild-type cells (SW620) and BAX frameshift mutated cells (LoVo, which carry a G7 allele at exon 3 and a G9 allele at exon 3 (codons 38 to 41) of the BAX gene), we determined the threshold of sensitivity to be greater than 10% of frameshift mutated cells. We also confirmed with Western blot analysis the absence of BAX protein expression in the case of BAX gene frameshift mutations at exon 3 (data not shown). One of the 41 metastases was positive for BAX frameshift mutation with a deletion of one deoxyguanosine in the G8 tract. In immunohistochemistry, this tumor was found to be "negative" for BAX protein expression (Fig 1). This confirms that BAX mutation results in loss of BAX expression in clinical samples. The patient was a 36-year-old female with a T3N1G1M1 tumor of the colon descendens, and survival was 30 months after surgery. This tumor was considered to carry the MMP+, because such frameshift mutations have been described previously only in the presence of this type of genetic instability.
Because the phenotype of MMP+ tumors (which have a generally more indolent clinical course) differs markedly from that of MMP- colorectal cancers, we excluded this tumor from survival analysis.
Analysis of BAX Expression Interestingly, there seems to be an association between the lymph node involvement (N stage) of the primary tumor and the BAX expression in the liver metastases. The number of BAX-expressing cells declined from stage N0 to N1 and N2. In parallel, increasing size of primary tumor (T stage) and higher grading of the tumor also showed a trend for lower BAX expression in the higher stages (Fig 2).
Follow-Up
Univariate Survival Analysis
The low BAX expression could be a consequence of a defective activation of the BAX gene. With regard to the described transcriptional regulation of the BAX gene by p53, we analyzed BAX expression in relation to the p53 mutational status (see below). However, no significant difference was seen between mean BAX expression levels (product of the number of BAX-expressing cells and the staining intensity) in p53 wild-type tumors as compared with p53 mutated tumors (P > .1; Fig 4A).
p53 Mutations and p53 Expression p53 mutations, as assessed by SSCP-PCR, were found in 14 of 40 patients (35%). Eight mutations were located in exon 7, four mutations were located in exon 5, two were located in exon 8, and one was located in exon 6. One tumor carried both mutations in exon 6 and exon 7. The p53 mutational status did not result in clear-cut changes of the mean p53 expression level (Fig 4B). There was no association between p53 mutational status and staining pattern: in the homogeneously nuclear p53-expressing subgroup, six of 23 tumors were mutated. In the heterogeneous subgroup, three of four were mutated. In the subgroup with no p53 expression, five of 13 were mutated.
p53 Status and Survival Overall survival was independent of p53 mutational status (Fig 5A) or p53 protein expression (Fig 5B). Interestingly, p53 mutational status and nuclear accumulation of p53 showed diverging trends with regard to their impact on survival. There was a nonsignificant trend for better survival with non-p53 accumulating tumors, whereas no such trend was observed in p53 wild-type tumors as compared with p53 mutated tumors.
p21 Expression and Survival
p21 staining was mainly nuclear. The mean percentage of p21-expressing cells was 6.0% ± 2.0% (range, 0% to 60%). Twenty-five of 40 tumors showed no nuclear expression of p21 at all. Five of 40 tumors showed
p53/BAX and Overall Survival
Univariate and Multivariate Regression Analysis of Survival
When the univariate analysis was performed for the subgroup of patients with p53 wild-type tumors, high grading (G3) and low BAX expression were associated with increased risk of early death. In the multivariate analysis, only low BAX expression was associated with a significantly increased risk for shorter survival time (relative risk = 8.21; P = .0095). The frequency distribution of the analyzed dichotomized variables in relation to BAX expression is shown in Table 3. Although we analyzed only a relatively small sample number, all the parameters show a random distribution, except the nodal status. Here we observed that low BAX expression is more frequent in the group with lymph node involvement of the primary tumor. Nevertheless, only BAX expression reveals some prognostic power in the multivariate analysis.
In the present study, we performed a pathway analysis of genes and proteins known to be activated in the p53-mediated response to genotoxic damage: BAX, a proapoptotic member of the bcl-2 family, and p21, a cyclin-dependent kinase inhibitor known to take part in the regulation of the G1-restriction point. p21 is transcriptionally activated by p53, and BAX is a downstream proapoptotic effector of p53, which may also be transcriptionally activated by p53. Mutations in the p53 gene may prevent activation of these downstream effectors. To this end, we analyzed expression and mutations of the proapoptotic BAX gene and its upstream regulator p53 with regard to their impact on overall survival in a cohort of 41 patients with metastatic colorectal cancer. All patients suffered from hepatic metastases and underwent R0 resection. The most striking finding in our p53/BAX pathway analysis is the potential prognostic value of the BAX protein, which is a key regulator of apoptosis. Patients with high BAX expression have a significantly better prognosis in the analysis of overall survival. Median overall survival is 18.2 months longer for patients with BAX-positive tumors compared with patients with BAX-negative tumors (53.6 v 35.4 months). This dichotomy in overall survival becomes even more striking when only patients with p53 wild-type lesions are analyzed. In p53 wild-type metastases, median survival is 30.7 months shorter for tumors with low BAX expression (54.0 v 23.3 months). In patients with p53 wild-type lesions, multivariate analysis showed an 8.2-fold increase in risk for shorter survival time for patients with tumors demonstrating low BAX expression. We analyzed a relatively small cohort of R0-resected liver metastases, but comparison to the recently published largest series of 456 consecutive resections of liver metastases reveals similar survival data11: 1-year survival of 88% (present cohort, 92%), 3-year survival of 59% (present cohort, 58%), and 5-year survival of 29% (present cohort, 30%). Therefore, selection bias with the gathering of patients for the present investigation seems unlikely as cause for the observed effect of BAX on survival. A possible bias in this small sample would have been present by analyzing hepatic metastases of MMP+ tumors. As many as 15% of sporadic colorectal cancers are MMP+, and half of them do exhibit a BAX frameshift mutation17 leading to a truncated protein, which may not be detected by the antibodies currently used in immunohistochemistry. We therefore at first screened all tumor samples for the presence of a frameshift mutation in the BAX gene. We found that only one sample of the 41 tested carried such a frameshift mutation, which is consistent with the reported frequency in colorectal cancer. We considered this tumor to belong to the MMP+ group and excluded this patient from all prognostic analysis, because MMP+ tumors are known to have a distinct phenotype. This is in line with the observation that in colorectal cancer with the MMP+ phenotype, BAX mutations and p53 mutations seem to be mutually exclusive events, as recently reported by Simms et al.22 In this study, univariate analysis identified BAX as a positive prognostic marker. In contrast, p53 overexpression or mutation showed only marginal influence on survival, and no effect was seen when p21 nuclear expression was analyzed. In the multivariate regression analysis, the prognostic value of BAX is also superior to conventional parameters (ie, tumor-node-metastasis staging and grading). There is increasing evidence from other tumor entities concerning the prognostic value of BAX. In patients with breast cancer, loss of BAX immunostaining is associated with a decreased response to chemotherapy and shorter survival.10 Recently, it was shown for ovarian cancer that high BAX expression is associated with significant improvement of the percentage of complete remissions after first-line chemotherapy with paclitaxel and a platinum analog. Survival is also prolonged in the BAX high-expressing group.23 In diffuse aggressive non-Hodgkin's lymphoma, reduced BAX expression was associated with a lower 8-year survival, although BAX expression was not an independent factor in the multivariate analysis in this cohort.24 From a functional point of view, BAX plays a central role in regulation and commitment to programmed cell death. BAX counteracts the apoptosis-preventing effect of Bcl-2 and may directly induce apoptosis.25,26 The proapoptotic BAX is located in the outer mitochondrial membrane.27 BAX overexpression induces mitochondrial permeability transition, which leads to the release of cytochrome c.28 Recently, BAX has been shown to release cytochrome c without mitochondrial swelling,29 which suggests that a different pathway of mitochondrial activation and cytochrome c release might exist as compared with other apoptosis signaling cascades.30 Cytochrome c, together with APAF-1, induces autocleavage of caspase-9, and the subsequent activation of the effector caspase-3. Cleavage of caspase substrates such as poly-(adenosine 5'-diphosphate-ribose) polymerase and DNA fragmentation factor,31,32 leading to the degradation of the chromosomal DNA, is believed to irreversibly trigger the execution phase of apoptosis. BAX-negative tumors, therefore, may be less susceptible to apoptosis and thus may have a growth advantage, despite intact p53. p53 mutations have been described in a wide variety of tumors.33 More than 90% of mutations occur in the DNA-binding site of p53, which is coded by exons 5 to 8. Wild-type p53 is upregulated after cellular stress such as DNA damage or hypoxia.34 It mediates pathways that lead to a G1 arrest and may allow mechanisms for DNA repair to be active before entering S phase or induces apoptotic pathways that drive cells to commit suicide. In colorectal cancer, p53 mutations occur with a frequency of 35% to 60%.35,36 Survival analysis involving p53 mutational status yielded diverging results, either demonstrating p53 mutations37-39 or accumulation40-42 to be of negative prognostic value or demonstrating p53 to not be of prognostic relevance.43-45 The question of p53 status in hepatic metastases as compared with the primary colorectal tumor was addressed in two previous studies. Belluco et al46 found an identical immunohistochemical staining pattern for p53 in 27 pairs of primary tumors and their corresponding hepatic metastases. A study analyzing 18 such tumor/metastasis pairs revealed an increase in p53 mutations in the hepatic metastases.47 We found a mutated p53 gene in 35% of the hepatic metastases. This is in the lower range of what is found in primary colorectal adenocarcinomas. p21 expression served as a control for the functional relevance (and completeness) of our p53 mutational analysis, because it has been shown that p21 is transcriptionally regulated by p53.48 The significant decrease of p21 expression, in our 14 patients with p53 mutations, offers some validation control at the functional level for the accuracy of our mutational analysis. Immunohistochemical analysis for nuclear p53 protein expression was thought to discriminate between wild-type p53 and mutated p53 because of the short half-life of wild-type p53 (no immunohistochemical detection) and the increase of half-life with mutations in the p53 gene.49 In our cohort, there is no correlation between p53 protein nuclear accumulation and p53 mutation analysis. This is not a surprising finding, because concordance between p53 nuclear overexpression and gene mutation (both positive and negative) was only 68% for colorectal carcinomas in a study by Soong et al.50 Another study of 109 colorectal adenocarcinomas performed by Dix et al51 showed only 69% concordance between the two technologies. Further studies comparing immunohistochemical detection of p53 protein (stabilization of p53 protein) and mutation analysis also showed that a substantial proportion of carcinomas with stabilized p53 protein as detected by immunohistochemistry do not contain mutations in exon 5 to 8, whereas some mutations are not associated with protein stabilization.52,53 Epigenetic phenomena, which may include posttranslational processing, may account for a significant proportion of p53-positive tumors as assessed by immunohistochemistry. Colorectal primary tumors have been extensively investigated for alterations in the p53 gene, either on the protein expression level or by the use of mutational analysis, mainly SSCP-PCR. By these analyses, which overall gave conflicting results, no clear prognostic value for p53 could be determined, nor was there a correlation between tumor size, stage, or grade. In contrast to p53, the lack of BAX protein leads to a clearly decreased survival. Our analysis shows that in p53 wild-type tumors, lack of BAX is an indicator for poor survival. Thus the defect of a common and central downstream regulator of apoptosis seems to be deleterious with regard to prognosis as compared with defects in p53. Interestingly, no decreased BAX expression was seen in patients carrying mutated p53. Thus, in case of a defective p53-to-BAX pathway (as in p53 mutated tumors), possibly redundant genes and proteins do bypass the defect in BAX activation by p53, thus allowing some cell homeostasis without the deleterious effect on survival of reduced BAX protein expression. What these redundant bypassing pathways are and how they are regulated is the subject of ongoing research. In contrast, tumor cells carrying frameshift mutations of the BAX gene were also BAX-negative in immunohistochemistry. Therefore, we believe that the analysis of BAX, in concert with p53, may prove valid in identifying patients who would benefit from aggressive treatment in colorectal cancer. Thus the prognostic value of BAX is currently analyzed in larger cohorts of colon cancer patients undergoing systemic chemotherapy.
Supported by the Deutsche Forschungsgemeinschaft grants no. SFB 273 and SFB 506, and by the European Union network grant, Regulation of Apoptosis in Tissue Homeostasis and Cancer, within the Training and Mobility of Researchers Program. We thank Carmen Hahstedt for expert technical assistance with the SSCP-PCR and fragment length analysis, Olrik Lischka for expert technical assistance in immunohistochemistry, and Bernd Schicke for help in statistical analysis and critical discussion.
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Copyright © 1999 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
10% stained cells was considered "low expressing." For p21, the cutoff value was set at 5% (due to lower p21 expression levels). Correlations with dichotomized immunostaining data (low expression/high expression) were calculated according to the Cox-Mantel log-rank test (which compares Kaplan-Meier plots with emphasis on the end points of the curve) and the Breslow-Gehan Wilcoxon test (which compares Kaplan-Meier plots with emphasis on the first half of the survival analysis). 
2 test were applied. 
