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© 2001 American Society for Clinical Oncology Analysis of p53/BAX/p16ink4a/CDKN2 in Esophageal Squamous Cell Carcinoma: High BAX and p16ink4a/CDKN2 Identifies Patients With Good PrognosisFrom the Department of Hematology, Oncology and Tumor Immunology, Charité - Campus Berlin-Buch, Humboldt University; Invitek GmbH; Theragen AG; and Max Delbrück Center for Molecular Medicine, Berlin-Buch; Institute of Pathology, Charité - Campus Berlin-Mitte, Berlin; and Department of General and Vascular Surgery, University Hospital, Johann Wolfgang Goethe University, Frankfurt am Main, 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: We have previously shown that loss of BAX expression is a negative prognostic factor in metastatic colorectal cancer. In the present study, we addressed the prognostic relevance of BAX and its upstream regulator p53 in squamous cell carcinoma (SCC) of the esophagus. Analysis of p16ink4a/CDKN2 was included because p16ink4a/CDKN2 and p53 were shown previously to cooperate during induction of cell cycle arrest and apoptosis. PATIENTS AND METHODS: Retrospective analysis of 53 patients with curative intended R0 resection of esophageal SCC was done. Protein expression of BAX, p53, and p16ink4a/CDKN2 was investigated by immunohistochemistry. In addition, tumor DNA was screened for BAX frameshift mutations by fragment length analysis and for p53 mutations by single-strand conformation polymorphism–polymerase chain reaction. RESULTS: Overall median survival was 13.7 months. Patients with high BAX protein expression had a median survival of 19.5 months versus 8.0 months with low BAX expression (P < .005). High p16ink4a/CDKN2 protein expression was associated with a median survival of 23.8 months versus 9.7 months with low p16ink4a/CDKN2 (P = .011). The best survival (median, 45.8 months) was seen in a subgroup of 12 patients whose tumors bore the combination of both favorite phenotypes (ie, high BAX and high p16ink4a/CDKN2 protein expression). CONCLUSION: In this retrospective investigation, the combined analysis of BAX and p16ink4a/CDKN2 shows subgroups in SCC of the esophagus with favorable (p16ink4a/CDKN2/BAX high expressing) or poor prognosis (loss of p16ink4a/CDKN2/loss of BAX). We suggest that such a multimarker analysis of apoptosis pathways could be useful for individualization of therapeutic strategies in the future, and suggest prospective studies to confirm these results.
ESOPHAGEAL CANCER is an aggressive disease with a generally poor prognosis. Frequently, patients present late with obstructive symptoms indicating advanced tumors. Therefore, cure after surgery is rare, and combined modalities such as radiochemotherapy are evaluated to improve prognosis. Despite the high risk of surgical treatment, the 2-year survival rate is still only in the 20% range.1 The principal treatment is surgery alone or in combination with radiotherapy or, more recently, radiochemotherapy. Nevertheless, progress in treatment results is more or less stagnating. Prediction of tumor aggressiveness by means of analysis of novel, molecularly defined prognostic markers might therefore yield strategies for individual escalation or de-escalation of antitumor therapy. To this end, there is increasing evidence that resistance toward apoptosis is not only involved in tumorigenesis, but also confers resistance to antitumor therapy.2,3 In this line, we previously observed a defect in expression of the proapoptotic BAX protein, a key promoter of apoptosis in breast cancer.4 Restoration of BAX expression in breast cancer cell lines inhibited tumorigenicity5 and increased sensitivity to cytotoxic drug therapy.6,7 We previously showed that overexpression of the BAX-related proapoptotic BIK/NBK could sensitize resistant tumor cells for drug-induced apoptosis8. In breast cancer patients, a reduced BAX expression correlates with a poor response to chemotherapy and shorter overall survival.9 In diffuse aggressive non-Hodgkin’s lymphoma,10 in ovarian cancer,11 and in pancreatic cancer,12 reduced BAX expression was shown to be a negative prognostic factor. In metastatic colorectal cancer, we recently found that the loss of BAX expression is most deleterious in those patients carrying the wild-type p53 gene.13 Previous data showed a cooperation between the cyclin-dependent kinase inhibitor p16ink4a/CDKN2 and p53 in p53-induced cell death.14 The inactivation of p16ink4a/CDKN2 has been correlated with a bad prognosis in malignant melanoma,15 pancreatic adenocarcinoma,16 leukemia,17,18 non–small-cell lung cancer,19 and squamous cell carcinoma (SCC) of the lung.20 In some cases of familial predisposition to melanoma and pancreatic adenocarcinoma, germ-line mutations of p16ink4a/CDKN2 could be identified.21,22 In esophageal SCC, it has been shown that inactivation of the p16ink4a/CDKN2 gene by homozygous deletion or hypermethylation is associated with advanced tumor stages,23 and that patients whose tumors exhibit loss of p16ink4a/CDKN2 protein expression have a significantly shorter survival.24 We therefore were interested in determining whether the combined analysis of p16ink4a/CDKN2 with p53 /BAX could identify patients with especially favorable or especially deleterious prognosis. The study presented here is a retrospective analysis. The aim of the study is to propose a hypothesis for further prospective studies.
Patients Fifty-three patients with curative intended R0 resection of SCC of the esophagus from a single institution (Department of General and Vascular Surgery, University Hospital, Frankfurt) were included in this retrospective analysis: 40 men and 13 women with a median age of 54.9 years (range, 39.6 to 70.2 years). Resection of the esophagus was performed between 1985 and 1995. No distant metastases were present at the time of operation. Overall, 63 patients with SCC of the esophagus were treated between 1985 and 1995 with upfront curative intended R0 resection (defined by tumor-free resection margins on pathology evaluation) of the esophagus. Paraffin-embedded resection material was available from 53 patients, and actual follow-up data concerning survival were assessed with the help of the patients’ general care physicians and the official administration department. The 10 patients that could not be investigated because of missing paraffin blocks were nine men and one woman with a median age of 46 years (range, 42 to 75 years), who underwent surgery between 1987 and 1991. The stage of primary tumor as well as further patient characteristics are listed in Table 1. Transthoracic esophagectomy was performed in the majority of the patients (33 of 53), and alternative transhiatal esophagectomy was performed in the remaining 20 patients. In 49 patients, a gastric conduit was used to reestablish gastrointestinal continuity. The remaining four had a conduit with colon or small intestine. Fifteen patients received radiation therapy or combined radiochemotherapy after surgery.
Immunohistochemistry for BAX, p53, and p16ink4a/CDKN2 For protein detection by immunohistochemistry, 4-µm slices from paraffin-embedded tissue were stained as described in detail elsewhere.13 The primary antibody was a rabbit polyclonal antibody for BAX (AB-1 [dilution 1:50], Oncogene Research Products, Cambridge, MA) and a mouse monoclonal antibody for p53 (clone DO-7 [dilution 1:75], Dako, Denmark) and p16ink4a/CDKN2 (clone G175-405 [dilution 1:150], Pharmingen, Hamburg, Germany). Analysis of slides was performed blinded, by two observers without knowledge of clinicopathologic data. Four high-power fields (x400) were evaluated for percentage positive cells (0% to 100% in 5% steps for BAX and p53, in 10% steps for p16ink4a/CDKN2), and staining intensity (0 to +++). For further analysis, we used the percentage of positive cells as well as a product of percentage positive cells and staining intensity. The results for both methods were comparable, and for clarity of data presentation, we decided to present "percentage of stained tumor cells" for all analyses. In addition to the primary tumors, paraffin-embedded lymph nodes from all 26 patients with N1 status were available for histologic investigation. In only 16 of the 26 patients was the amount of tumor cells in the lymph node considered evaluable by immunohistochemistry.
Mutation Analysis of p53 p53 mutations in the DNA-binding region were detected by single-strand conformation polymorphism–polymerase chain reaction (SSCP-PCR) analysis. Precise description and primer sequences of the method are given elsewhere.13 Briefly, exons 5 to 8 of the DNA-binding domain of the p53 gene were amplified, and for SSCP analysis,5 µL of the amplified fragments were diluted in 7 µL loading buffer (82% formamide, 10 mmol/L NaOH, 50 mmol/L EDTA, bromophenol blue, xylene cyanol 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 Multiphor electrophoresis chamber (Pharmacia, Freiburg, Germany) and were subsequently visualized by silver staining.
BAX Frameshift Mutations
Statistical Analysis
We examined the frequency distribution graph (in 10% steps) visually in order to find a "natural notch" in the distribution. Because there was no clear biphasic distribution for BAX, p53, and p16 expression, we considered the cutoff point applied in a previous investigation in metastasized colorectal carcinoma13 for BAX and p53. For both tumor series, the same method was applied. Because of a higher median expression of BAX and p53 in the SCC compared with the adenocarcinoma (BAX, 30% v 15%; p53, 50% v 35%), a higher cutoff in the SCC series was applied: 20% instead of 10% (ie,
For p16, we had no previous experimental experience; therefore, a new cutoff value had to be defined. This was done in 10% steps with the "minimum P value approach" (Pmin).26 This means that the cutoff point was searched for the value that would yield the best discrimination between the two Kaplan-Meier survival curves, as assessed by the repeated application of the log-rank test. The cutoff point found here could be the basis for further (prospective) studies. The chosen value of 70% (< 70% stained tumor cells are defined as "low expressing," For multivariate regression analysis of survival, the Cox model was developed in a backward and forward fashion (backward and forward stepwise selection of categorical variables) on the basis of changes in likelihood interactions between the different parameters, with the parameters listed in Table 2 and, in addition, with an interaction term for p53 status*BAX expression and p16ink4a/CDKN2 expression*BAX expression.
For intervariable assessment, the Mann-Whitney U test for continuous variables, the Wilcoxon signed rank test for paired analysis (comparison of primary tumor with lymph node metastasis), and the  2 test or Fisher’s exact test, where appropriate, were applied.
Follow-Up We analyzed patients who underwent curatively intended R0 resection of SCC of the esophagus. Data for age, gender, stage, grading, kind of operating procedure, and whether additional radiochemotherapy was given are listed in Table 1. At the end of the follow-up period, nine of 53 (17%) patients were still alive. Median follow-up after esophageal resection for the nine censored patients was 48 months (range, 24 to 144 months). Median overall survival was 13.7 months for the whole group. Including the 30-day perioperative mortality, the overall 1-, 2-, and 5-year survival rates were 52%, 24%, and 17%, respectively.
Analysis of p53, BAX, and p16ink4a/CDKN2 Expression We searched for an association between p53, BAX, or p16ink4a/CDKN2 expression (continuous values) and tumor stage, lymph node involvement, or grading (dichotomized values, see Patients and Methods) with the unpaired Mann-Whitney U test. We did not find significant differences for p53, BAX, or p16ink4a/CDKN2 expression ( Fig 1). There was a tendency for an increased percentage of p16INK4A/CDKN2 staining tumor cells in dedifferentiated tumors (G3 and G4 [n = 10]; median, 70% [range, 10% to 100%]; G1 and G2 [n = 43]; median, 35% [range, 0% to 90%]; P = .06), which was qualified when staining indices are compared (G3 and G4; median, 90 [range, 20 to 300]; G1 and G2; median, 70 [range, 0 to 270]; P = .2).
Fifteen patients received radiation therapy or combined radiochemotherapy after surgery. These patients were equally distributed in the BAXhigh (seven of 21 with additional therapy) and BAXlow groups (eight of 32 with additional therapy) and in the p16ink4a/CDKN2high (six of 17) and p16ink4a/CDKN2low groups (nine of 36) (Fisher’s exact test, P > .5 for both variables).
BAX and p16ink4a/CDKN2 Expression in Lymph Node Metastasis
Analysis of p53 and BAX Mutations p53 mutations in exons 5 to 8 of the p53 gene (encoding the DNA-binding domain, where > 95% of all mutations in the p53 gene in tumors are located) were found in 12 of 53 primary tumors (22.6%). Altogether, 21 mutations were detected: one was located in exon 5, two in exon 6, five in exon 7, and 13 in exon 8. Two primary tumors and one lymph node metastasis exhibited mutations in two exons. One additional lymph node metastasis showed a mutation in exon 8, which could not be detected in the corresponding primary tumor. p53 mutated tumors had only a tendency for increased p53 protein expression (median, 75% [range, 0% to 95%] v 50% [range, 1% to 100%); P = .19). Although p53 was described as a transcriptional activator of BAX expression, the median number of tumor cells with positive protein expression of BAX was unaffected by the p53 mutational status ( p53 mutated tumors: median BAX expression, 30% [range, 2% to 100%) v 30% [range, 0% to 100%]; P = .63 in the wild-type tumors), as was the p16 expression ( p53 mutated: median, 32.5% [range, 3% to 70%]; p53 wild-type: median, 48% [range, 0% to 100%]; P = .42). Since the differential BAX expression could also be the consequence of BAX frameshift mutations, which lead to the introduction of premature stop codons and loss of protein expression,13,25 we performed fragment length analysis of a BAX gene PCR product encompassing the G(8) tract in exon 3 of the BAX gene. Established cell lines of human gastrointestinal tumors were used as positive (LoVo, biallelic mutation, insertion and deletion) and negative (SW620, wild-type) control. None of the 53 primary tumors or the 26 lymph node metastases carried a BAX frameshift mutation.
Effect of BAX, p16ink4a/CDKN2, and p53 on Survival In the univariate survival analysis, patients with BAX-expressing tumors show a better overall survival ( Fig 3A). The median survival is 8.0 months for patients with low BAX expression but 19.5 months for those patients with BAX high expressing tumors (Cox-Mantel log-rank test, P = .0028). There was no prognostic impact of p53 gene mutation and protein expression alone in the survival analysis (Fig 3, B and C).
As shown above, patients who are negative for BAX expression have a decreased overall survival (Fig 3A). In a previous study on metastatic colorectal cancer, we demonstrated that the inclusion of the p53 mutational status to the survival analysis corroborates the power of BAX as a prognostic marker.13 Therefore, we performed Kaplan-Meier analyses for the four subgroups: p53 wild-type (WT)/BAXhigh, p53 WT/BAXlow, p53 mutation/BAXhigh, and p53 mutation/BAXlow. Figure 4 shows that the worst survival is observed in the group with disrupted p53 and BAX pathway ( p53 mutation/BAXlow; median survival, 3.4 months), whereas the longest survival is seen in the subgroup with intact p53 and BAX pathway ( p53 WT/BAXhigh; median survival, 14.6 months). A Cox-Mantel log-rank test for the comparison of the four curves (3 df) gives a P value of .0066 for a significant difference between the four curves. By inspection, it can be seen (Fig 4) that the p53 WT/BAXhigh subgroup differs from the remaining three subgroups.
P16ink4a/CDKN2 was shown previously to synergize with p53-induced apoptosis.14 We therefore included p16ink4a/CDKN2 in the present analysis. For patients with p16ink4a/CDKN2low-expressing tumors, median survival is 9.7 months versus 23.8 months for patients with p16ink4a/CDKN2high tumors (Pmin = .011, Pcorr = .116; see Patients and Methods) ( Fig 5A). There was no significant correlation between loss of BAX and loss of p16ink4a/CDKN2 expression (Fisher’s exact test, P = .37).
We next combined the information on BAX with the p16ink4a/CDKN2 status, and performed survival analysis for the four subgroups p16ink4a/CDKN2high/BAXhigh, p16ink4a/CDKN2high/BAXlow, p16ink4a/CDKN2low/BAXhigh, and p16ink4a/CDKN2low/BAXlow. Figure 5B shows that the worst survival is observed in the two groups with low BAX (median survival p16ink4a/CDKN2low/BAXlow, 8.2 months; p16 ink4a/CDKN2high/BAXlow, 7.7 months), whereas the longest survival is seen in the subgroup with intact p16ink4a/CDKN2 and intact BAX (p16ink4a/CDKN2high/BAXhigh median survival, 45.8 months). A Cox-Mantel log-rank test for the comparison of the four curves (3 df) gives a P value of .002 (Pcorr = .033; see Patients and Methods) for a significant difference between the curves. Inspection of Fig 5B shows that the group with the best survival is the p16ink4a/CDKN2high/BAXhigh subgroup. The corresponding 1-, 2-, and 5-year survival rates, as estimated with the Kaplan-Meier method, are listed in Table 3. p53 was not included because a further subdivision did not make sense with regard to the low number of patients in the subgroups.
Multivariate Regression Analysis of Survival In a multivariate regression model of survival with forward and backward stepwise selection out of the variables listed in Table 2 ([T3 and T4 v T1 and T2; N1 v N0; stage I and II v stage III; G3 v G1 and G2], age [> 55 years v  55 years]; additional therapy [v no additional therapy]; gender; kind of operation [transesophageal v transhiatal]; p53 mutation; and dichotomized variables for p53, BAX, and p16ink4a/CDKN2 expression) and, in addition, with an interaction term for p53 status*BAX expression and p16ink4a/CDKN2 expression*BAX expression, only BAX expression, lymph node involvement, and the interaction term for p16ink4a/CDKN2 expression*BAX expression remained after the stepwise procedure in the Cox model ( Table 4).
Of the "conventional" prognostic parameters, lymph node status especially is known to have prognostic implications. The 27 patients who presented with an N0 stage showed a median survival of 18.3 months, whereas the 26 patients with an N1 stage only had a median survival of 8.3 months (comparison of Kaplan-Meier curves with the Cox-Mantel log-rank test , P = .084) ( Fig 6). In the multivariate regression analysis of survival for all patients, low BAX expression (relative risk [RR] for earlier death, 2.3; P = .016) and—the only "conventional" parameter that entered the Cox model—lymph node involvement at the time of resection (RR, 1.97; P = .049 for earlier death) had an influence on survival, together with the interaction term for p16ink4a/CDKN2 expression*BAX expression (RR, 3.84; P = .0063 for earlier death). The parameter p16ink4a/CDKN2 as single parameter was not included by the Cox model, because of the superior influence of the interaction between p16ink4a/CDKN2 expression and BAX expression.
For the interaction of p53 gene status and BAX expression, we could not demonstrate this additive effect by the means of a Cox model, although the Kaplan-Meier curves differ significantly (Fig 4).
The proapoptotic member of the BCL-2 gene family BAX counteracts the apoptosis-preventing effect of BCL-227,28 and may actively induce apoptosis by initiating mitochondrial permeability shift transition.29 This leads to the activation of downstream apoptosis signaling pathways. Expression of BAX can be induced by p53 (eg, on DNA damage or other forms of cellular stress such as hypoxia). We previously demonstrated that the lack of BAX expression is a negative prognostic factor in metastatic colorectal cancer. This loss of BAX was especially deleterious in those patients carrying tumors with the wild-type p53 gene.13 In the present retrospective analysis, we show that the expression profile of BAX is also an important prognostic factor for survival for patients with SCC of the esophagus. To extend this study, we have investigated the effect of p53/p16ink4a/CDKN2 deregulation with respect to prognosis in SCC of the esophagus. P16ink4a/CDKN2 is involved in G1 cell cycle arrest and is—like p53—upregulated in cellular stress such as DNA damage. It may act as an important tumor suppressor.30 Inactivation of the p16ink4a/CDKN2 gene in human tumors is a frequent event and may occur as a result of various processes: on the gene level, homozygous deletion as well as heterozygous deletion have been described as well as point mutations or deletions in exon 1 or 2, leading to frameshift mutations and nonsense proteins. On the epigenetic level, hypermethylation of the CpG islands in the promoter region has been described. Most of these genetic or epigenetic alterations result in reduced or loss of p16ink4a/CDKN2 expression.31,32 Furthermore, from a therapeutic point of view, it is important that human cells lacking p16ink4a/CDKN2 are resistant to DNA damage–induced growth arrest compared with cells that retain p16ink4a/CDKN2 and Rb.33 Here, we show that the combination of an intact BAX with p16ink4a/CDKN2 expression describes a subgroup of patients with excellent prognosis for this disease. This is of potential clinical importance for the treatment of SCC of the esophagus because options are limited. To date, the treatment of choice is surgery when the tumor is resectable. The addition of radiotherapy with or without chemotherapy has not yet resulted in a major improvement of the generally poor outcome.1 Thus, new prognostic markers for therapeutic concepts and clinical decision-making are required. The analysis of apoptotic pathways in human tumors offers a new approach for individual risk assessment. It is recognized that radiotherapy as well as most chemotherapeutic agents induce apoptosis in responding tumors. It is also established that malignancy is associated—at least to some extent—with a decreased capacity to undergo apoptosis, which can be functionally separated from the deregulated cell cycling of cancer cells. Therefore, the investigation of apoptotic pathways and their defects in human tumors appears to be a promising new approach to identify novel prognostic and predictive factors in cancer. Much work has been done with p53, the "guardian of the genome."34 p53 is known to play a central role in sensing and signaling for growth arrest and apoptosis in cells on DNA damage. Mutations impairing p53 function are a frequent event in cancer, and mutations in the DNA-binding domain of the p53 gene in SCC of the esophagus have been described in 17%35 to 40%,36,37 reaching up to 50% in some studies.38,39 Our data support the assumption that p53 inactivation by itself is not decisive for the clinical prognosis in esophageal SCC.36,40-44 The cell death response that is activated by p53 on DNA damage is executed by the apoptosis-promoting BAX protein. We have previously shown that reduced expression of BAX is a negative prognostic factor in patients undergoing potentially curative resection of hepatic metastases of colorectal adenocarcinoma.13 In the present retrospective study, we see that, analogous to metastatic colorectal cancer, BAX expression is of potential prognostic value in SCC of the esophagus. These data are in concordance with observations in ovarian carcinoma,11 breast cancer,4,5,9 pancreatic cancer,12 SCC of the lung,20 and a subtype of diffuse large-cell lymphoma.10 Furthermore, we showed recently that BAX is lost in childhood acute lymphoblastic leukemia at the time of relapse.45 There are several possibilities why BAX is differentially expressed. It is known that some tumors show a frameshift mutation in one or both BAX alleles, thereby impairing BAX protein expression.13,25 The insertion or deletion of one guanosine into the G(8) tract results in a premature translational stop. These tumors do not produce BAX protein.13,25 We excluded this mechanism in esophageal cancer as cause for loss of BAX expression by frameshift analysis. In the present study in SCC, the BAX expression does not correlate with the mutational status of p53 . Thus, p53 does not appear to be the sole transcriptional activator of the BAX gene. Possibly, other members of the p53 family (such as p73 or p5146-48) may exert p53-like action in case of its loss of function. Nevertheless, in our analysis, the shortest survival was observed for tumors with both mutated p53 and loss of BAX expression, although this group consisted of only five patients. In addition, we also have to keep in mind that the sample size of 53 patients might have been too small—resulting in low statistical power—to detect differences that truly exist. It has been shown that p16ink4a/CDKN2 and p53 cooperate to induce apoptotic tumor cell death.14 In this study, we show that impaired expression of p16ink4a/CDKN2 is a negative prognostic factor. We furthermore observe that the five patients with high p16ink4a/CDKN2 expression but loss of BAX have a prognosis as bad as those with loss of BAX alone. Thus, high expression of p16ink4a/CDKN2 per se is not sufficient. We can offer only a speculative explanation: p16ink4a/CDKN2 might act via a BAX-dependent pathway, which would be in line with the cell experimental finding of p16ink4a/CDKN2 and p53 cooperating in apoptosis induction. It is known that part of the tumor suppressor properties of p53 are mediated by BAX. High p16ink4a/CDKN2 expression together with intact BAX—in our cohort, present in 22.6% of patients with esophageal SCC—could provide a tool for identification of a subgroup with excellent prognosis after surgical therapy. This subgroup might either not need any further radiotherapy after surgery with curative intention at all or might even further benefit from a more aggressive adjuvant therapy. This, however, is only a hypothesis that is generated from a relatively small patient group in a retrospective analysis. It remains to be elucidated in a prospective trial, preferentially with stratification for the p16ink4a/CDKN2/BAX status.
Supported by grant nos. SFB 273 and SFB 506 from the Deutsche Forschungsgemeinschaft, and by a network grant from the Regulation of Apoptosis in Tissue Homeostasis and Cancer by the European Union Training and Mobility of Researchers program. We thank Jana Roßius for expert technical assistance, Olrik Lischka for technical assistance in immunohistochemistry, and Dr I. Küchler and A. Fischer for help in statistical analysis.
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Copyright © 2001 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
20% stained cells in a tumor was considered "high expressing" or "positive," < 20% stained cells was considered "low expressing" or "negative"). 
-1.63*Pmin*(1 + loge Pmin), and give both values. 
hne CH, Wolff G, et al: Analysis of the p53/BAX pathway in colorectal cancer: Low BAX is a negative prognostic factor in patients with resected liver metastases. J Clin Oncol 17: 1364-1374, 1999
